# US 10,441,546 B2 · Bacterially derived intact minicells that encompass plasmid free DNA and methods of using the same

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- Number: US 10,441,546 B2
- Document ID: US10441546B2
- Status: Granted
- Kind: B2
- Application number: 15/604,555
- Filed: 2017-05-24
- Published: 2017-09-14
- Granted: 2019-10-15
- Earliest claimed: 2004-08-26
- Assignees: EnGenelC Molecular Delivery Pty Ltd
- Applicants: EnGeneIC Molecular Delivery Pty Ltd.
- Inventors: Himanshu Brahmbhatt; Jennifer MacDiarmid; Toby Hulf
- Web page: https://patentgrep.com/patent/US10441546B2

## CPC

- A61K 9/5068: {Cell membranes or bacterial membranes enclosing drugs (liposomes with additional exogenous lipids A61K9/127)}
- A61K 31/4745: condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines (yohimbine derivatives, vinblastine A61K31/475; ergoline derivatives A61K31/48)
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 39/39558: {against tumor tissues, cells, antigens}
- A61K 39/40: bacterial
- A61K 47/46: Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
- A61K 47/6901: {Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors}
- A61K 47/6923: {the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb}
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/1235: Salmonella (G)
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/40: against enzymes
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12N 15/1138: {against receptors or cell surface proteins}
- C07K 2317/31: multispecific
- C12N 2310/11: Antisense
- C12N 2310/111: spanning the whole gene, or a large part of it
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Intact, bacterially-derived minicells can safely introduce therapeutically effective amounts of plasmid-free functional nucleic acid to target mammalian cells. To this end, functional nucleic acid can be packaged into intact minicells directly, without resort to expression constructs, the expression machinery of the host cell, harsh chemicals or electroporation.

## Claims

### Claim 1

A composition comprising:
(a) a plurality of intact, bacterial minicells, each minicell of the plurality comprising at least one exogenous functional nucleic acid, wherein the exogenous functional nucleic acid is DNA; and
(b) a pharmaceutically acceptable carrier therefor.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the plurality comprises a therapeutically effective amount of the exogenous functional nucleic acid.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the exogenous functional nucleic acid is not chemically modified.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the DNA is single-, double-, or multi-stranded DNA.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the DNA is antisense oligonucleotide.

### Claim 6 (depends on 5)

The composition of claim 5, wherein:
(a) the antisense oligonucleotide targets the RNA transcript encoding a protein that contributes to drug resistance;
(b) the antisense oligonucleotide targets the RNA transcript encoding a protein that contributes to apoptosis resistance; or
(c) the antisense oligonucleotide targets a RNA transcript of encoding a protein that contributes to neoplasticity.

### Claim 7 (depends on 6)

The composition of claim 6 wherein:
(a) the protein that contributes to drug resistance of subsection (a) is P-glycoprotein, MDR-2, or MDR-3;
(b) the antisense oligonucleotide of subsection (a) targets the transcript of MRP2, BCR-ABL, STI-571 resistance-associated protein, lung resistance-related protein, cyclooxygenase-2, nuclear factor kappa, XRCCl, ERCCI, GSTPI, mutant f3-tubulin, or a growth factor;
(c) the antisense oligonucleotide of subsection (b) targets a transcript of Bcl-2, Bcl-XL, A1/Bfl 1, focal adhesion kinase or p53 protein; or
(d) the antisense oligonucleotide of subsection (c) targets a transcript of β-Catenin, PKC-α, C-RAF, K-Ras, DP97 Dead box RNA helicase, cdk1, DNMT1, FLIP, C-Sfc, 53BPI, Polycomb group protein EZH2, ErbB1, HPV-16 E5 and E7, Fortilin & MCI1P, KSP, DIPp13α, MBD2, p21, KLF4, tpt/TCTP, SPK1 & SPK2, P300, PLK1, Trp53, Ras, ErbB1, VEGF, or BAG-1.

### Claim 8 (depends on 1)

The composition of claim 1, further comprising a drug.

### Claim 9 (depends on 8)

The composition of claim 8, wherein the exogenous functional nucleic acid targets a transcript encoding a protein that contributes to resistance to the drug.

### Claim 10 (depends on 8)

The composition of claim 8, wherein the exogenous functional nucleic acid and the drug are packaged within the same intact minicell.

### Claim 11 (depends on 8)

The composition of claim 8, wherein the exogenous functional nucleic acid and the drug are packaged within different intact minicells.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the minicells further comprise a bispecific ligand.

### Claim 13 (depends on 12)

The composition of claim 12, wherein the bispecific ligand comprises an antibody or antibody fragment.

### Claim 14 (depends on 12)

The composition of claim 12, wherein the bispecific ligand comprises a first arm that carries specificity for a minicell surface structure and a second arm that carries specificity for a non-phagocytic mammalian cell surface receptor.

### Claim 15 (depends on 14)

The composition of claim 14, wherein:
(a) the minicell surface structure is an O-polysaccharide component of a lipopolysaccharide on the minicell surface; and/or
(b) the mammalian cell surface receptor is capable of activating receptor-mediated endocytosis of the minicell.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the composition comprises:
(a) fewer than about 1 contaminating parent bacterial cell per 107 minicells;
(b) fewer than about 1 contaminating parent bacterial cell per 108 minicells;
(c) fewer than about 1 contaminating parent bacterial cell per 109 minicells;
(d) fewer than about 1 contaminating parent bacterial cell per 1010 minicells; or
(e) fewer than about 1 contaminating parent bacterial cell per 1011 minicells.

