Systems and methods for diminishing cell growth and inducing selective killing of target cells
View Patent ↗The invention relates to a biological system for diminishing cell growth or inducing selective killing of target cells, in particular pathogenic bacterial or fungal cells, or cancer cells. The biological system is based on toxin-antitoxin systems, as found in prokaryotic plasmids and their host chromosomes. The biological system comprises a vehicle with a first nucleic acid sequence or amino acid sequence encoding for a prokaryotic toxin of a prokaryotic toxin-antitoxin pair, and a second nucleic acid sequence or amino acid sequence encoding for the corresponding prokaryotic antitoxin of the prokaryotic toxin-antitoxin pair. The system is characterized in that the toxin and/or the antitoxin is operably linked to a protein output modifier (POM) that comprises a nucleic acid sequence or amino acid sequence that modifies the relative rate of transcription, mRNA stability, mRNA translatability or protein stability of the toxin and/or antitoxin.
1. A biological system for diminishing cell growth or inducing selective killing of target cells comprising a vehicle with a first nucleic acid sequence or amino acid sequence encoding for a prokaryotic toxin of a prokaryotic toxin-antitoxin pair, and a second nucleic acid sequence or amino acid sequence encoding for the corresponding prokaryotic antitoxin of the prokaryotic toxin-antitoxin pair, characterized in that the toxin and/or the antitoxin is operably linked to a protein output modifier (POM) that comprises a nucleic acid sequence or amino acid sequence that modifies the relative rate of transcription, mRNA stability, mRNA translatability or protein stability of the toxin and/or antitoxin thereby changing the relative ratio in the concentration of the toxin and/or the antitoxin within the target cells and/or, where applicable, within non-target cells by decreasing the antitoxin outputs in the target cells relative to the toxin outputs, and/or decreasing the toxin outputs in the non-target cells relative to the antitoxin outputs.
2. The system according to claim 1 , wherein the protein output modifier (POM) is selected from the group consisting of promoter/operator sequence(s), miRNA target site(s), 3′-UTRs or ubiquitin ligase target domain(s).
3. The system according to claim 1 , wherein the protein output modifier (POM) contains one or more target sequences for a cellular POM interacting molecule that is (over)expressed in the target cells and/or, if applicable, in non-target cells, and that interacts with the one or more target sequences of the POM, thereby resulting in a decrease of the toxin outputs in the non-target cells and/or decrease of the antitoxin outputs in the target cells.
4. The system according to claim 3 , wherein the protein output modifier (POM) contains one or more PDZ domains from HR-HPV-E6 target cellular proteins and wherein the cellular POM interacting molecule is the E6 oncogene from High Risk HPV serotypes that interacts with said PDZ domain(s).
5. The system according to claim 2 , wherein the protein output modifier (POM) is a miRNA target site and the cellular POM interacting molecule is a miRNA that interacts with said miRNA target site.
6. The system according to claim 5 , wherein the miRNA target site is 100% complementary to the miRNA that targets it and wherein the miRNA target site is positioned immediately downstream of the target gene.
7. The system according to claim 1 , wherein the protein output modifier (POM) is a 3′-UTR sequence in the toxin or antitoxin nucleic acid sequence.
8. The system according to claim 1 , wherein the protein output modifier (POM) contains a promoter/operator sequence that decreases the rate of transcription of the antitoxin in the target cells, and/or decreases the rate of transcription of the toxin in the non-target cells.
9. The system according to claim 1 , wherein the toxin-antitoxin pair is selected from the group consisting of kid/kis, CcdB/CcdA, MazF/MazE, ChpBK/ChpBI, RelE/RelB, ParE/ParD, HipA/HipB, PhD/Doc, Hok/Sok, YafM/YoeB, YafN/YafO, YgjM/YgjN, YgiT/YgiU, DinJ/YafQ, VapB/VapC, HipB/HipA, HicB/HicA, and their homologs in other organisms.
10. The system according to claim 1 , wherein the protein output modifier (POM), the prokaryotic toxin and the prokaryotic antitoxin are contained in a single or in independent carrier plasmids or viruses, wherein the toxin and antitoxin are transcribed from the same single promoter or from independent promoters.
