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(12) Patent Application Publication (10) Pub. No.: US 2017/0196966 A1 HENDERSON (43) Pub US 2017.0196966A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0196966 A1 HENDERSON (43) Pub. Date: Jul. 13, 2017 (54) MICRONEEDLE COMPOSITIONS AND A6IR 9/00 (2006.01) METHODS OF USING SAME A6IR 48/00 (2006.01) (52) U.S. Cl. (71) Applicant: Verndari, Inc., Sacramento, CA (US) CPC .......... A61K 39/145 (2013.01); A61K 48/005 (2013.01); C12N 7/00 (2013.01); A61K 9/002I (72) Inventor: Daniel R. HENDERSON, Sacramento, (2013.01); CI2N 27.60/16(34 (2013.01); CI2N CA (US) 2760/16234 (2013.01); C12N 2770/36143 (2013.01); A61 K 2039/53 (2013.01) (21) Appl. No.: 15/403,989 (57) ABSTRACT (22) Filed: Jan. 11, 2017 Described herein, are microneedle devices comprising a recombinant alphavirus replicon encoding an exogenous Related U.S. Application Data polypeptide, wherein the recombinant alphavirus replicon is (60) Provisional application No. 62/277.312, filed on Jan. coated onto or embedded into a plurality of microneedles. 11, 2016. Also described herein are methods of preparing a microneedle device comprising a recombinant alphavirus replicon encoding an exogenous polypeptide. Also disclosed Publication Classification herein are methods of inducing an immune response in an (51) Int. Cl. individual comprising contacting the individual with a A6 IK 39/45 (2006.01) microneedle device comprising a recombinant alphavirus CI2N 7/00 (2006.01) replicon encoding an exogenous polypeptide. Patent Application Publication Jul. 13, 2017. Sheet 1 of 19 US 2017/O196966 A1 ---- Patent Application Publication Jul. 13, 2017. Sheet 2 of 19 US 2017/O196966 A1 F.G. 2 Patent Application Publication US 2017/O196966 A1 |?ON-1685(36/6) Patent Application Publication Jul. 13, 2017. Sheet 4 of 19 US 2017/O196966 A1 Patent Application Publication Jul. 13, 2017. Sheet 5 of 19 US 2017/O196966 A1 Patent Application Publication Jul. 13, 2017. Sheet 6 of 19 US 2017/O196966 A1 Patent Application Publication Jul. 13, 2017. Sheet 7 of 19 US 2017/O196966 A1 Patent Application Publication Jul. 13, 2017 Sheet 8 of 19 US 2017/O196966 A1 in Vitro Synthesis of Repticon miRNA ised to 2.2 ied c. 237 x On x 220 dose 230 iOSes Size f. k. Size 8.8 k Size 94 ki Patent Application Publication Jul. 13, 2017. Sheet 9 of 19 US 2017/0196966 A1 EGFR 24h 24h 24h 48h 48th 48 - ka 250 ... 50 Patent Application Publication Jul. 13, 2017. Sheet 10 of 19 US 2017/0196966 A1 kDa 150 100 75 50 37 HA-Contro 25 RPA Buffer, not ransfected RIPA Buffer, ag RNA 20 RIPA Buffer-DTT, ag RNA 10 Oid lysis Buffer, ag RNA adder FG. Patent Application Publication Jul. 13, 2017. Sheet 11 of 19 US 2017/0196966 A1 cy Kix 3.2. ce r Nig is re : ošs 35 3 s S. is Z SS s s 2Y: ifS a. cxs i. ww. r s - - ar S i. re: S. Ca e N 5- s: S, 8. ES S. W. war n E. o SB S N . N: . gsa 2.35"S 3. N SHSp. N. - as its* , wer ... i a 2. c re Fiorescence Patent Application Publication Jul. 13, 2017. Sheet 12 of 19 US 2017/0196966 A1 : Patent Application Publication Jul. 13, 2017. Sheet 13 of 19 US 2017/0196966 A1 s c w s ow s s w w s i. f s: (3. 8. s -a s g c c EC d c s cN Size n: Patent Application Publication Jul. 13, 2017. Sheet 14 of 19 US 2017/0196966 A1 92 a-a-a-a- se w s s ag ar 8: K .a. cs g S. : See ...g-SS E .32 se: . - S. C.S. As 2 32Y cs d g d C o c ad r N ar Yew ass () lara y Yser S. o K. S2 . g. N 2 seta. r c g Patent Application Publication Jul. 13, 2017. Sheet 16 of 19 US 2017/0196966 A1 Recovery of mEGFP off Array 100 88 OO 4. O 20 F.G. 6 O792 0.575 Patent Application Publication Jul. 13, 2017. Sheet 17 of 19 US 2017/O196966 A1 FG ... 8 FG, 9 Patent Application Publication Jul. 13, 2017. Sheet 18 of 19 US 2017/0196966 A1 FG, 2. Patent Application Publication Jul. 13, 2017. Sheet 19 of 19 US 2017/O196966 A1 1 ef US 2017/O 196966 A1 Jul. 13, 2017 MCRONEEDLE COMPOSITIONS AND antigen associated with a cancer. In some embodiments, the METHODS OF USING SAME foreign antigen is an antigen associated with an infectious agent. In some embodiments, the recombinant alphavirus CROSS REFERENCE TO RELATED replicon is present in an amount effective to induce an APPLICATIONS immune response to the foreign or self-antigen. In some 0001. This application claims the benefit of U.S. Provi embodiments, the exogenous polypeptide is an influenza sional Application No. 62/277.312, filed on Jan. 11, 2016, virus HA or NA polypeptide. In some embodiments, the which is incorporated by reference herein in its entirety. influenza virus HA polypeptide is an influenza A virus HA polypeptide or an influenza B virus HA polypeptide. In some SEQUENCE LISTING embodiments, the influenza