All Submissions | J. Saba Submissions | Physics Site Links | Home Page |
| Email: James Saba |
GSJ:Received February 1, 2005:
http://wbabin.net/saba/saba21.htm
Lysogenic Bacteria, Capable of Therapeutic Virus Liberation Subsequent to Tumor Cell Targeting
James Saba
Recently Yu, et al (1) have reported utilizing in vivo fluorescent to clearly show what had previous been realized, that certain bacteria can specifically localize to tumors. As stated to by these authors and others, such bacteria can be designed to carry multiple genes for detection and treatment of cancer.
Also, others have described the use of intracellular bacteria to deliver plasmids within eukaryotic cells, particular antigen encoding plasmids (2-8). Also thoroughly investigated is the variation of bacterial and eukaryotic viral outer coat proteins so as to facilitate eukaryotic internalization and expression of encapsulated nucleic acids (9-39).
Herein is a novel concept, wherein either a bacterial or eukaryotic cell capable of being localized near or within a target cell, liberates or can be induced to liberate multiple polypeptide-associated nucleic acids.
Polypeptide-congugated nucleic acids call be engineered in any way desirable, for example to produce a mRNA, ribozyme, or siRNA. They may also be capable of reproduction within a target cell.
Polypeptide-associated nucleic acids, subsequent to being taken up by the target cell cause a desirable effect therein. Such an effect can be beneficial to the target cell, induce an immune response, or can be devastating such as if transformed cells or autoimmune effecter cells were targeted.
Inspiration for using cells to to deliver therapeutic polypeptide-associated nucleic acids, particularly viruses, to tumor cells arose in part from the recently reported finding that cancer cells compress intratumor vessels (40). As such its reasonable that macromolecules and viruses may not be able to access all the cells. However, due to the ability of live cells to expansively grow and/or individually move within a tissue, it was assumed that such a means of delivering therapeutics would be advantageous.
The figure above is one preferred embodiment of the invention. We start with a lysogenic tumor-localizing bacteria cell which has found its targets. We next provide a small molecule inducer of virus production and liberation. The liberated viruses are internalized and destroy the transformed cell targets.
Analogously, one might envision the lysogenic targeting cells as 'arterially shells' which subsequent to being adequately positioned, are detonated to explosively liberate virion shrapnel. A formidable new weapon in the War on Cancer.
Also envisioned are a libraries of lysogenic cells, each member capable of producing a different polypeptide-associated nucleic acid, and screening processes therewith. Commonly, such a library is screened via first isolating the viruses. An interesting perhaps novel random array-based screening method utilizing a library of lysogenic bacterial cells is shown in the figure below.
Similar to the process of Jayawickreme, et al (44) a confluent layer of eukaryotic cells is covered with a thin layer of agarose within which individual members of a library of lysogenic bacteria are thinly dispersed. After an initial bacterial growth phase, the bacteria with each colony are induced to produce and liberate virus, which subsequently contacts local cells. The loci of cells which have take up virions and are effected indicate the viruses to be identified.
Speculating, perhaps such a screen, or another accomplishing the same objective, could be done for tumor cells of each individual patent so as to customize the therapy to that patent's particular tumor.
Several modifications can be envisioned. Of particular importance, the targeting cells as well as cells targeted could be either prokaryotic or eukaryotic.
Further, a plausible alternative to utilizing targeting cells which produce or can be induced to product virions, is to utilize targeting cells which are selectively permissive to a viral infection. Subsequent to targeting, these permissive cells are provided with infectious virus.
While this invention is currently only being conceptually developed, if it continues to prove novel it is considered exceptional valuable, and a patent application is anticipated to be filed within a year. However, it is hoped that others with adequate laboratory resources will meanwhile investigate the full potential of the invention.
Provisional Claims
2) The targeting cell of claim 1 wherein the polypeptide-associated nucleic acids are virions.
3) The targeting cell of claim 1 or 2 which is prokaryotic or eukaryotic, and wherein the cell it targets is eukaryotic.
4) A library of targeting cells as described in claim 1, 2 or 3, wherein the nucleic acid of each member's liberated polypeptide-associated nucleic acids is different.
5) A library of targeting cells as described in claim 1, 2 or 3, wherein at least one nucleic acid-associated polypeptide of each member's liberated polypeptide-associated nucleic acids is different.
