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WO2025122955A1 - Administration de virus oncolytiques avec une salmonelle modifiée - Google Patents

Administration de virus oncolytiques avec une salmonelle modifiée Download PDF

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Publication number
WO2025122955A1
WO2025122955A1 PCT/US2024/059002 US2024059002W WO2025122955A1 WO 2025122955 A1 WO2025122955 A1 WO 2025122955A1 US 2024059002 W US2024059002 W US 2024059002W WO 2025122955 A1 WO2025122955 A1 WO 2025122955A1
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cells
vds
salmonella
bacterial cell
cell
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Shradha KHANDUJA
Neil S. Forbes
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University of Massachusetts Amherst
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University of Massachusetts Amherst
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766

Definitions

  • VDS virus-delivering Salmonella
  • the VDS strain contains the PsseJ-lysE delivery circuit and has deletions in four homologous recombination genes ( ⁇ recB, ⁇ sbcB, ⁇ sbcCD and ⁇ recF) to preserve hairpins in the viral genome with a role in replication and infectivity.
  • VDS delivered the genome of a virus to multiple cancers, including breast, pancreatic carcinoma, and osteosarcoma. Viral delivery produced functional viral particles that were cytotoxic and infective to neighboring cells. The release of mature virions initiated new rounds of infection and amplified the infection.
  • Salmonella for delivery circumvents the limitations of oncolytic viruses and provides a novel therapy for cancer.
  • FIG.1 Bacterial delivery of viruses into the cancer cells.
  • VDS carrying the genome of MVMp, invades cancer cells.
  • Salmonella lysis releases the viral plasmid into Salmonella-containing vacuoles (SCVs).
  • SCV breakdown releases the plasmid into the cytoplasm.
  • FIGS.2A-2B ID Salmonella delivers encoding plasmids into cells.
  • CMV-GFP eukaryotic-expression plasmid
  • PsseJ-lysE intracellular delivering gene circuit
  • FIGS. 3A-3D Bacteria with PsseJ-lysE (ID-CMV-GFP) induced significantly more GFP expression than control CMV-GFP bacteria (***, P ⁇ 0.001).
  • FIGS. 3A-3D Parental Salmonella deleted the right-end hairpin and disabled viral replication.
  • A) The MVMp plasmid contains the full MVM genome including the left- and right-end hairpins. This plasmid replicates in Salmonella and produces viruses when in transfected into mammalian cells.
  • An SSPI restriction digest of the MVMp plasmid produced three fragments (left), 5078, 1395 and 732 bp, when maintained in SURE 2.
  • Strains CD, BB’CDF and BCDJ maintained the complete right-end hairpin similar to SURE 2. All of the other strains had a 97 bp deletion resulting in a shorter third fragment (635 bp).
  • FIGS.5A-5F Deletion of homologous recombination genes does not impair growth or cell invasion.
  • FIGS. 6A-6I VDS delivers functional virions that kill cancer cells.
  • VDS-B contains both the MVMp and PsseJ-lysE plasmids
  • BB’CDF contains only the PsseJ-lysE plasmid.
  • VDS-C was compared to strain CD, its bacterial control. After two hours, external bacteria were removed with gentamicin (green arrow). In parallel, positive control cells were directly transfected with MVMp using lipofectamine (bottom). After 36 hours, NS1 expression was measured by immunoblot. After a further 36 hours, cell viability and cell death were measured using crystal violet, ethidium homodimer and MTT.
  • VDS-B invaded cancer cells in culture, the cells produced viral NS1, similar to direct transfection with the MVMp plasmid. Cell invaded with VDS-C did not produce NS1.
  • VDS-B At 60 hours after invasion (or transfection), more dead cells (arrows, ethidium homodimer + ) started to appear in cultures treated with VDS-B or directly transfected with MVMp.
  • F With time, the area of cell death increased for VDS-B and MVMp transfection but remained constant for BB’CDF.
  • G Death caused by VDS-B was greater than BB’CDF (*, P ⁇ 0.05).
  • VDS-C did not increase cell death compared to CD.
  • FIGS. 7A-7C VDS delivers function virions that spread to neighboring cells.
  • conditioned media from VDS-treated cells was applied to cultures of na ⁇ ve cells. Cancer cells (4T1) were incubated (blue arrows) with VDS-B or bacterial controls (BB’CDF) and external bacteria were cleared after two hours with gentamicin (green arrow, top). Positive controls were cells directly transfected with the MVMp plasmid using lipofectamine (bottom). After 72 hours, the culture medium was collected and added to fresh media at a 40:60 ratio. Addition of 50 ⁇ g/mL gentamycin prevented bacterial growth.
  • This conditioned medium was added to na ⁇ ve cultures of (1) HEK- 293T cells to measure virus formation, and (2) 4T1 cancer cells to measure cytotoxicity.
  • VDS-B Treatment with VDS-B produced NS1 when applied to human embryonic kidney cells (HEK- 293T), human osteosarcoma cells (U2OS), and murine pancreatic ductal adenocarcinoma cells (KPCY). No NS1 was expressed in any of the cell lines after being treated with VDS-C.
  • FIGS. 9A-9B Deletion of homologous recombination genes maintains the right-end hairpin of H1PV.
  • FIGS.10A-10I VDS delivers functional virions that kill cancer cells. To measure virus delivery and induced lysis, VDS was applied to cultures of Human Hepatocellular carcinoma (HCC) cells. Human Hepatocellular carcinoma cells (HUH7) were incubated with VDS-B or bacterial controls (BB’CDF).
  • HCC Human Hepatocellular carcinoma
  • BB’CDF Human Hepatocellular carcinoma cells
  • VDS-B contains both the H1PV or MH1PV and PsseJ- lysE plasmids; BB’CDF contains only the PsseJ-lysE plasmid.
  • VDS-C was compared to strain CDS, its bacterial control and VDS-BS was compared to its bacterial control BB’CDFS. After two hours, external bacteria were removed with gentamicin. In parallel, positive control cells were directly transfected with H1PV or MH1PV using lipofectamine. After 36 hours, NS1 expression was measured by immunoblot. After a further 36 hours, cell viability and cell death were measured using MTT.
  • VDS virus delivery and induced lysis
  • HCC Murine Hepatocellular carcinoma
  • Hepa1-6 Murine Hepatocellular carcinoma cells
  • BB bacterial controls
  • VDS- C was compared to strain CDS, its bacterial control. After three hours, external bacteria were removed with gentamicin.
  • positive control cells were directly transfected with H1PV or MH1PV using lipofectamine. After 36 hours, NS1 expression was measured by immunoblot. After a further 36 hours, cell viability and cell death were measured using MTT.
  • FIGS. 12A-12E To measure virus delivery and induced lysis, VDS was applied to cultures of pancreatic ductal adenocarcinoma cells (KPCY). Murine pancreatic ductal adenocarcinoma cells (KPCY) were incubated with VDS-B or bacterial controls (BB’CDF).
  • VDS-C was compared to strain CDS, its bacterial control. After three hours, external bacteria were removed with gentamicin. In parallel, positive control cells were directly transfected with H1PV or MH1PV using lipofectamine. After 36 hours, NS1 expression was measured by immunoblot. After a further 36 hours, cell viability and cell death were measured using MTT.
  • FIGS. 13A-13K To measure virus delivery and induced lysis, VDS was applied to cultures of human embryonic kidney cells (HEK-293T) and human Mammary Adenocarcinoma (MCF 7). KPCY and MCF 7 were incubated with VDS-B or bacterial controls (BB’CDF).
  • VDS-C was compared to strain CDS, its bacterial control and VDS-BS was compared to its bacterial control BB’CDFS. After two hours, external bacteria were removed with gentamicin. In parallel, positive control cells were directly transfected with H1PV or MH1PV using lipofectamine. After a further 72 hours, cell viability and cell death were measured using MTT. For HEK-293T cells A) Cell viability, measured by MTT assay, decreased for H1PV VDS-B compared to BB’CDF (****, P ⁇ 0.0001). B) H1PV VDS-C did not increase cell death compared to CDS.
