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(12) Patent Application Publication (10) Pub. No.: US 2014/0364588 A1 Haugwitz Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2014/0364588 A1 Haugwitz Et Al US 2014036.4588A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0364588 A1 Haugwitz et al. (43) Pub. Date: Dec. 11, 2014 (54) PROTEIN ENRCHEDMCROVESCLES AND Publication Classification METHODS OF MAKING AND USING THE SAME (51) Int. Cl. CI2P 2L/00 (2006.01) (71) Applicant: Clontech Laboratories, Inc., Mountain (52) U.S. Cl. View, CA (US) CPC ...................................... CI2P21/00 (2013.01) USPC ........................ 530/350; 435/69.1:435/320.1 (72) Inventors: Michael Haugwitz, Mountain View, CA (US); Thomas Patrick Quinn, Pacifica, (57) ABSTRACT CA (US); Andrew Alan Farmer, Mountain View, CA (US) Protein enriched micro-vesicles and methods of making and using the same are provided. Aspects of the methods include (21) Appl. No.: 14/278,714 maintaining a cell having a membrane-associated protein comprising a first dimerization domain and a target protein (22) Filed: May 15, 2014 having a second dimerization domain under conditions Suf ficient to produce a micro-vesicle from the cell, wherein the Related U.S. Application Data micro-vesicle includes the target protein. Also provided are (60) Provisional application No. 61/872,115, filed on Aug. cells, reagents and kits that find use in making the micro 30, 2013, provisional application No. 61/833,880, vesicles, as well as methods of using the micro-vesicles, e.g., filed on Jun. 11, 2013. in research and therapeutic applications. Patent Application Publication US 2014/036.4588 A1 Patent Application Publication Dec. 11, 2014 Sheet 2 of 6 US 2014/036.4588 A1 k N CD i res & s Patent Application Publication Dec. 11, 2014 Sheet 3 of 6 US 2014/036.4588 A1 Patent Application Publication Dec. 11, 2014 Sheet 4 of 6 US 2014/036.4588 A1 Patent Application Publication Dec. 11, 2014 Sheet 5 of 6 US 2014/036.4588 A1 Patent Application Publication Dec. 11, 2014 Sheet 6 of 6 US 2014/036.4588 A1 s US 2014/03.64588 A1 Dec. 11, 2014 PROTEIN ENRCHEDMCROVESCLES AND the micro-vesicles, as well as methods of using the micro METHODS OF MAKING AND USING THE vesicles, e.g., in research and therapeutic applications. SAME BRIEF DESCRIPTION OF THE FIGURES CROSS REFERENCE TO RELATED APPLICATIONS 0007 FIG. 1 provides a schematic of the preparation and use micro-vesicles as described in the Experimental Section, 0001 Pursuant to 35 U.S.C. S 119 (e), this application below. claims priority to the filing date of the U.S. Provisional Patent 0008 FIGS. 2A to 2D illustrate various aspects and pro Application Ser. No. 61/872,115, filed Aug. 30, 2013 and vide results of an embodiment of the invention in which CRE U.S. Provisional Patent Application Ser. No. 61/833,880, recombinase micro-vesicles are produced. filed Jun. 11, 2013; the disclosure of which is herein incor 0009 FIG.3 provides the results of assay of micro-vesicle porated by reference. target protein content. INTRODUCTION DETAILED DESCRIPTION 0002 Cell modification finds use in a variety of different applications, including research, diagnostic and therapeutic 0010 Protein enriched micro-vesicles and methods of applications. Cell modification may beachieved using a num making and using the same are provided. Aspects of the ber of different approaches, including the introduction of methods include maintaining a cell having a membrane-as exogenous nucleic acids and/or proteins into a cell. Sociated protein comprising a first dimerization domain and a 0003 Protein delivery, which is known in the art as protein target protein having a second dimerization domain under transduction, is the process by which a peptide or protein conditions sufficient to produce a micro-vesicle from the cell, motif is delivered across the plasma membrane into the cell. wherein the micro-vesicle includes the target protein. Also Protein delivery methods include micro-injection and elec provided are cells, reagents and kits that find use in making troporation. Protein delivery methods also include: transfec the micro-vesicles, as well as methods of using the micro tion by forming complexes with lipid-based reagents; trans vesicles, e.g., in research and therapeutic applications. fection by forming complexes with polymer or peptide based 0011 Before the present invention is described in greater reagents; direct addition through inclusion of a peptide trans detail, it is to be understood that this invention is not limited duction domain (PTD) to the protein of interest; virus like to particular embodiments described, as Such may, of course, particle mediated introduction; and exosome mediated pro vary. The invention encompasses various alternatives, modi tein introduction. fications, and equivalents, as will be appreciated by those of 