6) A process of altering or killing a target cell comprising bringing into proximity with this cell, an engineered targeting cell which liberates or can be induced to liberate multiple polypeptide-associated nucleic acids.
7) The process of claim 6 wherein the targeting cell, constituently or subsequent to induction, liberates virions.
8) The process of claim 6 or 7 wherein the targeting cell is prokaryotic or eukaryotic and the cell targeted is eukaryotic.
9) The process of claim 8, wherein the eukaryotic cell targeted is a transformed (e.g. tumor) cell.
As far as I know, induced lysis of external bacteria to liberate naked nucleic acids, particular plasmids, in the vicinity of a targeted cell is novel, and as such embodied in the present invention. The following simple experiment is suggested to any who would like to investigate this concept.
2) Subsequent to tumor localization, induce the lysis and liberation of the plasmid, for example by penicillin.
3) Monitor fluorescence within target cells.
Addendum 2/6/05
While liberated polypeptide-associated nucleic acids (including viruses) are primarily useful after being taken up and expressed within target cells; a functional screen assay such as in Figure 2 would allow screening and identification of polypeptide-associated nucleic acids wherein the polypeptide, not the nucleic acid, effected a cellular response particularly via binding to one or more target cell surface receptors.
A variation of this would be to use a library of cells, which do not liberate polypeptide-associated nucleic acids, but which express only polypeptides, particular so as to be secreted or anchored on their surfaces. Recently Harvey, et al (45) utilized a method called Anchored Periplasmic Expression, which would provided the necessary library of polypeptide expressing targeting cells. That is instead of utilizing lysogenic bacteria as in Figure 2, the bacteria as described by Harvey, et al could be utilized. Yeast are also an attractive means of creating the appropriate library of polypeptide-expressing targeting cells (46).
Finally note that the array format as described above is not required in certain of these functional assays.
Claim
A multiplex screening process comprising
ii) optionally allowing clonal expansion of each different targeting cell,
iii) identifying affected target cells, and thereby
identify the particular targeting cell(s) which
effected the response.
------------
Feedback from a few researchers prominent in the field have indicated that the using cells to delivery virus is not new.
If anyone is aware of prior report of utilizing a
library of cells in a screening process, particular as
depicted in Figure 2, please, by all means, contact me.
------------
References
2) Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Low, et al Nat Biotechnol 1999 Jan;17(1):37-41
3) Delivery of polypeptide-encoding plasmid DNA into the cytosol of macrophages by attenuated listeria suicide bacteria. Goebel US Patent 6,143,551 Nov 7, 2000
4) Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery. Pilgrim, et al Gene Ther. 2003 Nov;10(24):2036-45
5) Wild-type intracellular bacteria deliver DNA into mammalian cells. Grillot-Courvalin, et al Cell Microbiol. 2002 Mar;4(3):177-86
6) Transfer of eukaryotic expression plasmids to mammalian hosts by attenuated Salmonella spp. Weiss S. Int J Med Microbiol. 2003 Apr;293(1):95-106
7) Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Hense, et al Cell Microbiol. 2001 Sep;3(9):599-609
8) Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier. Darji, et al FEMS Immunol Med Microbiol. 2000 Apr;27(4):341-9
9) How viruses enter animal cells (reveiw). Smith, et al Science 2004 Apr ;304(5668):237-42
10) Cell-type specific targeting and gene expression using a variant of polyoma VP1 virus-like particles. Gleiter, et al Biol Chem. 2003 Feb;384(2):247-55
11) Adenovirus targeting to c-erbB-2 oncoprotein by single-chain antibody fused to trimeric form of adenovirus receptor ectodomain. Kashentseva, et al Cancer Res. 2002 Jan 15;62(2):609-16
12) In vitro and in vivo characterisation of endothelial cell selective adenoviral vectors. Nicklin, et al J Gene Med. 2004 Mar;6(3):300-8
13) Specific binding of baculoviruses displaying gp64 fusion proteins to mammalian cells. Ojala, et al Biochem Biophys Res Commun. 2001 Jun 15;284(3):777-84
