(12) STANDARD PATENT (11) Application No. AU 2009212543 B2 (19) AUSTRALIAN PATENT OFFICE (54) Title Use of microvesicles in diagnosis, prognosis and treatment of medical diseases and conditions (51) International Patent Classification(s) C12Q 1/68 (2006.01) (21) Application No: 2009212543 (22) Date of Filing: 2009.02.02 (87) WIPONo: WO09/100029 (30) Priority Data (31) Number (32) Date (33) Country 61/100,293 2008.09.26 US 61/025,536 2008.02.01 US (43) Publication Date: 2009.08.13 (44) Accepted Journal Date: 2015.07.09 (71) Applicant(s) The General Hospital Corporation (72) Inventor(s) Russo, Leileata M.;Brown, Dennis;Skog, Johan Karl Olov;Miranda, Kevin C.;Breakefield, Xandra O. (74) Agent / Attorney Pizzeys, PO Box 291, WODEN, ACT, 2606 (56) Related Art WO 2003/076603 A2 (ANOSYS INC.) 18 September 2003 US 2007/0104738 A1 (TATISCHEFF, I., et al.) 10 May 2007 US 2006/0116321 A1 (ROBBINS, P. D., et al.) 01 June 2006 WO 2007/103572 A2 (AETHLON MEDICAL, INC.) 13 September 2007 WO 2009/015357 A1 (UNIVERSITY OF LOUISVILLE RESEARCH FOUNDATION, INC.) 29 January 2009 # Baj-Krzyworzeka, M., et al. 2006. Cancer Immunology, Immunotherapy, vol. 55, pages 808-818. WO 2009/021322 A1 (THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY) 19 February 2009 WO 2009/036236 A1 (THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION) 19 March 2009 WO 2005/121369 A2 (SOURCEPHARM, INC.) 22 December 2005 WO 2001/036601 A1 (CHIRON S.P.A.) 25 May 2001 WO 1994/022018 A1 (NYCOMED PHARMA A/S) 29 September 1994 CA 2543198 A1 (MIN, W-P and ICHIM, T. E.) 07 July 2005 WO 2007/126386 A1 (LOTVALL, J. O. and VALADI, H.) 08 November 2007 (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 2009/100029 Al 13 August 2009 (13.08.2009) PCT (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, PCT/US2009/032881 EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (22) International Filing Date: HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, 2 February 2009 (02.02.2009) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, (25) Filing Language: English NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, (26) Publication Language: English SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/025,536 1 February 2008 (01.02.2008) US (84) Designated States (unless otherwise indicated, for every 61/100,293 26 September 2008 (26.09.2008) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (71) Applicant (for all designated States except US): THE ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, GENERAL HOSPITAL CORPORATION [US/US]; TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, 55 Fruit Street, Boston, MA 02114 (US). ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, TR), (72) Inventors; and OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, (75) Inventors/Applicants for US only): SKOG, Johan [SE/ MR, NE, SN, TD, TG). US]; 218 Thorndike Street, Cambridge, Massachusetts 02141 (US). BREAKEFIELD, Xandra O. [US/US]; 127 Declarations under Rule 4.17: Homer Street, Newton, Massachusetts 02459 (US). — of inventorship (Rule 4.17(iv)) BROWN, Dennis [GB/US]; 3 Walnut Avenue, Natick, Massachusetts 01760 (US). MIRANDA, Kevin C. [AU/ Published: US]; 22 Bigelow Street, Apt. 3A, Cambridge, Mas­ — with international search report (Art. 21(3)) sachusetts 02139 (US). RUSSO, Leileata M. [AU/US]; 1 Oak Grove Avenue, Apt. 227, Melrose, Massachusetts — before the expiration of the time limit for amending the 02176 (US). claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) (74) Agent: RESNICK, David; Nixon Peabody Lip, 100 Summer Street, Boston, Massachusetts 02110-2131 (US). — with sequence listing part of description (Rule 5.2(a)) A l (54) Title: USE OF MICROVESICLES IN DIAGNOSIS, PROGNOSIS AND TREATMENT OF MEDICAL DISEASES AND CONDITIONS (57) Abstract: The presently disclosed subject matter is directed to methods of aiding diagnosis, prognosis, monitoring and evalu­ ation of a disease or other medical condition in a' subject by detecting a biomarker in microvesicles isolated from a biological, 20 09 /100sample 02 9 from the subject. Moreover, disclosed subject matter is directed to methods of diagnosis, monitoring a disease by deter­ mining the concentration of microvesicles within a biological sample; methods of delivering a nucleic acid or protein to a target all by administering microvesicles that contain said nucleic acid or protein; methods for performing a body fluid transfusion by in­ troducing a microvesicle-free or microvesicle enriched fluid fraction into a patient. WO WO 2009/100029 PCT/US2009/032881 USE OF MICROVESICLES IN DIAGNOSIS, PROGNOSIS AND TREATMENT OF MEDICAL DISEASES