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WO 2015/034962 Al 12 March 2015 (12.03.2015) P O P C T

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WO 2015/034962 Al 12 March 2015 (12.03.2015) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/034962 Al 12 March 2015 (12.03.2015) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 15/03 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/US2014/053954 KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (22) International Filing Date: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 3 September 2014 (03.09.2014) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (25) Filing Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (26) Publication Language: English ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 61/873,308 3 September 2013 (03.09.2013) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: BELL BIOSYSTEMS, INC. [US/US]; 530 TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, Lytton Ave., 2nd Floor, Palo Alto, CA 94301 (US). TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors: BELL, Caleb, B.; 334 N. Claremont St., San LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Mateo, CA 94401 (US). BAZAROV, Alexey; 36407 Ser- SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, eno Common, Fremont, CA 94536 (US). WAREEL, Ab¬ GW, KM, ML, MR, NE, SN, TD, TG). dul; 4988 Cappy Terrace, Fremont, CA 94555 (US). BAR- ROZO, Joyce; 150 Yorkshire Court, San Bruno, CA Published: 94066 (US). — with international search report (Art. 21(3)) (74) Agents: OH, Euk et al; 2479 East Bayshore Road, Suite — with sequence listing part of description (Rule 5.2(a)) 215, Palo Alto, CA 94303 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: HOST CELL MODIFICATION WITH ARTIFICIAL ENDOSYMBIONTS FIG. 10 © (57) Abstract: The present invention is directed generally to host cells with artificial endosymbionts, wherein the artificial endosym o biont and the host cell communicate with each other to alter a phenotype of the host cell. In some embodiments, the communication comprises the secretion of a polypeptide from the artificial endosymbiont into the host cell. The secreted polypeptide can be a select - o able marker, a reporter protein, a transcription factor, a signal pathway protein, a receptor, a growth factor, a cytokine, an effector molecule or other factors that can produce a phenotype in the host cell. HOST CELL MODIFICATION WITH ARTIFICIAL ENDOSYMBIONTS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of U.S. Provisional application Serial No. 61/873,308, filed September 3, 2013, the contents of which are incorporated herein by reference. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM [0002] The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name ofBELL.018_ST25.txt", a creation date of September 3, 2014, and a size of 962 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein. TECHNICAL FIELD [0003] The present invention relates generally to host cells with artificial endosymbionts. The artificial endosymbionts of the host cell communicates with the host cell and changes aspects of the host cell. BACKGROUND [0004] The advent of recombinant DNA technology created the ability to alter the genetic makeup of organisms, eliminating the natural barriers that prevented transmission of genetic material between unrelated organisms. The ease of growing and manipulating bacteria and the numerous techniques available for introducing heterologous genes makes bacteria model organisms for genetic manipulation using recombinant DNA technology. Gram-negative and Gram-positive bacteria have been subjected to successful manipulation with recombinant DNA technology. Because of the well developed technology for transforming bacterial cells, the relative ease of genetically manipulating different bacteria, short reproduction times, and the comparatively small genomes, bacteria have been used as vehicles for synthetic biology applications, which aims to create novel artificial biological pathways, organisms or devices, or the redesign of existing natural biological systems. Moreover, bacteria exist with a wide range of functionalities, such as unique metabolic pathways (e.g., photo- and chemoautotrophism), magnetotactic properties (Blakemore, R., "Magnetotactic bacteria," Science 24: 377-379, 1975), and extremophiles (e.g., thermophiles), which allows creation of recombinant bacteria with the properties present in the bacterial host cell. [0005] Recombinant DNA techniques have also been used to manipulate the genetic makeup of eukaryotic cells, transiently or as a heritable property. For example, homologous and targeted recombination allows the generation of recombinant animals containing heterologous genes. Despite the advances in technology, manipulation of eukaryotic cells by recombinant DNA techniques has greater challenges as compared to manipulation of bacteria. Some eukaryotic cells targeted for recombinant DNA manipulation, e.g. , mammalian cells, are slower to grow than bacterial cells, making selection of eukaryotic cells containing a heterologous nucleic acid more time consuming. Creation of eukaryotic cells containing longer lasting changes, including heritable changes, typically requires the use of homologous recombination, which despite the advances in technology, continue to have low efficiency rates. In addition, introduction of multiple, different heterologous nucleic acids is complicated by the higher complexity of eukaryotic cells (e.g. , presence of organelles), greater degree of unpredictability in responses to multiple heterologous factors, and the technological disadvantages of selecting eukaryotic cells having multiple genetic/phenotypic changes. Thus, it is desirable to find alternative methods of engineering eukaryotic cells for introducing new functionalities into the cells, where the methods can be applied independently of or in combination with recombinant DNA technology for modifying eukaryotic cells. SUMMARY [0006] The present invention is related to host cells that have been altered or reprogrammed by communication and/or transfer of chemical information with artificial endosymbionts. In some embodiments, the artificial endosymbionts of the invention secrete to and/or transport from the host cell polypeptides, nucleic acids, lipids, carbohydrates, amino acids, or other factors. In some embodiments, the artificial endosymbiont secretes a protein into the host cell. In some embodiments, the secreted protein is a heterologous protein to the artificial endosymbiont. In some embodiments, the secreted protein from the artificial endosymbiont causes a phenotype in the host cell. In some embodiments, the artificial endosymbiont of the invention secretes a nucleic acid into the host cell. In some embodiments, the nucleic acid s a recombinant nucleic acid. In some embodiments, the nucleic acid secreted from the artificial endosymbiont causes a phenotype in the host cell. [0007] The invention also relates to methods for changing a host cell by the introduction of an artificial endosymbiont that secretes to and/or transports from the host cell polypeptides, nucleic acids, lipids, carbohydrates, amino acids, or other factors. In some embodiments, the method introduces a protein into the host cell from the artificial endosymbiont. In some embodiments, the method introduces a nucleic acid into the host cell from the artificial endosymbiont. In some embodiments, the method produces a phenotype in the host cell resulting from the polypeptide and/or nucleic acid introduced from the artificial endosymbiont. [0008] In some embodiments, the host cells and methods of the invention are used to make medically and industrially important recombinant peptides/proteins that will be useful for therapeutic, biopharmaceutical, agricultural, and industrial applications. In some embodiments, the artificial endosymbionts and methods of the invention are used to introduce into host cells phenotypes that require the introduction of multiple factors and/or multiple genes. [0009] In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human, mouse, rat, canine, primate, or rodent cell. In some embodiments, the host cell is a fibroblast cell, epithelial cell, keratinocyte, hepatocyte, adipocyte or endothelial cell. In some embodiments, the host cell is a stem cell, or pluripotent ES cell, or pluripotent iPS cell, a multipotent mesenchymal stem cell or multipotent hematopoietic stem cell. In some embodiments, the host cell is a progenitor cell, such as for example, a neural progenitor cell, an angioblast, an osteoblast, a chondroblast, a pancreatic progenitor cell, or an epidermal progenitor cell. In some embodiments, the host cell is a solid tumor cell or a hematopoietic cancer cell. In some embodiments, the host cell is from a carcinoma, sarcoma,
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