14) Alphavirus-based expression vectors: strategies and applications. (review) Frolov, et al Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11371-7
15) Systemic tumor targeting and killing by Sindbis viral vectors. Tseng, et al Nat Biotechnol. 2004 Jan;22(1):70-7
16) Retroviral vectors bearing IgG-binding motifs for antibody-mediated targeting of vascular endothelial growth factor receptors. Masood, et al Int J Mol Med. 2001 Oct;8(4):335-43
17) Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Muller, et al Nat Biotechnol. 2003 Sep;21(9):1040-6
18) Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Barry, et al Nat Med. 1996 Mar;2(3):299-305
19) Targeting bacteriophage to mammalian cell surface receptors for gene delivery. Larocca, et al Hum Gene Ther. 1998 Nov 1;9(16):2393-9
20) Targeted gene delivery to mammalian cells by filamentous bacteriophage. Poul, et al J Mol Biol 1999 Apr 30;288(2):203-11
21) Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells. Kassner, et al Biochem Biophys Res Commun. 1999 Nov 2;264(3):921-8
22) Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage. Larocca, et al FASEB J. 1999 Apr;13(6):727-34
23) Uptake and intracellular fate of phage display vectors in mammalian cells. Ivanenkov, et al Biochim Biophys Acta. 1999 Jan 11;1448(3):450-62
24) Toward selection of internalizing antibodies from phage libraries. Becerril, et al Biochem Biophys Res Commun. 1999 Feb 16;255(2):386-93
25) Combining phage display and screening of cDNA expression libraries: a new approach for identifying the target antigen of an scFv preselected by phage display. Barth, et al J Mol Biol 2000 Aug 25;301(4):751-7
26) Selection of tumor-specific internalizing human antibodies from phage libraries. Poul, et al J Mol Biol 2000 Sep 1;301(5):1149-61
27) Targeting of phage display vectors to mammalian cells. Uppala, et al Comb Chem High Throughput Screen. 2000 Oct;3(5):373-92
28) Phage as gene delivery vectors. (review) Monaci, et al Curr Opin Mol Ther. 2001 Apr;3(2):159-69
29) Biological effects of anti-ErbB2 single chain antibodies selected for internalizing function. Neve, et al Biochem Biophys Res Commun. 2001 Jan 12;280(1):274-9
30) Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage. Urbanelli, et al J Mol Biol. 2001 Nov 9;313(5):965-76
31) Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library. Heitner, et al J Immunol Methods. 2001 Feb 1;248(1-2):17-30
32) Binding properties, cell delivery, and gene transfer of adenoviral penton base displaying bacteriophage. Di Giovine, et al Virology. 2001 Mar 30;282(1):102-12
33) Receptor-targeted gene delivery using multivalent phagemid particles. Larocca, et al Mol Ther. 2001 Apr;3(4):476-84
34) Evolving phage vectors for cell targeted gene delivery. Larocca, et al Curr Pharm Biotechnol. 2002 Mar;3(1):45-57
35) Enhanced phagemid particle gene transfer in camptothecin-treated carcinoma cells. Burg, et al Cancer Res. 2002 Feb 15;62(4):977-81
36) Cell targeted phagemid rescued by preselected landscape phage. Mount, et al Gene. 2004 Oct 27;341:59-65
37) Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells. He, et al Gene. 1995 Oct 16;164(1):75-9
38) Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells. Shadidi, et al FASEB J. 2003 Feb;17(2):256-8. Epub 2002 Dec 18
39) Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery. Burg, et al DNA Cell Biol. 2004 Jul;23(7):457-62
40) Pathology: cancer cells compress intratumor vessels. Padera, et al Nature 2004 Feb 19;427(6976):685
41) Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered fas-mediated apoptosis. Chattergoon, et al Nat Biotechnol 2000 Sep;18(9):974-9
42) Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Leitner, et al Nat Med. 2003 Jan;9(1):33-9
43) Eukaryotic promoters can direct protein synthesis in Gram-negative bacteria. Goussard, et al J Mol Microbiol Biotechnol. 2003;6(3-4):211-8
44) Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library. Jayawickreme, et al J Pharmacol Toxicol Methods. 1999 Dec;42(4):189-97
45) Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Harvey, et al Proc Natl Acad Sci U S A. 2004 Jun 22;101(25):9193-8. Epub 2004 Jun 14.
46) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Feldhaus, et al Nat Biotechnol. 2003 Feb;21(2):163-70. Epub 2003 Jan 21.