公开(公告)号：US20140256802A1

公开(公告)日：2014-09-11

申请号：US14279142

申请日：2014-05-15

Applicant: University of Florida Research Foundation, Inc.

Inventor： Sanford Leon Boye ,  Shannon Elizabeth Boye ,  Frank Markus Dyka ,  William W. Hauswirth

IPC: C07K14/47

CPC classification number: C07K14/47 ,  A61K48/0066 ,  C07K14/4716 ,  C12N15/86 ,  C12N2750/14143 ,  C12N2800/40 ,  C12N2830/50 ,  C12N2840/445 ,  C12N2999/007

Abstract: Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

Abstract translation: 公开了用于治疗哺乳动物眼睛疾病的材料和方法，特别是Usher综合征1B（USH1B）。 本发明提供了促进编码序列超过单个AAV载体的多核苷酸包装能力的全长蛋白质的表达的基于AAV的双载体系统。 在一个实施方案中，提供了载体系统，其包括i）第一AAV载体多核苷酸，其在多核苷酸的每个末端包括反向末端重复序列，以及合适的启动子，随后是部分编码序列，其编码全长序列的N末端部分， 长度多肽; 和ii）第二个AAV载体多核苷酸，其在多核苷酸的每个末端包括反向末端重复序列，以及编码全长多肽的C末端部分，任选地随后是多聚腺苷酸化（pA）信号序列的部分编码序列。 在另一个实施方案中，载体系统包括i）第一个AAV载体多核苷酸，其包含每个末端的反向末端重复序列，合适的启动子，随后是部分编码序列，其编码全长多肽的N-末端部分，随后是剪接 供体位点和内含子，以及ii）第二个AAV载体多核苷酸，其在每个末端包含反向末端重复序列，随后是内含子和内含子的剪接受体位点，随后是编码C端部分的部分编码序列 全长多肽，任选地随后是聚腺苷酸化（pA）信号序列。 第一和第二AAV载体中的编码序列或内含子序列优选包含重叠的序列区。

公开(公告)号：US20180057840A1

公开(公告)日：2018-03-01

申请号：US15550864

申请日：2016-02-16

Applicant: University of Florida Research Foundation, Inc.

Inventor： William W. Hauswirth ,  Sanford L. Boye ,  Daniel M. Lipinski ,  Michael E. Boulton ,  Shannon E. Boye

IPC: C12N15/86 ,  C07K14/005 ,  C07K14/705 ,  C12N9/78 ,  C12N7/00 ,  A61K48/00 ,  A61K38/50

CPC classification number: C12N15/86 ,  A61K38/00 ,  A61K38/50 ,  A61K48/0058 ,  C07K14/005 ,  C07K14/705 ,  C12N7/00 ,  C12N9/78 ,  C12N2750/14121 ,  C12N2750/14122 ,  C12N2750/14143 ,  C12N2830/008 ,  C12N2830/15 ,  C12N2830/85 ,  C12Y305/04016

Abstract: Disclosed are capsid-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations that include them. Also disclosed are methods of preparing and using novel capsid-protein-mutated rAAV vector constructs in a variety of diagnostic and therapeutic applications including, inter alia, as delivery agents for diagnosis, treatment, or amelioration of one or more diseases, disorders, or dysfunctions of the mammalian vascular system, and complications from Type I diabetes. Also disclosed are methods for systemic and tissue-localized delivery of therapeutic rAAV-based gene expression cassettes to vascular endothelial cells, tissues, and organs, as well as use of the disclosed compositions in the manufacture of medicaments for a variety of in vitro and/or in vivo applications including the treatment of vasculitis, and complications arising from Type I diabetes, such as macular edema, nephropathy, diabetic retinopathy, and the like.

公开(公告)号：US20170348387A1

公开(公告)日：2017-12-07

申请号：US15445776

申请日：2017-02-28

Applicant: The Trustees of the University of Pennsylvania ,  University of Florida Research Foundation, Inc.

Inventor： Gustavo Aguirre ,  William A. Beltran ,  Samuel G. Jacobson ,  Artur V. Cideciyan ,  William W. Hauswirth ,  Sanford L. Boye

IPC: A61K38/17 ,  A61B3/12 ,  A61B3/14 ,  C07K14/005 ,  A61B3/00 ,  C12N15/86 ,  C12N7/00 ,  C07K14/47 ,  C12Q1/68 ,  A61K48/00

CPC classification number: A61K38/1709 ,  A61B3/0025 ,  A61B3/12 ,  A61B3/14 ,  A61K48/0008 ,  A61K48/0058 ,  A61K48/0075 ,  C07K14/005 ,  C07K14/47 ,  C12N7/00 ,  C12N15/86 ,  C12N2750/14121 ,  C12N2750/14122 ,  C12N2750/14143 ,  C12N2750/14171 ,  C12N2810/6027 ,  C12N2830/008 ,  C12Q1/6883 ,  C12Q2600/156

Abstract: Described herein are methods of preventing, arresting progression of or ameliorating vision loss and other conditions associated with Leber congenital amaurosis (LCA) in a subject. The methods include administering to said subject an effective concentration of a composition comprising a recombinant adeno-associated virus (AAV) carrying a nucleic acid sequence encoding a normal NPHP5 protein, or fragment thereof, under the control of regulatory sequences which express the NPHP5 protein in the photoreceptor cells of the subject, and a pharmaceutically acceptable carrier.

