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WO 2018/009838 Al 11 January 2018 (11.01.2018) W !P O PCT

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WO 2018/009838 Al 11 January 2018 (11.01.2018) W !P O PCT (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 2018/009838 Al 11 January 2018 (11.01.2018) W !P O PCT (51) International Patent Classification: Declarations under Rule 4.17: C12N 5/075 (2010.01) — as to applicant's entitlement to apply for and be granted a (21) International Application Number: patent (Rule 4.1 7(H)) PCT/US2017/041 155 — as to the applicant's entitlement to claim the priority of the earlier application (Rule 4.17(Hi)) (22) International Filing Date: 07 July 2017 (07.07.2017) Published: — with international search report (Art. 21(3)) (25) Filing Language: English — before the expiration of the time limit for amending the (26) Publication Langi English claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) (30) Priority Data: — with sequence listing part of description (Rule 5.2(a)) 62/359,416 07 July 2016 (07.07.2016) US (71) Applicant: RUBIUS THERAPEUTICS, INC. [US/US]; 620 Memorial Dr #100W, Cambridge, MA 02139 (US). (72) Inventors; and (71) Applicants: HARANDI, Omid [US/US]; 39 Rowena Road, Newton, MA 02459 (US). KHANWALKAR, Ur- jeet [IN/US]; 2 11 Elm Street, Apt. 3, Cambridge, MA 02139 (US). HARIHARAN, Sneha [IN/US]; 18 Hamilton Road, Apt. 407, Arlington, MA 02472 (US). (72) Inventors: KAHVEJIAN, Avak; 2 Beverly Road, Arling ton, MA 02474 (US). MATA-FINK, Jordi; 8 Windsor Rd #1, Somerville, MA 02144 (US).DEANS, Robert, J.; 1609 Ramsgate Court, Riverside, CA 92506 (US). (74) Agent: ZHANG, Wei et al; Lando & Anastasi, LLP, River front Office Park, One Main Street, Suite 1100, Cambridge, MA 02 142 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 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, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 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, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 00 TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, oo KM, ML, MR, NE, SN, TD, TG). © © 00 (54) Title: COMPOSITIONS AND METHODS RELATED TO THERAPEUTIC CELL SYSTEMS EXPRESSING EXOGENOUS o RNA (57) Abstract: The invention includes compositions and methods related to erythroid cells comprising exogenous RNA encoding a protein. The exogenous RNA can comprise a heterologous untranslated region comprising a regulatory element. Alternatively or in combination, the exogenous RNA can comprise chemical modifications. COMPOSITIONS AND METHODS RELATED TO THERAPEUTIC CELL SYSTEMS EXPRESSING EXOGENOUS RNA RELATED APPLICATIONS This application claims priority to U.S. Serial No. 62/359416 filed July 7, 2016, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 7, 2017, is named R2081-7015WO_SL.txt and is 921 bytes in size. BACKGROUND Red blood cells have been considered for use as drug delivery systems, e.g., to degrade toxic metabolites or inactivate xenobiotics, and in other biomedical applications. There is a need in the art for improved red blood cell based drug delivery systems. SUMMARY OF THE INVENTION The invention includes compositions and methods related to erythroid cells comprising exogenous RNA (e.g., exogenous RNA encoding a protein). The exogenous RNA can comprise a coding region and a heterologous untranslated region (UTR), e.g., a UTR comprising a regulatory element. Alternatively or in combination, the exogenous RNA can comprise chemical modifications. Alternatively or in combination, the exogenous RNA can be a regulatory RNA such as a miRNA. While not wishing to be bound by theory, in some embodiments the exogenous RNA has improved parameters, such as stability or increased translation, relative to a control. In certain aspects, the present disclosure provides an enucleated erythroid cell comprising: an exogenous mRNA comprising a coding region operatively linked to a heterologous untranslated region (UTR). In certain aspects, the present disclosure provides an enucleated erythroid cell, comprising: an exogenous mRNA comprising a coding region operatively linked to a heterologous untranslated region (UTR), wherein the heterologous UTR comprises a regulatory element. In certain aspects, the present disclosure provides an erythroid cell, e.g., an enucleated erythroid cell, comprising an exogenous mRNA that comprises one or more chemically modified nucleotides (e.g., one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof). The disclosure also provides a method of producing an erythroid cell, e.g., enucleated erythroid cell, comprising: a) contacting an erythroid cell, e.g., a nucleated erythroid cell, with an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element, (e.g., isolated RNA or in vitro transcribed RNA), and b) maintaining the contacted erythroid cell under conditions suitable for uptake of the exogenous mRNA, thereby producing the erythroid cell, e.g., an enucleated erythroid cell. The disclosure also provides a method of producing an erythroid cell, e.g., enucleated erythroid cell, comprising: a) contacting an erythroid cell, e.g., a nucleated erythroid cell, with an exogenous mRNA comprising one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof; and b) maintaining the contacted erythroid cell under conditions suitable for uptake of the exogenous mRNA, thereby producing the erythroid cell, e.g., an enucleated erythroid cell. The disclosure further provides a method of producing an exogenous protein in an enucleated erythroid cell: a) providing an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element, (e.g., isolated RNA or in vitro transcribed RNA), and b) culturing the erythroid cell under conditions suitable for production of the exogenous protein, thereby producing the exogenous protein. The disclosure further provides a method of producing an exogenous protein in an enucleated erythroid cell: a) providing an erythroid cell, e.g., a nucleated erythroid cell, comprising one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof, and b) culturing the erythroid cell under conditions suitable for production of the exogenous protein, thereby producing the exogenous protein. In certain aspects, the disclosure provides a method of providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject, comprising administering to the subject: an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element, (e.g., isolated RNA or in vitro transcribed RNA), thereby providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject. In certain aspects, the disclosure provides a method of providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject, comprising administering to the subject: an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof, thereby providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject. In some aspects, the disclosure provides a method of evaluating an erythroid cell, e.g., enucleated erythroid cell (or a batch of such cells) comprising: a) providing an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element (or a batch of such cells), and b) evaluating the erythroid cell, e.g., the nucleated erythroid cell (or batch of such cells) for a preselected parameter, thereby evaluating
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