AND CONDITIONS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to US provisional applications 61/025,536, filed February 1, 2008 and 61/100,293, filed September 26, 2008, each of which is incorporated herein by reference in its entirety. GOVERNMENTAL SUPPORT [0002] This invention was made with Government support under grants NCI CA86355 and NCI CA69246 awarded by the National Cancer Institute. The Government has certain rights in the invention. FIELD OF THE INVENTION [0003] The present invention relates to the fields of medical diagnosis, patient monitoring, treatment efficacy evaluation, nucleic acid and protein delivery, and blood transfusion. BACKGROUND OF THE INVENTION [0004] Glioblastomas are highly malignant brain tumors with a poor prognosis despite intensive research and clinical efforts (Louis et al., 2007). The invasive nature of this tumor makes complete surgical resection impossible and the median survival time is only about 15 months (Stupp et al., 2005). Glioblastoma cells as well as many other tumor cells have a remarkable ability to mold their stromal environment to their own advantage. Tumor cells directly alter surrounding normal cells to facilitate tumor cell growth, invasion, chemo- resistance, immune-evasion and metastasis (Mazzocca et al., 2005; Muerkoster et al., 2004; Singer et al., 2007). The tumor cells also hijack the normal vasculature and stimulate rapid formation of new blood vessels to supply the tumor with nutrition (Carmeliet and Jain, 2000). Although the immune system can initially suppress tumor growth, it is often progressively blunted by tumor activation of immunosuppressive pathways (Gabrilovich, 2007). [0005] Small microvesicles shed by cells are known as exosomes (Thery et al., 2002). Exosomes are reported as having a diameter of approximately 30-100 nm and are shed from many different cell types under both normal and pathological conditions (Thery et al., 2002). These microvesicles were first described as a mechanism to discard transferrin-receptors 1 WO 2009/100029 PCT/US2009/032881 from the cell surface of maturing reticulocytes (Pan and Johnstone, 1983). Exosomes are formed through inward budding of endosomal membranes giving rise to intracellular multivesicular bodies (MVB) that later fuse with the plasma membrane, releasing the exosomes to the exterior (Thery et al., 2002). However, there is now evidence for a more direct release of exosomes. Certain cells, such as Jurkat T-cells, are said to shed exosomes directly by outward budding of the plasma membrane (Booth et al., 2006). All membrane vesicles shed by cells are referred to herein collectively as micro vesicles. [0006] Microvesicles in Drosophila melanogaster, so called argosomes, are said to contain morphogens such as Wingless protein and to move over large distances through the imaginal disc epithelium in developing Drosophila melanogaster embryos (Greco et al., 2001). Microvesicles found in semen, known as prostasomes, are stated to have a wide range of functions including the promotion of sperm motility, the stabilization of the acrosome reaction, the facilitation of immunosuppression and the inhibition of angiogenesis (Delves et al., 2007). On the other hand, prostasomes released by malignant prostate cells are said to promote angiogenesis. Microvesicles are said to transfer proteins (Mack et al., 2000) and recent studies state that microvesicles isolated from different cell lines can also contain messenger RNA (mRNA) and microRNA (miRNA) and can transfer mRNA to other cell types (Baj-Krzyworzeka et al., 2006; Valadi et al., 2007). [0007] Microvesicles derived from B-cells and dendritic cells are stated to have potent immuno-stimulatory and antitumor effects in vivo and have been used as antitumor vaccines (Chaput et al., 2005). Dendritic cell-derived microvesicles are stated to contain the co­ stimulatory proteins necessary for T-cell activation, whereas most tumor cell-derived microvesicles do not (Wieckowski and Whiteside, 2006). Microvesicles isolated from tumor cells may act to suppress the immune response and accelerate tumor growth (Clayton et al., 2007; Liu et al., 2006a). Breast cancer microvesicles may stimulate angiogenesis, and platelet-derived micro vesicles may promote tumor progression and metastasis of lung cancer cells (Janowska-Wieczorek et al., 2005; Millimaggi et al., 2007). [0008] Cancers arise through accumulation of genetic alterations that promote unrestricted cell growth. It has been stated that each tumor harbors, on average, around 50-80 mutations that are absent in non-tumor cells (Jones et al., 2008; Parsons et al., 2008; Wood et al., 2007).