US 20150232862A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0232862 A1 Graves et al. (43) Pub. Date: Aug. 20, 2015 (54) NUCLECACID VECTORS AND USES filed on Feb. 14, 2014, provisional application No. THEREOF 62/013,852, filed on Jun. 18, 2014. (71) Applicant: Symvivo Corporation, Burnaby (CA) Publication Classification (72) Inventors: Herbert Alexander Graves, Vancouver (51) Int. C. (CA); Mark Andrew Fox, Vancouver CI2N 15/74 (2006.01) (CA) (52) U.S. C. CPC .................................... CI2N 15/746 (2013.01) (21) Appl. No.: 14/621,308 (57) ABSTRACT There are disclosed nucleic acid vectors for use in both gram (22) Filed: Feb. 12, 2015 positive and gram negative bacteria. In embodiments the vec tors comprise a prokaryotic expression cassette and in Related U.S. Application Data embodiments comprise a eukaryotic expression cassette. In (60) Provisional application No. 61/940,274, filed on Feb. embodiments the vectors encode a hybrid protein comprising 14, 2014, provisional application No. 61/940,258, a DNA binding domain, a CPP domain and a signal sequence. Patent Application Publication Aug. 20, 2015 Sheet 1 of 10 US 2015/0232862 A1 ErA 2.0S Patent Application Publication Aug. 20, 2015 Sheet 2 of 10 US 2015/0232862 A1 prest.s-Sir issip pr8G 5. Sh Patent Application Publication Aug. 20, 2015 Sheet 3 of 10 US 2015/0232862 A1 Figars. 3. Siti and $2 firiteract with pisikA2, arc resuit in gei-shift with increasing to centratics of petities Patent Application Publication Aug. 20, 2015 Sheet 4 of 10 US 2015/0232862 A1 is 4: Patent Application Publication Aug. 20, 2015 Sheet 5 of 10 US 2015/0232862 A1 EK-293 ceis transfected with 8 RA3.8 positively express &FP & 88: &: is.& ... : -... tieta teiis transfected with 8R&2.8 positiwety express &f -3.3 Patent Application Publication Aug. 20, 2015 Sheet 6 of 10 US 2015/0232862 A1 3. 8 Patent Application Publication Aug. 20, 2015 Sheet 7 of 10 US 2015/0232862 A1 Patent Application Publication Aug. 20, 2015 Sheet 8 of 10 US 2015/0232862 A1 Patent Application Publication Aug. 20, 2015 Sheet 9 of 10 US 2015/0232862 A1 Patent Application Publication Aug. 20, 2015 Sheet 10 of 10 US 2015/0232862 A1 ::K-233:reats: $88.8 g.:38&2.3-883 &833xxxx xas:s:y 8xxess. 388 88.888& 88:8: 3888.8:883:38 88.82.8883 :388.888 & 388.83.3828 :888&s 388x8y &xg:888 (388 s:::::::::::38:8 88.88: 8883.88:88-83888. Figure it herapetities raiectates p38A2.0-Ski and p38A2.8-S2 extapiexes cast transfect and express if is attalian ceilies. US 2015/0232862 A1 Aug. 20, 2015 NUCLECACID VECTORS AND USES domain, at least one cell penetrating peptide (CPP) domain, THEREOF and at least one secretion signal sequence. 0016. In alternative embodiments, the DNA binding PRIORITY CLAIMS domain is one of a Zinc finger DNA binding domain, a 0001. This application claims priority under 35 USC S119 homeobox DNA binding domain, and a MerR DNA binding (e) of U.S. provisional patent application No. 61/940,274, domain, or combinations thereof. filed Feb. 14, 2014, U.S. provisional patent application No. 0017. In alternative embodiments, the CPP comprises a 61/940,258 filed Feb. 14, 2014; and U.S. provisional patent TAT domain, a V22 protein of Herpes Simplex Virus, or the application No. 62/013,852 filed Jun. 18, 2014. The specifi protein transduction domain of the Antennapedia (Antp) pro cations of which are hereby incorporated herein by reference tein, or combinations thereof. 0018. In alternative embodiments the secretion signal wherever permissible by law. sequence is alpha-L-arabinosidase signal sequence, alpha BACKGROUND amylase signal sequence or a truncated alpha amylase signal Sequence. 0002 1. Field 0019. In alternative embodiments, the nucleic acid vector 0003. The subject matter disclosed generally relates to may further comprise at least one DNA motif that binds to the novel shuttle vectors suitable for propagation in both gram at least one DNA binding domain of the hybrid protein. positive and gram negative bacteria and comprising a eukary 0020. In alternative embodiments, the nucleic acid vector otic expression cassette. may further comprise a eukaryotic expression cassette for 0004 2. Related Art expressing a second cargo sequence in a eukaryotic cell. 0005. A variety of vectors and methods are known in the 0021. In alternative embodiments, the second cargo art for propagating nucleic acid sequences in bacteria and for sequence is selected from the group consisting of an onco introducing Such sequences into eukaryotic cells. The follow gene, a tumour Suppressor, a growth factor, a growth factor ing publications are of note: receptor, and a marker protein. 0006 Salomone, F., et al., “A novel cell-penetrating pep 0022. In alternative embodiments, the second cargo tide with membrane disruptive properties for efficient sequence is a fluorescent protein. endosomal escape” (2010), Journal of controlled release 0023. In alternative embodiments, at least one DNA motif 163 (293-303) has at least 90% sequence identity to SEQID NO: 41 or 43. 