### Claim 17

A method of delivering an exogenous nucleic acid to a target cell, comprising:
(a) providing a plurality of intact, bacterial minicells in a pharmaceutically acceptable carrier, each minicell of the plurality comprising an exogenous functional nucleic acid, wherein the exogenous functional nucleic acid is DNA; and
(b) bringing the minicells into contact with a mammalian target cell such that the mammalian target cell engulfs the minicells, whereby the exogenous functional nucleic acid is released into the cytoplasm of the mammalian target cell.

### Claim 18 (depends on 17)

The method of claim 17, wherein contact between the minicells and the mammalian target cell occurs in vitro.

### Claim 19 (depends on 17)

The method of claim 17, wherein contact between the minicells and the mammalian target cell occurs in vivo.

### Claim 20 (depends on 17)

The method of claim 17, wherein at least a portion of the plurality of minicells comprises a therapeutically effective amount of the exogenous functional nucleic acid.

### Claim 21 (depends on 17)

The method of claim 17, wherein the DNA is antisense oligonucleotide.

### Claim 22 (depends on 21)

The method of claim 21, wherein:
(a) the antisense oligonucleotide targets the RNA transcript encoding a protein that contributes to drug resistance;
(b) the antisense oligonucleotide targets the RNA transcript encoding a protein that contributes to apoptosis resistance; or
(c) the antisense oligonucleotide targets a RNA transcript of encoding a protein that contributes to neoplasticity.

### Claim 23 (depends on 17)

The method of claim 17, further comprising a step (c) of delivering a drug to the target mammalian cell.

### Claim 24 (depends on 23)

The method of claim 23, wherein the exogenous functional nucleic acid targets a transcript encoding a protein that contributes to resistance to the drug.

### Claim 25 (depends on 22, 24)

The method of claim 22 wherein:
(a) the protein that contributes to drug resistance of subsection (a) is P-glycoprotein, MDR-2 or MDR-3;
(b) the antisense oligonucleotide of claim 24 subsection (a) targets the transcript of MRP2, BCR-ABL, STI-571 resistance-associated protein, lung resistance-related protein, cyclooxygenase-2, nuclear factor kappa, XRCC1, ERCC1, GSTP1, mutant β-tubulin, or a growth factor;
(c) the antisense oligonucleotide of claim 24 subsection (b) targets a transcript of Bcl-2, Bcl-XL, A1/Bfl 1, focal adhesion kinase or p53protein; or
(d) the antisense oligonucleotide of claim 24 subsection (c) targets a transcript of β-Catenin, PKC-α, C-RAF, K-Ras, DP97 Dead box RNA helicase, cdk1, DNMT1, FLIP, C-Sfc, 53BPI, Polycomb group protein EZH2, ErbB1, HPV-16 E5 and E7, Fortilin & MCI1P, DIP13α, KSP, MBD2, p21, KLF4, tpt/TCTP, SPK1 & SPK2, P300, PLK1, Trp53, Ras, ErbB1, VEGF, or BAG-1.

### Claim 26 (depends on 23)

The method of claim 23, wherein the exogenous functional nucleic acid and the drug are packaged within the same intact minicell.

### Claim 27 (depends on 23)

The method of claim 23, wherein the exogenous functional nucleic acid and the drug are packaged within different intact minicells.

### Claim 28 (depends on 17)

The method of claim 17, wherein:
(a) the drug is delivered concurrently with step (b); or
(b) the drug is delivered subsequently to steps (a) and (b).

### Claim 29 (depends on 17)

The method of claim 17, wherein step (b) entails bringing a bispecific ligand into contact with at least some of the minicells and mammalian target cell, such that the bispecific ligand causes the minicells to bind to the mammalian target cell.

### Claim 30 (depends on 29)

The method of claim 29, wherein:
(a) the mammalian target cell is a non-phagocytic cell; and/or
(b) the mammalian cell is phagocytosis- or endocytosis-competent.

### Claim 31 (depends on 29)

The method of claim 29, wherein the bispecific ligand comprises an antibody or antibody fragment.

### Claim 32 (depends on 29)

The method of claim 29, wherein the bispecific ligand comprises a first arm that carries specificity for a minicell surface structure and a second arm that carries specificity for a non-phagocytic mammalian cell surface receptor.

### Claim 33 (depends on 32)

The method of claim 32, wherein:
(a) the minicell surface structure is an O-polysaccharide component of a lipopolysaccharide on said minicell surface; and/or
(b) the mammalian cell surface receptor is capable of activating receptor-mediated endocytosis of said minicell.

### Claim 34 (depends on 1)

A method for formulating a minicell of the composition of claim 1, comprising co-incubating a plurality of intact, bacterially-derived minicells with the exogenous nucleic acid in a buffer.

### Claim 35 (depends on 34)

The method of claim 34, wherein the co-incubation comprises gentle shaking.

### Claim 36 (depends on 34)

The method of claim 34, wherein the buffer comprises buffered saline.

### Claim 37 (depends on 36)

The method of claim 36, wherein the buffered saline is in gelatin form.

### Claim 38 (depends on 36)

The method of claim 36, wherein the co-incubation is conducted at a temperature of:
(a) about 4° C. to about 37° C.;
(b) about 20° C. to about 30° C.;
(c) about 25° C.; or
(d) about 37° C.

### Claim 39 (depends on 36)

The method of claim 36, wherein:
(a) the co-incubation lasts at least about 0.5 hour; or
(b) the co-incubation lasts at least about 1.0 hour.

### Claim 40 (depends on 34)

The method of claim 34, wherein the buffer comprises a 1× phosphate buffer solution.

### Claim 41 (depends on 34)

The method of claim 34, wherein the co-incubation comprises about 107, about 108, about 109, about 1010, about 1011, about 1012 or about 1013 minicells.

### Claim 42 (depends on 41)

The method of claim 41, wherein the co-incubation comprises about 1010 minicells.