  • H1PV VDS-BS did not increase cell death compared to BB’CDFS.
  • H) H1PV VDS-BS did not increase cell death compared to BB’CDFS.
  • FIGS. 14A-14H Salmonella delivers functional H1PV virions that are cytotoxic to liver cancer cells: A) Similar to the SURE 2 strain of E. coli, the engineered BB’CDF and CDS strains preserved H1PV genome integrity as shown by both DNA bands after restriction digestion.
  • VDS-B and VDS-C delivered functional virus into Hepa 1-6 mouse hepatoma cells as shown by expression of NS1.
  • H Delivery of H1PV with VDS-B induced significant cellular cytotoxicity compared to the bacterial control (*, P ⁇ 0.05).
  • FIGS.16A-16E Figure 3: VDS-B inhibits the growth of liver cancer in vivo.
  • VDS-B mice injected with VDS-B exhibited minimal tumor growth as compared to mice injected with the BB’CDF control strain.
  • FIGS. 17A-17F. Intravenous injection is similarly efficacious compared to intratumoral injection of VDS-B.
  • VDS-B generates an innate immunostimulatory response against tumors.
  • VDS-B generates an anti-tumor adaptive immune response.
  • D) The ratio of CD8+ T cells to regulatory T cells increased 12-fold after treatment with VDS-B as compared BB’CDF (*, P ⁇ 0.05).
  • mice were challenged with hepa1-6 tumor cells two weeks after splenocyte transfer and monitored for tumor growth for two 66 days.
  • J 75% of na ⁇ ve mice grafted hepa1-6 tumor cells as compared to 0% of mice with adoptively transferred splenocytes from VDS-B treated, tumor bearing mice.
  • K After tumor cell injection, mice with adoptively transferred splenocytes from VDS-B treated mice exhibited no tumor growth, in contrast to the control mice.
  • Engineered Salmonella deliver functional H1PV in liver tumors in vivo.
  • A) C57BL/6 mice with subcutaneous Hepa1-6 tumors were intravenously injected with BB’CDF.
  • OVs Oncolytic viruses
  • OVs When injected systemically, OVs are cleared from the blood and do not effectively reach tumors (1, 5, 7-20). This problem could be overcome with a carrier with tumor specific tropism. It has been shown that bacterial therapies predominantly accumulate in tumors after intravenous injections (21-30). Developing a bacterial system to deliver viruses into cancer cells would couple the benefits of these two microbial therapies and focus treatment specifically to cancers.
  • the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”
  • the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.”
  • the term “about” means plus or minus 10% of the indicated value. For example, about 100 means from 90 to 110.
  • Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.”
  • the terms "individual,” “subject,” and “patient,” are used interchangeably herein and refer to any subject for whom diagnosis, treatment, or therapy is desired, including a mammal. Mammals include, but are not limited to, humans, farm animals, sport animals and pets. A “subject” is a vertebrate, such as a mammal, including a human. Mammals include, but are not limited to, humans, farm animals, sport animals and companion animals.
  • animal is dog, cat, fish, gerbil, guinea pig, hamster, horse, rabbit, swine, mouse, monkey (e.g., ape, gorilla, chimpanzee, orangutan) rat, sheep, goat, cow and bird.
  • treatment e.g., ape, gorilla, chimpanzee, orangutan
  • rat sheep, goat, cow and bird.
  • treatment e.g., ape, gorilla, chimpanzee, orangutan
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • various clinical and scientific methodologies and assays may be used to assess the development or progression of a disorder, and similarly, various clinical and scientific methodologies and assays may be used to assess the reduction, regression, or remission of a disorder or its symptoms.
  • treatment can be applied to a subject or to a cell culture (in vivo or in vitro).
  • inhibitor refers to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, group of cells, protein or its expression.
  • the inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.
  • “Expression” refers to the production of RNA from DNA and/or the production of protein directed by genetic material (e.g., RNA (mRNA)).
  • mRNA RNA
  • Inducible expression is expression which only occurs under certain conditions, such as in the presence of specific molecule (e.g., arabinose) or an environmental que.
  • nucleic acid or a protein
  • a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid.
  • a non-naturally occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature.
  • a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature.
  • any vector, autonomously replicating plasmid, or virus e.g., retrovirus, adenovirus, or herpes virus
  • genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally occurring nucleic acid since they exist as separate molecules not found in nature.
  • an exogenous sequence may therefore be integrated into the genome of the host. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally occurring nucleic acid.
  • a nucleic acid that is naturally occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.
  • endogenous as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature.
  • a cell “endogenously expressing” a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature.
  • a host “endogenously producing” or that "endogenously produces” a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.
  • Engineered Salmonella could be any strain of Salmonella designed to lyse and deliver protein intracellularly.
  • the engineered Salmonella is non-pathogenic.
  • the term "contacting” refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.
  • An "effective amount” is an amount sufficient to effect beneficial or desired result, such as a preclinical or clinical result. An effective amount can be administered in one or more administrations.
  • an effective amount means the quantity necessary to render the desired therapeutic result.
  • an effective amount is a level effective to treat, cure, or alleviate the symptoms of a disorder and/or disease for which the therapeutic compound, biologic or composition is being administered.
  • Amounts effective for the particular therapeutic goal sought will depend upon a variety of factors including the disorder being treated and its severity and/or stage of development/progression; the bioavailability, and activity of the specific compound, biologic or pharmaceutical composition used; the route or method of administration and introduction site on the subject; the rate of clearance of the specific compound or biologic and other pharmacokinetic properties; the duration of treatment; inoculation regimen; drugs used in combination or coincident with the specific compound, biologic or composition; the age, body weight, sex, diet, physiology and general health of the subject being treated; and like factors well known to one of skill in the relevant scientific art.
  • dosage can occur depending upon the condition of the subject being treated, and the physician or other individual administering treatment will, in any event, determine the appropriate dose for an individual patient.
  • disorder refers to a disorder, disease or condition, or other departure from healthy or normal biological activity, and the terms can be used interchangeably. The terms would refer to any condition that impairs normal function.
  • the condition may be caused by sporadic or heritable genetic abnormalities.
  • the condition may also be caused by non- genetic abnormalities.
  • the condition may also be caused by injuries to a subject from environmental factors, such as, but not limited to, cutting, crushing, burning, piercing, stretching, shearing, injecting, or otherwise modifying a subject's cell(s), tissue(s), organ(s), system(s), or the like.
  • the terms “cell,” “cell line,” and “cell culture” as used herein may be used interchangeably. All of these terms also include their progeny, which are any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • a “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
  • “Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position.
  • nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).
  • nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).
  • base pairing specific hydrogen bonds
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • an “essentially pure” preparation of a particular protein or peptide is a preparation wherein at least about 95%, and preferably at least about 99%, by weight, of the protein or peptide in the preparation is the particular protein or peptide.
  • a “fragment” or “segment” is a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide.
  • the terms “fragment” and “segment” are used interchangeably herein.
  • a “functional” biological molecule is a biological molecule in a form in which it exhibits a property by which it is characterized.
  • a functional enzyme for example, is one which exhibits the characteristic catalytic activity by which the enzyme is characterized.
  • “Homologous” as used herein refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
  • the DNA sequences 3’ATTGCC5’ and 3’TATGGC share 50% homology.
  • “homology” is used synonymously with “identity.” The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877).
  • This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site having the universal resource locator using the BLAST tool at the NCBI website.
  • NCBI National Center for Biotechnology Information
  • BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).
  • PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the length of the formed hybrid, and the G:C ratio within the nucleic acids.
  • an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the peptide of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein.