virus HA polypeptide is from a 0002 The instant application contains a Sequence Listing viral strain of a group 1 influenza A virus Subtype selected which has been submitted electronically in ASCII format from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and is hereby incorporated by reference in its entirety. Said or H18. In some embodiments, the influenza virus HA ASCII copy, created on Jan. 31, 2017, is named polypeptide is from a viral strain of a group 2 influenza A 47750701201 SL.txt and is 514 bytes in size. virus subtype selected from H3, H4, H7, H10, H14, or H15. In some embodiments, the influenza virus HA polypeptide is BACKGROUND OF THE INVENTION from a viral strain of an influenza B virus. In some embodi ments, the influenza virus HA polypeptide is from a viral 0003 Delivery of compositions to a target cell or tissue strain of an influenza A virus H1 subtype. In some embodi faces various transport barriers. Nucleic acids that encode ments, the influenza virus HA polypeptide is from a viral gene products, such as proteins, and non-coding RNA (e.g., strain of an influenza A virus H3 subtype. In some embodi siRNAs) can be delivered directly to a desired vertebrate ments, the influenza virus HA polypeptide is from a viral subject, or can be delivered ex vivo to cells obtained or strain of an influenza B virus Yamagata or Victoria lineage. derived from the subject, and the cells can be re-implanted In some embodiments, the recombinant alphavirus replicon into the subject. Delivery of such nucleic acids to a verte encodes an exogenous polypeptide comprising: (a) an HA brate subject is desirable for many purposes, such as, for polypeptide from a viral strain of an influenza A virus H1 gene therapy, to induce an immune response against an subtype; (b) an HA polypeptide from a viral strain of an encoded polypeptide, or to regulate the expression of endog influenza A virus H3 subtype; (c) an HA polypeptide from a enous genes. The use of this approach has been hindered viral strain of an influenza B virus Yamagata lineage; (d) an because free DNA is not readily taken up by cells and free HA polypeptide from a viral strain of an influenza B virus RNA is rapidly degraded in vivo. Moreover, delivery can Victoria lineage; or (e) any combinations thereof. In some also be problematic. For instance, Subcutaneous or intra embodiments, the recombinant alphavirus replicon encodes muscular injections using hypodermic needles can cause at least two exogenous polypeptides comprising: (a) an HA pain, trauma, and anxiety in a Subject. polypeptide from a viral strain of an influenza A virus H1 0004 Delivery of one or more polypeptides, whether subtype; (b) an HA polypeptide from a viral strain of an directly as protein or indirectly by an encoding polynucle influenza A virus H3 subtype; (c) an HA polypeptide from a otide, has many useful applications, including vaccination. viral strain of an influenza B virus Yamagata lineage; or (d) Vaccination has proven an effective means to fight and even an HA polypeptide from a viral strain of an influenza B virus eradicate infectious diseases. The influenza vaccine, for Victoria lineage. In some embodiments, each of the exog example, is currently recommended by the CDC as the enous polypeptides are encoded on a single recombinant primary method for preventing influenza. However, influ alphavirus replicon. In some embodiments, the exogenous enza virus has a high rate of mutation and antigenic variation polypeptides are encoded on different recombinant alphavi and a new vaccine is typically produced each year based rus replicons. In some embodiments, the exogenous poly upon the predicted circulating pathogenic strains. This poses peptide is a hepatitis B virus Surface antigen (HBSAg). In a number of challenges. For instance, the effectiveness of the Some embodiments, the recombinant alphavirus replicon vaccine is only as good as the prediction. If the prediction of encodes an exogenous polypeptide comprising: (a) an anti the dominant strain is incorrect, the vaccine will have gen from a polio virus; (b) an antigen from Clostridium limited effectiveness for most people. Further, it can take tetani; (c) an antigen from a rabies virus; or (d) any months to produce enough influenza vaccine to vaccinate a combinations thereof. In some embodiments, the recombi population. nant alphavirus replicon encodes an exogenous polypeptide comprising: (a) an antigen from a polio virus; (b) an antigen SUMMARY OF THE INVENTION from Clostridium tetan; and (c) an antigen from a rabies 0005 Disclosed herein, in some embodiments, are virus.
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