公开(公告)号：US10426844B2

公开(公告)日：2019-10-01

申请号：US15246385

申请日：2016-08-24

Applicant: University of Florida Research Foundation, Inc.

Inventor： Mavis Agbandje-McKenna ,  William W. Hauswirth ,  Arun Srivastava ,  Li Zhong

IPC: C12N15/86 ,  A61K48/00 ,  C07K14/705 ,  A61K45/06 ,  C07K14/72

Abstract: Disclosed are capsid-mutated rAAV vectors and methods for their use in gene therapy, and particularly for use in delivering therapeutic transgenes to treat a variety of mammalian diseases and disorders, including dysfunctions and abnormal conditions of the human eye. VP3 capsid proteins comprising a modification of one or more of the surface-exposed tyrosine residues are disclosed, and in particular, VP3 capsid protein comprising tyrosine-to-phenylalanine mutations at positions corresponding to Y444F, Y500F, and Y730F of the wild-type AAV2 sequence. Also provided are rAAV virions and viral particles that comprise such a mutated AAV capsid protein and a nucleic acid molecule that expresses one or more selected therapeutic or reporter transgenes in one or more mammalian cells of interest. Advantageously, the capsid-mutated rAAV vectors and virions disclosed herein afford improved transduction efficiency in a variety of cells, tissues and organs of interest, when compared to their unmodified (i.e., wild-type) rAAV vector counterparts.

公开(公告)号：US20170007720A1

公开(公告)日：2017-01-12

申请号：US15120425

申请日：2015-02-18

Applicant: UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.

Inventor： Shannon E. Boye ,  Frank Dyka ,  Sanford L. Boye ,  William W. Hauswirth

IPC: A61K48/00 ,  C12N15/86 ,  C12N7/00 ,  A61K45/06 ,  A61K38/17

CPC classification number: A61K48/0058 ,  A61K38/1709 ,  A61K45/06 ,  A61K48/0075 ,  A61K48/0083 ,  C12N7/00 ,  C12N15/86 ,  C12N2750/14122 ,  C12N2750/14123 ,  C12N2750/14143 ,  C12N2750/14145 ,  C12N2750/14171 ,  C12N2830/008

Abstract: Disclosed are capsid-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations that include them. Also disclosed are methods of preparing and using novel capsid-protein-mutated rAAV vector constructs in a variety of diagnostic and therapeutic applications including, inter alia, as delivery agents for diagnosis, treatment, or amelioration of one or more diseases, disorders, or dysfunctions of the mammalian eye. Also disclosed are methods for intravitreal delivery of therapeutic gene constructs to retinal neuron cells, and specifically to ON bipolar cells, of the mammalian eye, as well as use of the disclosed compositions in the manufacture of medicaments for a variety of in vitro and/or in vivo applications including the treatment of retinitis pigmentosa, melanoma-associated retinopathy, and congenital stationary night blindness.

Abstract translation: 公开了衣壳修饰的rAAV表达载体，以及感染性病毒粒子，组合物和包括它们的药物制剂。 还公开了在各种诊断和治疗应用中制备和使用新的衣壳蛋白突变的rAAV载体构建体的方法，包括特别地，作为用于诊断，治疗或改善一种或多种疾病，障碍或功能障碍的递送剂 的哺乳动物眼睛。 还公开了将治疗性基因构建体玻璃体内递送给视网膜神经元细胞，特别是哺乳动物眼睛的ON双极细胞的方法，以及所公开的组合物在制备多种体外和/或 体内应用，包括治疗色素性视网膜炎，黑素瘤相关性视网膜病变和先天性静止性夜盲。

公开(公告)号：US10214572B2

公开(公告)日：2019-02-26

申请号：US14279142

申请日：2014-05-15

Applicant: University of Florida Research Foundation, Inc.

Inventor： Sanford Leon Boye ,  Shannon Elizabeth Boye ,  Frank Markus Dyka ,  William W. Hauswirth

IPC: C07K14/47 ,  A61K48/00 ,  C12N15/86

Abstract: Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

公开(公告)号：US09375491B2

公开(公告)日：2016-06-28

申请号：US14354471

申请日：2012-10-29

Applicant: UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.