  • the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains the identified compound invention or be shipped together with a container which contains the identified compound.
  • the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
  • nucleic acid typically refers to large polynucleotides.
  • nucleic acid is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
  • phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridge
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
  • nucleic acid encompasses RNA as well as single and double stranded DNA and cDNA.
  • nucleic acid encompasses RNA as well as single and double stranded DNA and cDNA.
  • nucleic acid encompasses RNA as well as single and double stranded DNA and cDNA.
  • DNA DNA
  • RNA and similar terms also include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.
  • peptide nucleic acids which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • nucleic acid is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
  • phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridge
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).
  • bases other than the five biologically occurring bases
  • Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5’-direction.
  • the direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • nucleic acid construct encompasses DNA and RNA sequences encoding the particular gene or gene fragment desired, whether obtained by genomic or synthetic methods. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • Nucleotide sequences that encode proteins and RNA may include introns.
  • the term “oligonucleotide” typically refers to short polynucleotides, generally, no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”
  • “Substantially homologous nucleic acid sequence” means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur.
  • the substantially identical nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence.
  • the percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more.
  • Substantial identity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm.
  • Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50°C; preferably in 7% (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C; preferably 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C; and more preferably in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.
  • Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, GCS program package (Devereux et al., 1984 Nucl. Acids Res. 12:387), and the BLASTN or FASTA programs (Altschul et al., 1990 Proc. Natl. Acad. Sci. USA.1990 87:14:5509-13; Altschul et al., J. Mol. Biol. 1990215:3:403-10; Altschul et al., 1997 Nucleic Acids Res.25:3389-3402). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the present invention.
  • two polynucleotides as “operably linked” is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other.
  • a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region.
  • pharmaceutically acceptable carrier means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.
  • “Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary application.
  • “pharmaceutical compositions” include formulations for human and veterinary use.
  • the term “purified” and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term “purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process.
  • a “highly purified” compound as used herein refers to a compound that is greater than 90% pure.
  • purified sperm cell DNA refers to DNA that does not produce significant detectable levels of non-sperm cell DNA upon PCR amplification of the purified sperm cell DNA and subsequent analysis of that amplified DNA.
  • a “significant detectable level” is an amount of contaminate that would be visible in the presented data and would need to be addressed/explained during analysis of the forensic evidence.
  • “Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • a host cell that comprises a recombinant polynucleotide is referred to as a “recombinant host cell.”
  • a gene which is expressed in a recombinant host cell wherein the gene comprises a recombinant polynucleotide produces a “recombinant polypeptide.”
  • a “recombinant polypeptide” is one which is produced upon expression of a recombinant polynucleotide.
  • a “recombinant cell” is a cell that comprises a transgene.
  • Such a cell may be a eukaryotic or a prokaryotic cell.
  • the transgenic cell encompasses, but is not limited to, an embryonic stem cell comprising the transgene, a cell obtained from a chimeric mammal derived from a transgenic embryonic stem cell where the cell comprises the transgene, a cell obtained from a transgenic mammal, or fetal or placental tissue thereof, and a prokaryotic cell comprising the transgene.
  • the term “regulate” refers to either stimulating or inhibiting a function or activity of interest.
  • binds to when a compound or ligand functions in a binding reaction or assay conditions which is determinative of the presence of the compound in a sample of heterogeneous compounds, or it means that one molecule, such as a binding moiety, e.g., an oligonucleotide or antibody, binds preferentially to another molecule, such as a target molecule, e.g., a nucleic acid or a protein, in the presence of other molecules in a sample.
  • a binding moiety e.g., an oligonucleotide or antibody
  • telomere binding domain a structure allowing recognition and binding to a specific protein structure within a binding partner rather than to molecules in general.
  • a ligand is specific for binding pocket "A,” in a reaction containing labeled peptide ligand "A” (such as an isolated phage displayed peptide or isolated synthetic peptide) and unlabeled "A" in the presence of a protein comprising a binding pocket A the unlabeled peptide ligand will reduce the amount of labeled peptide ligand bound to the binding partner, in other words a competitive binding assay.
  • labeled peptide ligand "A” such as an isolated phage displayed peptide or isolated synthetic peptide
  • unlabeled peptide ligand will reduce the amount of labeled peptide ligand bound to the binding partner, in other words a competitive binding assay.
  • standard refers to something used for comparison.
  • Standard can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function.
  • Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured.
  • Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker. Methods involving conventional molecular biology techniques are described herein.
  • Bacteria Bacteria useful in the invention include, but are not limited to, Clostridium, Bifidus, Escherichia coli or Salmonella, T3SS-dependent bacteria, such as shigella, salmonella and Yersinia Pestis. Further, E.
  • Salmonella coli can be used if the T3SS system is place in E. Coli.
  • Salmonella examples of Salmonella strains which can be employed in the present invention include Salmonella typhi (ATCC No. 7251) and S. typhimurium (ATCC No. 13311). Attenuated Salmonella strains include S. typhi-aroC-aroD (Hone et al. Vacc.9:810 (1991) S. typhimurium- aroA mutant (Mastroeni et al. Micro. Pathol. 13:477 (1992)) and Salmonella typhimurium 7207.
  • Additional attenuated Salmonella strains that can be used in the invention include one or more other attenuating mutations such as (i) auxotrophic mutations, such as aro (Hoiseth et al. Nature, 291:238-239 (1981)), gua (McFarland et al Microbiol. Path., 3:129-141 (1987)), nad (Park et al. J. Bact, 170:3725-3730 (1988), thy (Nnalue et al. Infect.
  • auxotrophic mutations such as aro (Hoiseth et al. Nature, 291:238-239 (1981)), gua (McFarland et al Microbiol. Path., 3:129-141 (1987)), nad (Park et al. J. Bact, 170:3725-3730 (1988), thy (Nnalue et al. Infect.
  • the attenuating mutations can be either constitutively expressed or under the control of inducible promoters, such as the temperature sensitive heat shock family of promoters (Neidhardt et al. supra), or the anaerobically induced nirB promoter (Harbome et al. Mol. Micro., 6:2805-2813 (1992)) or repressible promoters, such as uapA (Gorfinkiel et al. J. Biol. Chem., 268:23376-23381 (1993)) or gcv (Stauffer et al. J. Bact, 176:6159-6164 (1994)).
  • inducible promoters such as the temperature sensitive heat shock family of promoters (Neidhardt et al. supra), or the anaerobically induced nirB promoter (Harbome et al. Mol. Micro., 6:2805-2813 (1992)
  • repressible promoters
  • the bacterial delivery system is safe and based on a non-toxic, attenuated Salmonella strain that has a partial deletion of the msbB gene. This deletion diminishes the TNF immune response to bacterial lipopolysaccharides and prevents septic shock. In another embodiment, it also has a partial deletion of the purI gene. This deletion makes the bacteria dependent on external sources of purines and speeds clearance from non-cancerous tissues (13). In mice, the virulence (LD 50 ) of the therapeutic strain is 10,000-fold less than wild-type Salmonella (72, 73). In pre-clinical trials, attenuated Salmonella has been administered systemically into mice and dogs without toxic side effects (17, 27).
  • the strain of bacteria is VNP20009, a derivative strain of Salmonella typhimurium. Deletion of two of its genes - msbB and purI -resulted in its complete attenuation (by preventing toxic shock in animal hosts) and dependence on external sources of purine for survival. This dependence renders the organism incapable of replicating in normal tissue such as the liver or spleen, but still capable of growing in tumors where purine is available. Further, insertion of a failsafe circuit into the bacterial vector prevents unwanted infection and defines the end of therapy without the need for antibiotics to remove the bacteria (e.g., salmonella). II.
  • DNA, RNA (e.g., a nucleic acid-based gene interfering agent) or protein may be produced by recombinant methods.
  • the nucleic acid is inserted into a replicable vector for expression.