11. A pharmacological composition, comprising a vehicle with a first nucleic acid sequence or amino acid sequence encoding for a prokaryotic toxin of a prokaryotic toxin-antitoxin pair, and a second nucleic acid sequence or amino acid sequence encoding for the corresponding prokaryotic antitoxin of the prokaryotic toxin-antitoxin pair, wherein the toxin and/or the antitoxin is operably linked to a protein output modifier (POM) that comprises a nucleic acid sequence or amino acid sequence that modifies the relative rate of transcription, mRNA stability, mRNA translatability or protein stability of the toxin and/or antitoxin thereby changing the relative ratio in the concentration of the toxin and/or the antitoxin within the target cells and/or, where applicable, within non-target cells by decreasing the antitoxin outputs in the target cells relative to the toxin outputs and/or decreasing the toxin outputs in the non-target cells relative to the antitoxin outputs, and a pharmaceutical carrier.
12. A biological system, comprising a vehicle with a first nucleic acid sequence or amino acid sequence encoding for a prokaryotic toxin of a prokaryotic toxin-antitoxin pair, and a second nucleic acid sequence or amino acid sequence encoding for the corresponding prokaryotic antitoxin of the prokaryotic toxin-antitoxin pair, wherein the toxin and/or the antitoxin is operably linked to a protein output modifier (POM) that comprises a nucleic acid sequence or amino acid sequence that modifies the relative rate of transcription, mRNA stability, mRNA translatability or protein stability of the toxin and/or antitoxin thereby changing the relative ratio in the concentration of the toxin and/or the antitoxin within the target cells and/or, where applicable, within non-target cells by decreasing the antitoxin outputs in the target cells and/or decreasing the toxin outputs in the non-target cells relative to the antitoxin outputs, for use in the treatment of a pathological bacterial or fungal disease, or cancer.
13. The system according to claim 12 , wherein the vehicle is delivered to the target cells by means of nanocells (minicells).
14. A drug delivery system for delivering a vehicle to target cells, wherein the vehicle comprises a first nucleic acid sequence or amino acid sequence encoding for a prokaryotic toxin of a prokaryotic toxin-antitoxin pair, and a second nucleic acid sequence or amino acid sequence encoding for the corresponding prokaryotic antitoxin of the prokaryotic toxin-antitoxin pair, wherein the toxin and/or the antitoxin is operably linked to a protein output modifier (POM) that comprises a nucleic acid sequence or amino acid sequence that modifies the relative rate of transcription, mRNA stability, mRNA translatability or protein stability of the toxin and/or antitoxin thereby changing the relative ratio in the concentration of the toxin and/or the antitoxin within the target cells and/or, where applicable, within non-target cells by decreasing the antitoxin outputs in the target cells relative to the toxin outputs, and/or decreasing the toxin outputs in the non-target cells relative to the antitoxin outputs, wherein the drug delivery system comprises nanocells containing the vehicle, characterized in that the nanocells are coated with one or more antibodies that recognize antigens specifically expressed by said target cells by exposing multiple copies of the Fc binding domain of Protein G, or protein A, protein A-G fusions, or Fc-receptors (FcR) to the external medium.
15. The drug delivery system according to claim 14 , wherein the Fc binding domain of Protein G is anchored to the outer membrane of the nanocells via an invasin protein fragment covering amino acids 1-796 of Yersinia pseudotuberculosis.
16. The drug delivery system according to claim 14 , wherein the nanocells are produced from lipid A minus bacterial strains.
17. The drug delivery system according to claim 14 , wherein the Fc binding domain of Protein G is a polypeptide fragment containing amino acids 191-384 of Streptococcal Protein G.
18. The drug delivery system according to claim 14 , wherein the nanocells are coated with binding molecules that bind to antigens that have therapeutic or diagnostic properties.
19. The drug delivery system according to claim 14 for use in therapy, diagnosis or theranosis.
20. A method for delivery of a biological system to target cells, comprising the steps of
producing the biological system of claim 1 in a nanocell parental bacterial strain and producing nanocells from said biological system-producing parental bacteria cells, or incubating the biological system with nanocells produced from parental bacteria cells,
coating the nanocells with one or more antibodies that recognize antigens specifically expressed by said target cells by exposing multiple copies of the Fc binding domain of protein G to the external medium,
exposing the target cells to said nanocells.