Inventor： Sanford Leon Boye ,  Frank Markus Dyka ,  William W. Hauswirth

IPC: A61K48/00 ,  C12N15/86

CPC classification number: A61K48/005 ,  C12N15/86 ,  C12N2750/14143 ,  C12N2830/008 ,  C12N2830/85

Abstract: The subject invention concerns materials and methods for providing for cone cell specific expression of a polynucleotide in a human or animal. One aspect of the invention concerns a polynucleotide promoter sequence that directs expression of an operably linked polynucleotide in cone cells. In one embodiment, a polynucleotide of the invention comprises a nucleotide sequence of an interphotoreceptor retinoid-binding protein (IRBP) gene that is positioned upstream of a promoter nucleotide sequence of a cone transducin alpha-subunit (GNAT2) gene. Another aspect of the subject invention concerns methods for expressing a selected polynucleotide in cone cells. The selected polynucleotide can be provided in a polynucleotide of the invention wherein the selected polynucleotide is operably linked to a polynucleotide promoter sequence of the invention. In one embodiment, the selected polynucleotide sequence is provided in a polynucleotide vector of the invention. The vector comprising the selected polynucleotide is then introduced into a cell. The selected polynucleotide is expressed only in cone cells, with very little, if any, expression in rods or other cells. A selected polynucleotide can be one that encodes, for example, a therapeutic protein or a functional protein that is defective or underexpressed in the targeted cone cells.

Abstract translation: 本发明涉及用于在人或动物中提供多核苷酸的锥细胞特异性表达的材料和方法。 本发明的一个方面涉及指导可操作地连接的多核苷酸在锥细胞中的表达的多核苷酸启动子序列。 在一个实施方案中，本发明的多核苷酸包含位于锥体转导蛋白α-亚基（GNAT2）基因的启动子核苷酸序列上游的光受体类维生素A结合蛋白（IRBP）基因的核苷酸序列。 本发明的另一方面涉及在锥细胞中表达选定的多核苷酸的方法。 所选择的多核苷酸可以提供在本发明的多核苷酸中，其中所选择的多核苷酸可操作地连接到本发明的多核苷酸启动子序列。 在一个实施方案中，所选择的多核苷酸序列在本发明的多核苷酸载体中提供。 然后将包含所选多核苷酸的载体导入细胞。 所选择的多核苷酸仅在锥细胞中表达，在棒或其它细胞中表达很少（如果有的话）。 所选择的多核苷酸可以是编码例如在靶向锥细胞中有缺陷或低表达的治疗性蛋白质或功能性蛋白质的多核苷酸。

公开(公告)号：US20140275231A1

公开(公告)日：2014-09-18

申请号：US14354471

申请日：2012-10-29

Applicant: UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.

Inventor： Sanford Leon Boye ,  Frank Markus Dyka ,  William W. Hauswirth

IPC: A61K48/00

CPC classification number: A61K48/005 ,  C12N15/86 ,  C12N2750/14143 ,  C12N2830/008 ,  C12N2830/85

Abstract: The subject invention concerns materials and methods for providing for cone cell specific expression of a polynucleotide in a human or animal. One aspect of the invention concerns a polynucleotide promoter sequence that directs expression of an operably linked polynucleotide in cone cells. In one embodiment, a polynucleotide of the invention comprises a nucleotide sequence of an interphotoreceptor retinoid-binding protein (IRBP) gene that is positioned upstream of a promoter nucleotide sequence of a cone transducin alpha-subunit (GNAT2) gene. Another aspect of the subject invention concerns methods for expressing a selected polynucleotide in cone cells. The selected polynucleotide can be provided in a polynucleotide of the invention wherein the selected polynucleotide is operably linked to a polynucleotide promoter sequence of the invention. In one embodiment, the selected polynucleotide sequence is provided in a polynucleotide vector of the invention. The vector comprising the selected polynucleotide is then introduced into a cell. The selected polynucleotide is expressed only in cone cells, with very little, if any, expression in rods or other cells. A selected polynucleotide can be one that encodes, for example, a therapeutic protein or a functional protein that is defective or underexpressed in the targeted cone cells.

Abstract translation: 本发明涉及用于在人或动物中提供多核苷酸的锥细胞特异性表达的材料和方法。 本发明的一个方面涉及指导可操作地连接的多核苷酸在锥细胞中的表达的多核苷酸启动子序列。 在一个实施方案中，本发明的多核苷酸包含位于锥体转导蛋白α-亚基（GNAT2）基因的启动子核苷酸序列上游的光受体类维生素A结合蛋白（IRBP）基因的核苷酸序列。 本发明的另一方面涉及在锥细胞中表达选定的多核苷酸的方法。 所选择的多核苷酸可以提供在本发明的多核苷酸中，其中所选择的多核苷酸可操作地连接到本发明的多核苷酸启动子序列。 在一个实施方案中，所选择的多核苷酸序列在本发明的多核苷酸载体中提供。 然后将包含所选多核苷酸的载体导入细胞。 所选择的多核苷酸仅在锥细胞中表达，在棒或其它细胞中表达很少（如果有的话）。 所选择的多核苷酸可以是编码例如在靶向锥细胞中有缺陷或低表达的治疗性蛋白质或功能性蛋白质的多核苷酸。