  • the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence and coding sequence.
  • the gene and/or promoter may be integrated into the host cell chromosome or may be presented on, for example, a plasmid/vector.
  • Expression vectors usually contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
  • Expression vectors can contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid sequence, such as a nucleic acid sequence coding for an open reading frame. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription of particular nucleic acid sequence to which they are operably linked. In bacterial cells, the region controlling overall regulation can be referred to as the operator.
  • Promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, hybrid promoters such as the tac promoter, and starvation promoters (Matin, A.
  • bacterial promoters are also suitable. Such nucleotide sequences have been published, thereby enabling a skilled worker to operably ligate them to a DNA coding sequence. Promoters for use in bacterial systems also can contain a Shine-Dalgarno (S.D.) sequence operably linked to the coding sequence. Construction of suitable vectors containing one or more of the above-listed components employs standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required.
  • the expression vector is a plasmid or bacteriophage vector suitable for use in Salmonella, and the DNA, RNA and/or protein is provided to a subject through expression by an engineered Salmonella (in one aspect attenuated) administered to the patient.
  • plasmid refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids.
  • the nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
  • One embodiment provides a Salmonella strain comprising a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter.
  • One embodiment provides an attenuated Salmonella strain comprising a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter.
  • the lysis cassette is Lysin E from phage phiX174, phage iEPS5, or lambda phage.
  • the promoter is a promoter for one of the genes in Salmonella pathogenicity island 2 type III secretion system (SPI2-T3SS) selected from the group SpiC/SsaB (accession no. CBW17423.1), SseF (accession no.
  • SpiC/SsaB accession no. CBW17423.1: 1 MSEEGFMLAV LKGIPLIQDI RAEGNSRSWI MTIDGHPARG EIFSEAFSIS LFLNDLESLP 61 KPCLAYVTLL LAAHPDVHDY AIQLTADGGW LNGYYTTSSS SELIAIEIEK HLALTCILKN 121 VIRNHHKLYS GGV (SEQ ID NO: 1) SseF (accession no.
  • Oncolytic Virus Oncolytic viruses are an emerging class of cancer therapeutics that offer the benefits of selective replication in tumor cells, delivery of multiple eukaryotic transgene payloads, induction of immunogenic cell death and promotion of antitumor immunity, and a tolerable safety profile that largely does not overlap with that of other cancer therapeutics, such as immune checkpoint inhibitors (ICIs) or chimeric antigen receptors (CARs).
  • ICIs immune checkpoint inhibitors
  • CARs chimeric antigen receptors
  • the first oncolytic virus immunotherapy to be approved by the FDA was T-VEC, which is used to treat metastatic melanoma.
  • T-VEC is a herpes virus that has been engineered to be less likely to infect healthy cells. OVs directly kill cancer cells.
  • OVs replicate inside cancer cells, causing them to burst and release materials that the immune system can recognize.
  • OVs can be modified to carry genes that boost the treatment's effectiveness.
  • OVs can be engineered to express immune regulators that enhance antitumor immunity.
  • Oncolytic viruses include, but are not limited to, DNA (ssDNA) viruses, double-stranded DNA (dsDNA) viruses, single-stranded RNA (ssRNA) viruses, double-stranded RNA (dsRNA) viruses or a combination thereof.
  • viruses for use in the invention can include, but are not limited to, adenoviruses, coxsackievirus, herpes simplex virus (HSV), maraba viruses, measles, Newcastle disease virus, picornavirus, reovirus, respiratory syncytial virus, vesicular stomatitis virus, parvoviruses, poxviruses, such as vaccinia virus (VACV) and myxoma virus (MYXV), minute virus of mice (MVM), talimogene laherparepvec (T-VEC, or Imlygic®), and/or poliovirus (such as PVS- RIPO). Any of these viruses can be used in the methods herein.
  • HSV herpes simplex virus
  • MYXV myxoma virus
  • MYXV minute virus of mice
  • T-VEC talimogene laherparepvec
  • Imlygic® Imlygic®
  • poliovirus such as PVS
  • Bacteria such as Salmonella, Clostridium and Bifidobacterium have a natural tropism for cancers, such as solid tumors.
  • Types of cancer that can be treated using the methods of the invention include, but are not limited to, solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cyst
  • the subject is treated with radiation, immunotherapy and/or chemotherapy before, after or during administration of the bacterial cells described herein.
  • Administration includes administration of the attenuated bacteria, such as Salmonella, strains described herein and methods for preparing pharmaceutical compositions and administering such as well. Such methods comprise formulating a pharmaceutically acceptable carrier with one or more of the attenuated Salmonella strains described herein.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • buffers such as
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of other (undesired) microorganisms.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients discussed above.
  • dispersions are prepared by incorporating the active compound into a vehicle which contains a basic dispersion medium and various other ingredients discussed above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously.
  • Oral compositions generally include an inert diluent or an edible carrier. For example, they can be enclosed in gelatin capsules.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the bacteria are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the attenuated Salmonella When administered to a patient the attenuated Salmonella can be used alone or may be combined with any physiological carrier.
  • the dosage ranges from about 1.0 c.f.u./kg to about 1x10 12 c.f.u./kg; optionally from about 1.0 c.f.u./kg to about 1x10 10 c.f.u./kg; optionally from about 1.0 c.f.u./kg to about 1x10 8 c.f.u./kg; optionally from about 1x10 2 c.f.u./kg to about 1x10 8 c.f.u./kg; optionally from about 1x10 4 c.f.u./kg to about 1x10 8 c.f.u./kg; optionally from about 1x10 5 c.f.u./kg to about 1x10 12 c.f.u./kg; optionally from about 1x10 5 c.f.u./kg to about 1x10 10 c
  • EXAMPLE The following examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
  • Example I Introduction Many of the current problems associated with cancer treatment, e.g., metastatic disease and refractory tumors, could be overcome with microbial therapies.
  • Oncolytic viruses (OVs) have the potential to treat many tumors by directly lysing cancer cells and stimulating immune responses that eliminate cancer cells regardless of their location in the body (1-6). Despite this potential, oncolytic viruses have not been effective at treating internal solid tumors (5, 7-10). When injected systemically, OVs are cleared from the blood and do not effectively reach tumors (1, 5, 7-20). This problem could be overcome with a carrier with tumor specific tropism.
  • ID Salmonella intracellular delivering
  • ID Salmonella is a bacterial system recently developed to deliver macromolecules into cancer cells in tumors (31).
  • ID Salmonella contain a genetic circuit (PsseJ-lysE) that controls the release of plasmids and macromolecules (31). In tumors, Salmonella naturally invades cancer cells (31).
  • MMM Minute virus of mice
  • the prototype strain (MVMp) does not integrate into the genome, is non-infectious, and is non-pathogenic in humans (36, 38, 39).
  • NS1 is the initiator protein that creates a site-specific nick in the single-stranded DNA and serves as a helicase to initiate DNA replication (34, 38, 40, 42).
  • This DNA template is used to produce more single-stranded viral genomes, which are assembled in the nucleus into new virus particles (36, 43, 44).
  • Cell lysis kills cells and releases new viral particles that infect neighboring cells and initiates new rounds of infection (5, 10, 20, 45, 46). Because of its small size, the MVMp genome can be included in a standard cloning plasmid (47).
  • MVMp When mammalian cells are transfected with an MVMp-containing plasmid, they transcribe NS1 and produce viral particles, similar to natural infection (47, 48). Bacterial delivery of viral DNA is complicated by hairpins in the genomes of many viruses. These hairpins have a role in DNA replication, packaging, and viral infectivity. Palindromic sequences are not well tolerated in Salmonella and are subject to partial or complete deletion (49, 50). MVMp has two imperfect palindromes at the 3’ and 5’ termini that form hairpins in the DNA of the viral genome (32, 34, 41, 43, 47, 51-53). These palindromic regions are primers for replication and are used in virus formation and function (33, 41, 43, 47, 51-53).