0004 One drawback of the current methods of protein skill in the art. It is also to be understood that the terminology delivery is the requirement to produce a stock of purified used herein is for the purpose of describing particular protein for transfection into the desired target cell. Standard embodiments only, and is not intended to be limiting, since methods for the production of recombinant protein can the scope of the present invention will be limited only by the present issues with Solubility, yield, correct folding and post appended claims. translational modifications. These methods also do not allow 0012. Where a range of values is provided, it is understood for the delivery of recombinant membrane proteins. Many of that each intervening value, to the tenth of the unit of the lower these factors are important because they directly relate to the limit unless the context clearly dictates otherwise, between activity of the protein to be transfected. The activity of the the upper and lower limit of that range and any other stated or protein has the highest priority for direct delivery so that the intervening value in that stated range, is encompassed within delivered protein will exert an effect on a cell. the invention. The upper and lower limits of these smaller 0005. Another drawback to the current methods lies in the ranges may independently be included in the Smaller ranges delivery itself. Both the lipid and polymer/peptide based and are also encompassed within the invention, Subject to any transfection methods have issues with protein specific pack specifically excluded limit in the stated range. Where the aging efficiency due to unfavorable charge differences as well stated range includes one or both of the limits, ranges exclud as inefficient delivery and toxicity. Electroporation also has ing either or both of those included limits are also included in been shown to have issues with toxicity, high level of incon the invention. sistency and a lack of control over the protein amount deliv 0013 Certain ranges are presented herein with numerical ered. Inclusion of a PTD is known to cause aggregation and values being preceded by the term “about.” The term “about precipitation which can adversely affect delivery efficiency. is used herein to provide literal support for the exact number Lastly, delivery of proteins in virus like particles (VLPs) that it precedes, as well as a number that is near to or approxi requires that immune response-generating viral capsid pro mately the number that the term precedes. In determining teins are used for packaging. whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number SUMMARY may be a number which, in the context in which it is pre 0006 Protein enriched micro-vesicles and methods of sented, provides the Substantial equivalent of the specifically making and using the same are provided. Aspects of the recited number. methods include maintaining a cell having a membrane-as 0014. It is noted that, as used herein and in the appended Sociated protein comprising a first dimerization domain and a claims, the singular forms “a”, “an', and “the include plural target protein having a second dimerization domain under referents unless the context clearly dictates otherwise. It is conditions Sufficient to produce a micro-vesicle from the cell, further noted that the claims can be drafted to exclude any wherein the micro-vesicle includes the target protein. Also optional element. As such, this statement is intended to serve provided are cells, reagents and kits that find use in making as antecedent basis for use of Such exclusive terminology as US 2014/03.64588 A1 Dec. 11, 2014 “solely.” “only' and the like in connection with the recitation dimerization domain contacts the cytosol of the micro of claim elements, or use of a “negative' limitation. vesicle. Membrane-associated proteins may vary in size, 0015. As will be apparent to those of skill in the art upon ranging in some instances from 5 kDa to 250 kDa, Such as 10 reading this disclosure, each of the individual embodiments kDa to 100 kDa and including 12 kDa to 50 kDa. described and illustrated herein has discrete components and 0021. The membrane-associated protein may be modified features which can be readily separated from or combined to include a single dimerization domain or two or more dimer with the features of any of the other several embodiments ization domains (e.g., as described in greater detail below). In without departing from the scope or spirit of the present Some cases, two or more membrane-associated proteins may invention. Any recited method can be carried out in the order be included in the subject cells and micro-vesicles. Each of of events recited or in any other order which is logically the two or more membrane-associated proteins may
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