0007 Stentz, R. et al., “Controlled release of protein from 0024. In alternative embodiments, the at least one selec viable Lactococcus cells” (2010) Applied and Environ tion marker is resistance to an antibiotic effective against both mental Microbiology 76,3026-3031. gram positive and gram negative bacteria. 0008 Christy, B., and Nathans, D. “DNA binding site of 0025. In an embodiment there is disclosed a method for the growth factor-inducible protein Zif268” (1989) 86 transforming a eukaryotic target cell with a candidate DNA Proc. Nat. Acad. Sci. 8737-8741. sequence, the method comprising the steps of expressing in 0009 Khokhlova, E. V., et al., “Bifidobacterium Longum a first cell the hybrid protein from the first expression cassette Modifed recombinant HU protein as vector for nonviral of a nucleic acid vector disclosed herein, to form a complex delivery of DNA to HEK293 Human cell culture” (2011) between the hybrid protein and the nucleic acid vector; con 151 Bulletin of Experimental Biology and Medicine 717 tacting the target cell with the formed hybrid protein and 721. nucleic acid vector complex, the candidate DNA sequence is the second cargo sequence. SUMMARY 0026. In an embodiment there is disclosed a method for transforming a eukaryotic target cell with a candidate DNA 0010. In an embodiment there is disclosed a nucleic acid sequence, the method comprising the steps of contacting the vector comprising: an origin of replication for replication in eukaryotic target cell with a hybrid protein and nucleic acid gram-positive bacteria and gram-negative bacteria, at least vector complex formed by the expression of a hybrid protein one selection marker for selection in both gram-positive and from the first expression cassette of a nucleic acid vector gram-negative bacteria; and at least one expression cassette. disclosed herein, in a prokaryotic cell, the candidate DNA 0011. In alternative embodiments, the nucleic acid vector sequence is the Second cargo sequence. may comprise a first origin of replication functional in a 0027. In an embodiment there is disclosed a method for gram-negative bacteria and a second origin of replication transforming a prokaryotic target cell with a candidate DNA functional in a gram-positive bacteria. sequence, the method comprising the steps of providing a 0012. In alternative embodiments, the first origin of repli nucleic acid vector disclosed herein the candidate DNA cation is functional in E. coli and the second origin of repli sequence is the first cargo sequence; binding the nucleic acid cation is functional in at least one of Staphylococcus and vector to a hybrid protein comprising at least one DNA bind Bifidobacteria. ing domain Suitable to bind to the nucleic acid vector, at least 0013. In alternative embodiments, the first and second one cell penetrating peptide (CPP) domain, and at least one origins of replication are comprised within a single bifunc secretion signal sequence, to form a complex between the tional origin. hybrid protein and the nucleic acid vector, contacting the 0014. In alternative embodiments, the nucleic acid vector prokaryotic target cell with the complex formed by the hybrid may further comprise a prokaryotic expression cassette Suit protein and the nucleic acid vector. able to express a first cargo sequence in the gram-positive 0028. In an embodiment there is disclosed a method for bacteria. transforming a prokaryotic target cell with a candidate DNA 0015. In alternative embodiments, the first cargo sequence sequence, the method comprising the steps of contacting the is a hybrid protein comprising at least one DNA binding prokaryotic target cell with a hybrid protein and nucleic acid US 2015/0232862 A1 Aug. 20, 2015 vector complex formed by contacting with a hybrid protein 0042 FIG. 4 shows the results of transforming E. coli with comprising at least one DNA binding domain Suitable to bind pBRA2.0-SHT vector according to an embodiment of the to the nucleic acid vector, at least one cell penetrating peptide present subject matter, to confirm the effectiveness of the E. (CPP) domain, and at least one secretion signal sequence, a coli (pUC) origin of replication. nucleic acid vector disclosed herein the candidate DNA 0043 FIG. 5 shows the results of transfecting HEK-293 sequence is the first cargo sequence. and HeLa cells with pBRA2.0 SHT vector according to an 0029. In an embodiment there is disclosed a bacterial cell embodiment of the present subject matter. containing the nucleic acid vector disclosed herein. 0044 FIG. 6 shows the secretion of pBRA2.0 SHT from 0030. In an embodiment there is disclosed a eukaryotic Bifidobacterium longum cells hosting the vector. cell containing the nucleic acid vector disclosed herein.