  • homologous recombination When transformed into cloning strains of E. coli, homologous recombination forms site-specific deletions in the right-end hairpin of plasmids containing the MVMp genome (51). This deletion is prevented in SURE 2 (‘stop unwanted rearrangement events’ 2) E. Coli ( ⁇ recB, ⁇ recJ, and ⁇ sbcC), which is restriction minus, endonuclease deficient, and recombination deficient.
  • Salmonella contains two systems for repairing DNA damage and regulating homologous recombination: one for double-stranded breaks (DSBs) and another for single- stranded gap (SSG) repairs (54-56). These systems maintain genetic stability but also modify foreign DNA (54).
  • RecBCD A key component of the DSB repair system is RecBCD, which is a helicase- nuclease complex that initiates repair by unwinding double-stranded DNA (dsDNA) and creating a single-stranded tail for RecA to bind (54, 57-61).
  • RecA is a central protein in homologous recombination that compares and exchanges complementary DNA sequences (54, 60).
  • SbcB and SbcCD Two additional components of the DSB repair system are SbcB and SbcCD (62).
  • SbcCD is a nuclease that cleaves double stranded hairpins (59-61).
  • the primary component of SSG repair system is RecFOR (54, 57).
  • the bacterial vector must (1) preserve the viral genome and (2) deposit it intact into cancer cells. It was hypothesized that 1) DNA delivered by ID Salmonella is expressed by cancer cells, 2) removal of homologous recombination genes from Salmonella prevents hairpin deletion and stabilize the MVMp genome, 3) Salmonella delivery of the MVMp genome produces functional viral particles, and 4) the produced viral particles are infective and kill cancer cells. To test these hypotheses, seven strains of Salmonella were generated with deletions in six genes: recA, recB, recF, sbcB, sbcCD, and recJ. For H1PV and MH1PV sseJ knockouts were created in the strains which could stabilize the hairpins.
  • VDS Virus- delivering Salmonella
  • HRDS homologous-recombination- deficient Salmonella
  • HRDS homologous-recombination- deficient Salmonella
  • Fluorescence microscopy was used to measure the delivery of plasmid DNA to cancer cells with ID Salmonella.
  • Gentamicin invasion assay was used to determine the viability and invasiveness of the HRDS strains.
  • Cancer cells were treated with VDS to measure the production of NS1, the initiation of virus formation, and the cytotoxicity of the delivered virus. Finally, cells were retreated with conditioned media to quantify the infectiveness of released virions.
  • VDS By delivering OVs, VDS can expand the effectiveness of microbial therapy to include more solid tumors and be effective for a broader range of cancer patients.
  • Materials and Methods Bacterial culture All bacterial cultures (SURE 2, Salmonella) were grown in LB (10 g/L sodium chloride, 10 g/L tryptone, and 5 g/L yeast extract). Resistant strains of bacteria were grown in the presence of 100 ⁇ g/mL of carbenicillin, 33 ⁇ g/mL chloramphenicol, or 50 ⁇ g/mL of kanamycin.
  • Bacterial strains and plasmids The MVMp plasmid was provided by Dr. Peter Tattersall (Yale School of Medicine, New Haven, CT).
  • the plasmid is ampicillin resistant and has a ColE1 origin of replication.
  • the ptac-GFP plasmid contains green fluorescent protein (GFP) under control of the constitutive bacterial promoter ptac and a Lysin gene E from ⁇ X174 bacteriophage under the control of intracellular responsive Salmonella promoter PsseJ (31).
  • This plasmid is chloramphenicol resistant and has a p15A origin of replication.
  • HEK-293T Human embryonic kidney cells
  • RRID:CVCL_0063 human osteosarcoma cells
  • U2OS RRID:CVCL_0042
  • KPCY murine pancreatic ductal adenocarcinoma cells
  • DMEM low glucose Dulbecco’s modified Eagle medium
  • FBS fetal bovine serum
  • Murine mammary carcinoma cells (4T1, RRID:CVCL 0125) were maintained in Roswell Park Memorial Institute (RPMI 1640; Sigma Aldrich, St. Louis, MO) medium supplemented with 2 g/l of sodium bicarbonate and 10% FBS at 37 °C and 5% CO2.
  • RPMI 1640 Roswell Park Memorial Institute
  • FBS FBS
  • DMEM Roswell Park Memorial Institute
  • FBS FBS
  • GFP expression from a eukaryote promoter after delivery with Salmonella To measure plasmid and gene delivery, two plasmids for delivery and eukaryotic GFP expression were transformed into parental Salmonella ( ⁇ msbB, ⁇ purI, ⁇ xyl) and termed as ID- CMV-Sal.
  • the GFP expression plasmid contained the gene for green fluorescent protein (GFP) under control of the cytomegalovirus (CMV) mammalian expression promoter.
  • the delivery plasmid contained the PsseJ-lysE circuit.
  • Control bacteria were parental Salmonella transformed with CMV-GFP but not the PsseJ-lysE lysis plasmid. Salmonella strains were grown to an OD 600 between 0.8-1.0 and added to cultures of 4T1 cells for 2 hours. During this period, the bacteria invaded into the cancer cells.
  • strains A: ⁇ recA; B: ⁇ recB; F: ⁇ recF; CD: ⁇ sbcCD; BB’F: ⁇ recB, ⁇ sbcB and ⁇ recF; BCDJ: ⁇ recB, ⁇ sbcCD and ⁇ recJ; and BB’CDF: ⁇ recB, ⁇ sbcB, ⁇ sbcCD and ⁇ recF, CDS: ⁇ sbcCD ⁇ sseJ, BB’CDFS: ⁇ recB, ⁇ sbcB, ⁇ sbcCD and ⁇ recF ⁇ sseJ).
  • Salmonella was transformed with pkd46 (Yale CGSC E. Coli stock center), grown to OD6000.1, and induced with 10 mM arabinose. Once the OD600 reached 0.6-0.8, the bacteria were centrifuged at 3000 ⁇ g for 15 minutes. The pellet was washed twice with ice-cold water. A PCR product for the in-frame deletion of the gene using specific primers (Supplemental Table S1) was amplified from the pkd4 plasmid (accession AY048743.1) containing the FRT- KAN-FRT sequence. The PCR product, which contains 50 bp homology for the gene, was transformed into Salmonella using electroporation.
  • the recovery was plated on kanamycin plates (50 ⁇ g/ml) and grown overnight.
  • the colonies were screened for deletion by performing a colony PCR of the junction sites of the inserted PCR amplified products. Colonies with a successful knockout were grown overnight at 43 °C to eliminate pkd46.
  • the antibiotic resistance was removed by transforming the pcp20 plasmid into the deleted Salmonella. The colonies were grown overnight at 43 °C to eliminate pcp20. After the elimination of the plasmid, the overnight culture was diluted to approximately 100 CFU/ml and plated on hektoen plates. The colonies which were dark green were confirmed as Salmonella.
  • the colonies were sequentially streaked on hektoen, kanamycin, and chloramphenicol agar to verify the removal of the antibiotics. Hairpin stability and bacterial growth To test hairpin stability, the MVMp plasmid was transformed into each knockout strain by electroporation. Electroporation was performed in 1 mm cuvettes at 1800 V and 25 ⁇ F with a time constant of 5 msec.
  • Plasmids were extracted using the ZymoPURE plasmid miniprep kit per the manufacturer’s instructions (Irvine, CA) and sent for sequencing (Massachusetts General Hospital CCIB DNA core facility) To further test hairpin stability, isolated plasmids were digested with SSPI restriction enzyme (New England Biolabs, Ipswich, MA). Strains BB’CDF and CD strains were transformed with a plasmid containing the PsseJ-lysE cassette to deliver the MVMp plasmid and were termed virus-delivering Salmonella (VDS-B and VDS- C, respectively).
