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US 2016/0361439 A1 AGBANDUE-MCKENNA Et Al US 20160361439A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0361439 A1 AGBANDUE-MCKENNA et al. (43) Pub. Date: Dec. 15, 2016 (54) CAPSD-MUTATED RAAV VECTORS AND Publication Classification METHODS OF USE (51) Int. Cl. (71) Applicant: University of Florida Research A6IR 48/00 (2006.01) Foundation, Inc., Gainesville, FL (US) CI2N 5/86 (2006.01) (52) U.S. Cl. (72) Inventors: Mavis AGBANDJE-MCKENNA, CPC ........... A61K 48/0058 (2013.01): CI2N 15/86 Gainesville, FL (US); William W. (2013.01); C12N 27.10/10043 (2013.01); C12N HAUSWIRTH, Gainesville, FL (US); 2830/008 (2013.01) Arun SRIVASTAVA, Gainesville, FL (US); Li ZHONG, Boxborough, MA (57) ABSTRACT (US) Disclosed are capsid-mutated ra AV vectors and methods for their use in gene therapy, and particularly for use in deliv (21) Appl. No.: 15/246,385 ering therapeutic transgenes to treat a variety of mammalian diseases and disorders, including dysfunctions and abnormal conditions of the human eye. VP3 capsid proteins compris (22) Filed: Aug. 24, 2016 ing a modification of one or more of the Surface-exposed tyrosine residues are disclosed, and in particular. VP3 capsid Related U.S. Application Data protein comprising tyrosine-to-phenylalanine mutations at positions corresponding to Y444F, Y500F, and Y730F of the (63) Continuation of application No. 14/298,553, filed on wild-type AAV2 sequence. Also provided are ra AV virions Jun. 6, 2014, which is a continuation-in-part of ap and viral particles that comprise Such a mutated AAV capsid plication No. 12/993,092, filed on Jun. 10, 2011, now protein and a nucleic acid molecule that expresses one or abandoned, filed as application No. PCT/US09/.44753 more selected therapeutic or reporter transgenes in one or on May 20, 2009. more mammalian cells of interest. Advantageously, the (60) Provisional application No. 61/054,571, filed on May capsid-mutated ra AV vectors and virions disclosed herein 20, 2008, provisional application No. 61/199,241, afford improved transduction efficiency in a variety of cells, filed on Nov. 14, 2008, provisional application No. tissues and organs of interest, when compared to their 61/200,430, filed on Nov. 26, 2008. unmodified (i.e., wild-type) ra AV vector counterparts. Patent Application Publication Dec. 15, 2016 Sheet 6 of 13 US 2016/0361439 A1 GGCTAAACTT TTAAATCATG AATGAAGAG GCT,-- - - TTT- d. '''A CATA, ATC ATGTTTTC, CCTTTAACT - ACAG T CCI AAACA. TACACCCCC--- AAATCGAA CGCTAAAGT: , , , - TACATCCTACA . CAC^ - (SEQ ID NO 7) F. 6 Patent Application Publication Dec. 15, 2016 Sheet 8 of 13 US 2016/0361439 A1 i Patent Application Publication Dec. 15, 2016 Sheet 9 of 13 US 2016/0361439 A1 Patent Application Publication Dec. 15, 2016 Sheet 10 of 13 US 2016/0361439 A1 Subretina injection intravitreal injection r (AAV5 wild type) (AAV5 wild type) F.G. (A F.G. B Subretina injection intravitreal injection f (AAV5 wild type) F. C. F.G. O. Sibretina injection d6 (AAV8 t?33 mutant) F. E Patent Application Publication Dec. 15, 2016 Sheet 11 of 13 US 2016/0361439 A1 F. A FG. B F.G. C. F.G. D. FG. E. F.G. F Patent Application Publication Dec. 15, 2016 Sheet 12 of 13 US 2016/0361439 A1 F. 12A. F. 2A-2 reated - Untreated - . Wild type oceanoes 2 A is 8 to 2 14 a on a 5 raining Session 3 5 ------- rhG -/- shair injecte F. 12B 3 widtype 2 5 2 5 Photons f im2 x 3.5) F. 12C Patent Application Publication Dec. 15, 2016 Sheet 13 of 13 US 2016/0361439 A1 US 2016/0361439 A1 Dec. 15, 2016 CAPSD-MUTATED RAAV VECTORS AND facilitating or inhibiting the flow of positive or negative ions METHODS OF USE through cell membranes, the cell can be briefly depolarized, depolarized and maintained in that State, or hyperpolarized. CROSS-REFERENCE TO RELATED Thus, the control of the depolarization of cells can be APPLICATIONS beneficial for a number of different purposes, including 0001. The present application is a continuation of U.S. visual, muscular and sensory control. Light-sensitive protein patent application Ser. No. 14/298,533, filed Jun. 6, 2014 channels, pumps, and receptors can permit millisecond (Atty. Dkt. No. 3.6689.370; pending), which is a continua precision optical control of cells. Although light-sensitive tion-in-part of U.S. patent application Ser. No. 12/993,092, proteins in combination with light can be used to control the filed Jun. 10, 2011 (Atty. Dkt. No. 3.6689.358; now aban flow of ions through cell membranes, targeting and delivery doned), which was the U.S. national-stage filing of PCT Intl. remain to be addressed for specific diseases, disorders, and Pat. Appl. No. PCT/US2009/044753, filed May 20, 2009 circuits. (now abandoned); which claims priority to U.S. Prov. Pat. Appl. No. 61/054,571, filed May 20, 2008 (expired); U.S. BRIEF SUMMARY OF THE INVENTION Prov. Pat. Appl. No. 61/199,241, filed Nov. 14, 2008 (ex 0008. In one aspect, the invention provides a recombinant