  • Salmonella strains were transformed with a plasmid containing EGFP under a constitutive promoter and grown to an OD600 between 0.8-1.0. Salmonella were added to the cell cultures for 2 hours. Following this invasion period, cultures were rinsed with 1X PBS five times and incubated in RPMI with 2 g/L sodium bicarbonate, 10% FBS, and 50 ⁇ g/mL gentamicin. Cells were stained with 10 ⁇ g/mL Hoechst 33342 (Invitrogen, Waltham, MA). Multiple regions in a well were randomly selected, and 30 cells/regions were randomly selected and scored based on EGFP expression to quantify the percentage of the cells that were invaded.
  • VDS-B and VDS-C were added to the HEK- 293T cells at an MOI of 20. After two hours of invasion, extracellular bacteria were removed with gentamicin.
  • the positive control was the MVMp plasmid transfected into na ⁇ ve cells using Lipofectamine 3000 per manufacturer’s protocol (Thermo Fisher, Waltham, MA). Thirty-six hours after infection or transfection, cells were scraped from the flasks. Cells were collected and pelleted by centrifugation at 1000g for 5 minutes. Cells were suspended in Laemmli SDS 6x sample buffer (Alfa Aesar, Haverhill, MA).
  • VDS-B or VDS- C Bacteria (VDS-B or VDS- C) were added to cultures of 4T1 cells at an MOI of 100. Strains without the MVMp plasmid (CD or BB’CDF) were negative controls. The positive control was 4T1 cells transfected with the MVMp plasmid using Lipofectamine 3000. After two hours of invasion, bacteria were cleared with gentamicin. Seventy-two hours after infection/transfection, the cells were fixed with 10% formaldehyde in water for 1 hour at room temperature. Dead cell plaques were stained with crystal violet staining, dead cells were stained with ethidium homodimer and cell viability was determined by MTT assay.
  • crystal violet staining the formaldehyde was removed and the cells were stained with 1% crystal violet stain (Acros Organics, Waltham, MA). Cells were incubated in crystal violet for 15 minutes and rinsed with water. Cells were imaged on a Zeiss Axio Observer Z.1 microscope using a 5x objective lens. Images were tiled to image the entire surface. The area of unstained cells was analyzed using MATLAB. To quantify cell death, 1 ⁇ g/mL ethidium homodimer (Invitrogen, Waltham, MA) was added to cells 72 hours after infection or transfection. Live cell images were acquired every 10 minutes. The experiment was performed in triplicate, and death was determined in three areas per well.
  • the extent of cell death was determined in ImageJ by measuring the area with red fluorescence compared to the area covered by cells at the time of invasion/transfection.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Themo Fisher, Waltham, MA
  • VDS VDS-B
  • BB BB
  • CDF bacterial negative control
  • ID Salmonella was transformed with a plasmid containing GFP under control of the cytomegalovirus (CMV) promoter. This promoter only initiates transcription in eukaryotic cells (65). Salmonella with CMV-GFP did not express GFP. In addition to CMV-GFP, ID-CMV-GFP Salmonella contained the PsseJ-lysE circuit, which induces lysis after cell invasion (31). Control Salmonella contained CMV-GFP, but not PsseJ- lysE. When incubated with murine 4T1 mammary cancer cells, ID-CMV-GFP produced GFP (arrows, FIG 2A).
  • CMV cytomegalovirus
  • ID-CMV-GFP produced significantly more GFP (P ⁇ 0.001, FIG. 2B). This difference indicates that the lysis circuit is needed for delivering plasmids into cancer cells. GFP expression also indicates that after bacterial lysis, released plasmids were transported to the nucleus and delivered genes were translated by recipient cells.
  • Parental Salmonella disables MVMp replication When not modified, parental Salmonella ( ⁇ msbB, ⁇ purI, ⁇ xyl) deletes 97 base pairs from the right-end hairpin of MVMp (FIG. 3). The genome of MVMp contains two palindromes at either end that are primers for replication and are needed for virus formation.
  • the genome for MVMp is on a plasmid that also contains a ColE1 prokaryotic origin of replication, and ampicillin resistance (FIG.3A). Transcription of MVMp is initiated by the P4 promoter within its genome in a mammalian nucleus (44). Replicating this plasmid in SURE 2 recombination-deficient E. coli ( ⁇ recB, ⁇ recJ, and ⁇ sbcC), maintained both hairpins (FIG. 3B). SSPI digestion of the MVMp plasmid produced three fragments: 5078, 1395 and 732 bp (FIG. 3B, left). The smallest fragment contains the right-end hairpin.
  • CDS For H1PV and MH1PV plasmids two more strains with sseJ knockout were created in CD and BB’CDF and were termed as CDS: ( ⁇ sbcCD ⁇ sseJ), BB’CDFS ( ⁇ recB, ⁇ sbcB, ⁇ sbcCD and ⁇ recF ⁇ sseJ).
  • BB CDFS
  • ⁇ recB, ⁇ sbcB, ⁇ sbcCD and ⁇ recF ⁇ sseJ When the MVMp plasmid was replicated in each of these seven bacterial strains, four of the strains deleted 97 bp from the right-end hairpin (A, B, F and BB’F), as shown by SSPI restriction digest (FIG. 4A) and DNA sequencing (FIG. 4B).
  • FIGS.4A, B Three of the knockout stains maintained the hairpin (CD, BCDJ and BB’CDF; FIGS.4A, B).
  • the MVMp plasmid was replicated in CD and BB’CDF, purified, and transfected into HEK-293T cells. After transfection, all of these cells formed NS1 (FIG. 4C), demonstrating the initiation of virus formation. To show the durability of virus replication, the MVMp plasmid was maintained in CD, BB’CDF and SURE 2 for three passages.
  • both CD and BB’CDF maintained the full-length plasmid and the right- end hairpin (similar to SURE 2, FIG. 4D).
  • Deletion of homologous recombination genes does not impair growth or invasion
  • deletion of homologous recombination genes did not affect bacterial growth or invasiveness into cancer cells (FIG.5).
  • the growth of strain CD and BB’CDF were comparable with parental Salmonella (FIG. 5A).
  • the growth of strain BCDJ was impaired.
  • the deletions in BCDJ delayed growth by more than six hours (FIG. 5A).
  • the density of BCDJ was 4.3 times lower than parental Salmonella (P ⁇ 0.0001, FIG. 5B).
  • the densities of CD and BB’CDF were comparable to parental Salmonella at nine hours (FIG.5B).
  • the CD and BB’CDF strains invaded into cancer cells at comparable rates to parental Salmonella (FIG.5C-F).
  • the Salmonella strains (CD, BB’CDF, BCDJ and parental) were co-cultured with 4T1 murine mammary cancer cells for two hours and extracellular bacteria were cleared with gentamicin. After four hours, bacteria were inside cells (arrows) for the CD, BB’CDF and parental strains (FIG. 5C).
  • VDS-B BB’CDF
  • VDS-C VDS-C
  • both strains of VDS were applied to cultures of 4T1 cells (FIG. 6A). After two hours of co-culture, extracellular bacteria were removed with gentamicin (FIG.6A). After a further 36 hours to allow for plasmid release, transport to the nucleus and gene expression, the cells cultured with VDS-B produced the viral NS1 protein (FIG. 6B). NS1 production was comparable to positive control cells that were directly transfected with the MVMp plasmid using lipofectamine (FIG. 6B).
  • the bacterial control was the BB’CDF strain, which did not contain the MVMp plasmid but was transformed with the delivery (PsseJ-lysE) plasmid, and had the same knockouts ( ⁇ recB, ⁇ sbcB, ⁇ sbcCD and ⁇ recF) as VDS-B. Seventy-two hours after bacterial invasion, cells treated with VDS-B formed virus-infected, dead cell plaques (FIG. 6C). The area of the plaques was significantly more than bacterial controls (P ⁇ 0.05, FIG. 6D). Over time, VDS-B caused death similarly to transfection controls (arrows in FIGS. 6E-F).
  • VDS-C VDS-C
  • strain HRDS-CD its bacterial control
  • FIG.6I Direct transfection of MVMp caused more cell death than VDS-C (P ⁇ 0.001, FIG. 6I).
  • Lack of virus formation and undetectable cell death shows limited utility for VDS-C (compared to VDS-B).
  • VDS creates functional virus particles that infect new cells Delivery of MVMp by VDS forms functional and infective virions (FIG.7).
  • VDS was applied to cultures of 4T1 cancer cells for two hours (FIG. 7A). After this time for invasion, the cells were washed and fed with complete media containing gentamicin.
  • VDS-mediated cell death is independent of cancer type When applied to multiple cancer cell types, VDS induced the formation of cytotoxic viral particles that kill cells and infect neighboring cells (FIG.8).
  • VDS-B was applied to HEK-293T human embryonic kidney cells, U2OS human osteosarcoma cells, and KPCY murine pancreatic ductal adenocarcinoma cells (FIG. 8A). A similar procedure was used as FIG. 6A.
  • NS1 was produced by cells that were directly transfected with the MVMp plasmid using lipofectamine (FIG. 8A). When VDS-B was applied for two hours, these cells also produce NS1 (FIG. 8A). VDS-C did not produce NS1 in any of the cell types.
  • the delivery of MVMp with VDS killed osteosarcoma and pancreatic ductal adenocarcinoma cells (FIG. 8B). At 72 h after infection of KPCY cells, VDS-B significantly reduced viability compared to bacterial controls (strain BB’CDF; P ⁇ 0.01, FIG.8B).
  • VDS-B In U2OS cell, MVMp delivered by VDS-B also significantly reduced viability compared to bacteria controls (P ⁇ 0.0001, FIG.8B). As with results in FIG 6, VDS-C did not induce death in either of these cell lines.
  • VDS-C When applied to osteosarcoma and pancreatic ductal adenocarcinoma cells, VDS produced infective viral particles (re-infection, FIG.8C).
  • FIG. 7A VDS was added to cell cultures for 2 hours, extracellular bacterial were killed with gentamicin, and conditioned media was added to cultures of HEK-293T cells. After 48h in conditioned media, the HEK-293T cells that received media from VDS-B cultures produced NS1 (FIG. 8C).
  • Virus was also present in the culture media from cells transfected with the MVMp plasmid (FIG. 8C). No virus was produced by cultures treated with VDS-C. Viewed together, these data demonstrate that MVMp delivery by VDS resulted in the production of viable virus particles that kill infected cancer cells, independent of cell type. Discussion A bacterial vector was created that delivers oncolytic viruses into cancer cells. The genome for the virus (MVMp) was contained on a plasmid that the bacteria propagated without deleting palindromic sections that are essential for virus infection and replication (FIGS. 3-5). When these virus-delivering Salmonella (VDS) encounter cancer cells, they invade, and deposit the plasmid into the cellular cytoplasm.
  • the cell expresses the viral genes (FIGS. 2, 6).
  • Production of initiating proteins begins the process of forming and assembling viral particles in the cells (FIG.6). Once formed, these virions kill the invaded cancer cells (FIG.6) and spread into the environment. There the viruses invade and kill na ⁇ ve cells (FIG.7).
  • the release of virions by infected cells perpetuates and amplifies the infection.
  • the results show that deletion of homologous recombination genes enables Salmonella to carry and maintain the MVMp genome (FIGS. 3-5). This parvovirus genome contains a right-end hairpin that is unstable in Salmonella (FIG. 3). VDS was designed to prevent this deletion.
  • VDS delivers functional viruses to multiple cancer types, including breast carcinoma, pancreatic carcinoma, and osteosarcoma (FIG. 8).
  • VDS was used to deliver MVMp, which is a single-stranded DNA (ssDNA) virus.
  • VDS could also deliver double- stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) viruses.
  • ssDNA single-stranded DNA
  • ssRNA single-stranded RNA
  • dsRNA double-stranded RNA
  • VDS enables oncolytic virus treatment of solid tumors.
  • VDS After systemic injection, VDS would preferentially accumulate in tumors over other organs, similar to other Salmonella vectors (31, 67). Delivery of the viral genome would produce infective virus particles that can initiate successive rounds of infection (FIG. 7) and mediate a potent bystander effect. Salmonella delivery would increase the delivery of viruses to the tumor microenvironment. Combining the benefits of Salmonella delivery and oncolytic virus therapy can provide an effective therapy for many cancers.
  • Salmonella delivers functional H1PV virions that are cytotoxic to liver cancer cells: Two strains of homologous recombination deficient Salmonella, BB’CDF ( ⁇ recB, ⁇ sbcB, ⁇ sbcCD and ⁇ recF) and CDS ( ⁇ sbcCD and ⁇ sseJ) maintained the H1PV right-end hairpin. Two test the stability of the plasmid, SSPI and EcoRI restriction digest was performed and the size of the two bands were same as from the plasmid maintained in the SURE 2 strain (FIG. 14A). To test whether, the plasmid from these strains could generate functional virus, plasmid was transfected into HEK-293T cells.
  • BB’CDF and CDS strains of Salmonella was transformed with the H1PV plasmid and a plasmid containing the intracellular delivery circuit.
  • the strains containing both the plasmid were termed as H1PV VDS-B when transformed in BB’CDF and H1PV VDS-C when transformed into CDS.
  • H1PV VDS-B and VDS-C were co-cultured with HUH7 (human liver cancer cells) at a Multiplicity of infection (MOI) of 100.2 hours post infection, the cells were washed 3X with PBS and added new media with gentamycin.36 hours post cells were scraped and looked for NS1 protein. Both the strains of Salmonella were able to deliver the viral plasmid in human liver cancer cell similar to the transfection of the viral plasmid. (FIG. 14C).
  • H1PV plasmid delivery by VDS-B at a MOI of 100 resulted in the generation of NS1 protein (FIG. 14D).
  • BB’CDF was better at delivering the viral plasmid in the cancer cells than the CDS. Therefore, BB’CDF strain was carried forward for all the in vivo experiments. This delivery resulted in a significant decrease in the cell viability in all the above cell lines.
  • Engineered Salmonella delivers H1PV in tumors that creates functional virions: 1*10 7 CFU of H1PV VDS-B was injected intravenously (IV) into tumor free mice. The mice injected regained their weights in three days after injection (FIG.15A). After 7 days blood was collected from mice injected with H1PV VDS-B and saline and the serum was analyzed for complete cytokine panel.
  • mice injected with H1PV VDS-B and saline There was no difference in the cytokine levels between mice injected with H1PV VDS-B and saline (FIG.15B).
  • 4*10 7 CFU of bacteria was injected in mice intratumorally (IT) in subcutaneous liver hepatoma (Hepa1-6) tumors between 250-450mm 3 .
  • 5 days post injection tumor was harvested, processed, and stained for viral protein, NS1 on a western blot (FIG.15C).
  • mice with tumors injected with 4*10 7 CFU of H1PV VDS-B were harvested and processed.
  • Functional virions were harvested from the tumors and the supernatant containing the virus was put on HEK-293T cells. 72 hours post infection, the HEK-293T cells were analyzed for the NS1 viral protein (FIG. 15E).
  • H1PV VDS-B had NS1 band when the supernatant from these tumors were put on HEK-293T cells whereas the supernatant from tumors injected with saline and H1PV VDS-C did not show up a NS1 band on the immunoblot (FIG.15F).
  • BB’CDF was used as a control and the same amount was injected.
  • the tumors were measured every three days until the maximum tumor burden was reached (1000mm 3 ). Injections were given until the last control died, a total of six injections were given every six days to both control and experimental group (FIG.16A).