pired); and U.S. Prov. Pat. Appl. No. 61/200,430, filed Nov. nucleic acid comprising a nucleic acid encoding a light 26, 2008 (expired); the contents of each of which is spe sensitive protein operatively linked to a metabotropic glu cifically incorporated herein in its entirety by express ref tamate receptor 6 (mGluR6) regulatory sequence or frag erence thereto. ment thereof. In one embodiment, the light-sensitive protein can be selected from the group consisting of ChR1, ChR2, STATEMENT REGARDING FEDERALLY VChR1, ChR2C128A, ChR2 C128S, ChR2 C128T, ChR1 SPONSORED RESEARCH OR DEVELOPMENT ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, 0002 This invention was made with government support eNpHR, melanopsin, and variants thereof. In another under Grant Nos. DK-058327 GM-082946, HL-076901, and embodiment the light-sensitive protein is ChR2 or a light HL-097088 awarded by the National Institutes of Health. sensitive protein that is at least about 70%, at least about The government has certain rights in the invention. 80%, at least about 90% or at least about 95% identical to ChR2. In another embodiment the mGluR6 regulatory NAMES OF THE PARTIES TO A JOINT sequence fragment comprises less than about 1000, less than RESEARCH AGREEMENT about 750, less than about 500, less than about 250, or less than about 100 base pairs. In a related embodiment, the 0003) Not Applicable. mGluR6 regulatory sequence or fragment thereof is a mGluR6 promoter or enhancer. In a specific related embodi BACKGROUND OF THE INVENTION ment, the nucleic acid further comprises a sequence that 0004 Field of the Invention encodes a green fluorescent protein. In another embodiment, 0005. The present disclosure relates to the fields of the nucleic acid is encapsidated within a recombinant adeno molecular biology, ophthalmology, and gene therapy. In associated virus (AAV). In certain embodiments, the recom particular embodiments, capsid-mutated ra AV vectors and binant AAV is of a combinatorial hybrid of 2, 3, 4, 5, 6, 7, methods for their use in gene therapy are disclosed. In 8, 9, 10, 11, 12, 13, 14, 15 or more serotypes or mutants exemplary embodiments, capsid proteins comprising a thereof. In certain embodiments, the recombinant adeno modification of one or more of the Surface-exposed tyrosine associated virus is of a serotype selected from the group residues are disclosed, including VP3 capsid proteins that consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, include tyrosine-to-phenylalanine mutations at positions AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids corresponding to Y444F,Y500F, and Y730F of the wild-type thereof. In a related embodiment, the nucleic acid is encapsi AAV2 sequence. Also provided are ra AV virions and viral dated within a recombinant virus selected from the group particles that comprise Such a mutated AAV capsid protein consisting of recombinant adeno-associated virus (AAV), and a nucleic acid molecule that expresses one or more recombinant retrovirus, recombinant lentivirus, and recom selected therapeutic or reporter transgenes in one or more binant poxvirus. mammalian cells of interest. 0009. In another aspect, the invention provides a vector 0006. Description of Related Art comprising a nucleic acid encoding a light-sensitive protein, 0007. A gene-delivery therapy to treat a disease or dis wherein the nucleic acid is operatively linked to a metabo order independent of treating an underlying mutation could tropic glutamate receptor 6 (mGluR6) regulatory sequence have potential value. Methods capable of controlling, regu or fragment thereof. In one embodiment, the light-sensitive lating, and/or driving specific neural circuits so as to mediate protein is selected from the group consisting of ChR1, naturalistic neural responses and high resolution perception ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2C128T, and control could also be of enormous potential therapeutic ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, value. Neurons are an example of a type of cell that uses the NpHR, eNpHR, melanopsin, and variants thereof. In a electrical currents created by depolarization to generate related embodiment the light-sensitive protein is ChR2 or a communication signals (e.g., nerve impulses). Other elec light-sensitive protein that is at least about 70%, at least trically excitable cells include skeletal muscle, cardiac about 80%, at least about 90% or at least about 95% identical muscle, and endocrine cells. Neurons use rapid depolariza to ChR2. In another embodiment, the mGluR6 regulatory tion to transmit signals throughout the body and for various sequence fragment is less than about 1000, less than about purposes, such as motor control (e.g., muscle contractions), 750, less than about 500, less than about 250, or less than sensory responses (e.g., touch, hearing, and other senses) about 100 base pairs.
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