  • Intravenous (IV) delivery of H1PV VDS-B is as efficacious as intratumoral (IT) delivery: 4*10 7 CFU of H1PV VDS-B or BB’CDF was injected IV in Hepa1-6 tumors between the size of 50-180mm 3 .6 injections were given, six days apart and tumors were measured every three days until maximum tumor burden was reached (FIG.17A).
  • the efficacy was stopped when the first control mice died.
  • the mice in experimental also survived significantly longer than the mice in the control group (P ⁇ 0.05) (FIG.17E).
  • IV injected mice were compared with IT injected mice of H1PV VDS-B there was no difference in the tumor volumes (FIG.
  • H1PV delivery by Salmonella causes reduction in tumor weights, cancer cells and increases immunostimulatory innate immune cells: 4*10 7 CFU of H1PV VDS-B was injected IT in Hepa1-6 tumor model with BB’CDF as a control. 3 injections were given, six days apart and five days after the last injection mice were euthanized and tumor were harvested (FIG.18A).
  • the tumor was then stained for Epcam positive cells which is pan cancer cell marker. There was over a 2-fold reduction in the Epcam positive cells in mice injected with H1PV VDS-B as compared to mice injected with BB’CDF (P ⁇ 0.05) (FIG.18D). The tumor was also stained for all the innate cells; Natural killer cells (Npk46 marker), Macrophages (F4/80 marker). There was a significant increase in the NK cells (P ⁇ 0.05) (FIG. 17E) and Macrophages (P ⁇ 0.05) (FIG. 18F) in the experimental group as compared to the control group.
  • the H1PV VDS-B tumors also have a significant decrease in the CD45 + cells as compared to the control tumors which is a pan immune cell marker (P ⁇ 0.05) (FIG.18G). Since all the other cells (NK cells, Macrophages and CD3 + cells) had a significant increase in mice injected with H1PV VDS-B. This reduction could be attributed to the reduction in neutrophils. So, there was a significant reduction in the neutrophil population in the experimental group as compared to the control group (P ⁇ 0.01) (FIG. 18H). The increase in neutrophils is associated with tissue damage and poor prognosis of the tumor.
  • H1PV delivery be engineered Salmonella activates adaptive immunity and generates tumor memory: 4*10 7 CFU of H1PV VDS-B was injected IT with Hepa1-6 tumor model with BB’CDF as a control.
  • FIG.19A 3 injections were given, six days apart and five days after the last injection mice were euthanized and tumor were harvested (FIG.19A).
  • the tumor was processed and stained for pan T cells (CD3 marker), cytotoxic T cells (CD8 marker), Helper T cells (CD4 marker), Regulatory T cells (CD25 marker).
  • Mice injected with H1PV VDS-B had over a 4-fold increase in the CD3 + cells as compared to the bacterial control group (P ⁇ 0.05) (FIG. 19B).
  • the cells were then stained for CD8 T cells and regulatory T cells (FIG. 19C). There was a 12-fold increase in the ratio of CD8 + to CD4 + CD25 + cells (P ⁇ 0.05) (FIG. 19D).
  • the virus delivery can recruit both virus specific T cells and cancer specific T cells. To check whether the T cells recruited in the tumor microenvironment were cancer specific.
  • the mice were injected with H1PV VDS-B three times, six days apart with 4*10 7 CFU. Five days post the last injection, spleens were then extracted from the mice. The spleen was processed and the splenocytes were then injected intraperitonially (IP) in the na ⁇ ve mice. Two weeks post the injection of splenocytes na ⁇ ve mice were rechallenged with 2.5 million Hepa1-6 cells (FIG. 19I). To determine the take rate of the Hepa1-6 cells, a million cells were injected in the na ⁇ ve mice.
  • mice not injected with splenocytes derived from H1PV VDS-B mice tumors took in 75% mice of the mice whereas the mice injected with the splenocytes no mice grafted tumors (FIG.19J).
  • the tumors injected were monitored for 66 days. All the tumors in the mice injected with saline grew while the mice injected with the splenocytes had pin pricks but then regressed and the mice remained tumor free for 66 days (FIG.19K).
  • Engineered Salmonella colonize tumor and is safe 1*10 7 CFU BB’CDF strain of Salmonella with the intracellular delivery plasmid was injected intravenously in the mice having a Hepa1-6. 72 hours post injection tumor along with liver, spleen, lungs, kidney, heart, and brain were harvested to determine the colonization of bacteria in different organs (FIG.20A).
  • Engineered Salmonella colonized tumors significantly higher than any other healthy organs (FIG. 20B). The colonization in the tumors was over 50 million-fold higher than in liver and spleen (FIG.20B).
  • BB’CDF and saline There was no difference in the cytokine levels between BB’CDF and saline (FIG.20F). There was a significant difference in the levels of IL-6 (P ⁇ 0.05) (FIG.20G) and TNF ⁇ (P ⁇ 0.01) (FIG. 20H) between BB’CDF and parental Salmonella, the two most important cytokines responsible for cytokine storm.
  • MTD maximum tolerable dose
  • BB’CDF and parental Salmonella strain were injected IV in mice. 5*10 7 CFU of BB’CDF bacteria was very well tolerated in mice with weight loss of around 10% but with the parental strain the weight loss was around 15% (FIG. 20I). After 7 days the mice were rechallenged with another same dose of the bacteria.
  • mice After the second dose of parental Salmonella, mice died whereas mice injected with BB’CDF regained their weights after 3 days of injection (FIG. 20I). Mice were then injected with 1*10 8 CFU of BB’CDF strain. After the first injection, mice lost around 12% of their body weights but after second injection they lost about 18% of their body weights (FIG.20I). Taken together, all these results indicate that the engineered Salmonella strain colonizes tumors and clears out of healthy organs better than the parental strain. The strain is also significantly safer than the parental strain.

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Abstract

L'invention concerne des méthodes et des compositions pour le traitement du cancer. Une composition comprend une cellule bactérienne modifiée comprenant : a) un gène de lyse ou une cassette de lyse fonctionnellement liée à un promoteur de salmonelle induit de manière intracellulaire ; b) un ou plusieurs des gènes de la cellule bactérienne choisis dans le groupe constitué par recA, recB, recF, sbcB, sbcCD, red, sseJ ou toute combinaison de ceux-ci sont inactivés ; et c) une séquence d'acide nucléique codant pour un génome de virus oncolytique.
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US20100178684A1 (en) * 2006-12-21 2010-07-15 Woo Savio L C Transgenic oncolytic viruses and uses thereof
WO2022174226A1 (fr) * 2021-02-09 2022-08-18 University Of Massachusetts Administration intracellulaire de protéines thérapeutiques conçues pour envahir et lyser de manière autonome et procédés d'utilisation associés
US20230132250A1 (en) * 2020-03-11 2023-04-27 Nature Technology Corporation Bacterial host strains

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US20100178684A1 (en) * 2006-12-21 2010-07-15 Woo Savio L C Transgenic oncolytic viruses and uses thereof
US20230132250A1 (en) * 2020-03-11 2023-04-27 Nature Technology Corporation Bacterial host strains
WO2022174226A1 (fr) * 2021-02-09 2022-08-18 University Of Massachusetts Administration intracellulaire de protéines thérapeutiques conçues pour envahir et lyser de manière autonome et procédés d'utilisation associés

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MARCHINI ANTONIO, DAEFFLER LAURENT, POZDEEV VITALY I., ANGELOVA ASSIA, ROMMELAERE JEAN: "Immune Conversion of Tumor Microenvironment by Oncolytic Viruses: The Protoparvovirus H-1PV Case Study", FRONTIERS IN IMMUNOLOGY, vol. 10, 1 August 2019 (2019-08-01), pages 1848, XP055803151, DOI: 10.3389/fimmu.2019.01848 *

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