Nuclear localization signals, compositions formed therefrom, and methods of use thereof for delivery of cargo to the nucleus
Inventors
Hansen, James E. • RATTRAY, Zahra
Assignees
Publication Number
US-12304970-B2
Publication Date
2025-05-20
Expiration Date
2039-02-01
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Abstract
Amino acid sequences capable of enhancing nuclear translocation are provided. Also referred to a nuclear localization signals (NLS) the sequences can be linked to or grafted into antibodies or fragments or fusion proteins thereof to enhance nuclear translocation of the antibody. Compositions and antibodies including an NLS conjugated or otherwise linked directly or indirectly to an active agent cargo are also provided. Exemplary cargo includes proteins, peptides, carbohydrates, polysaccharides, nucleic acid molecules, inorganic molecules, organic molecules, and diagnostic agents. Pharmaceutical compositions further including a pharmaceutically acceptable carrier are also provided. Methods of delivering the composition or antibody alone or with a cargo linked thereto to the nucleus of cell, methods of selecting or screening for compositions having a desired cellular activity, and methods of treating diseases and disorders are also provided.
Core Innovation
The invention provides amino acid sequences, referred to as nuclear localization signals (NLS), capable of enhancing nuclear translocation. These sequences can be grafted into or linked to antibodies, fragments, or fusion proteins to improve their ability to translocate to the nucleus of cells. The NLS sequences identified, such as those corresponding to SEQ ID NOS:50-53, are used to engineer compositions that facilitate transport of various cargos, including proteins, peptides, carbohydrates, nucleic acids, inorganic molecules, organic molecules, and diagnostic agents, into the cell nucleus.
A central challenge addressed by the invention stems from the need to effectively deliver antibodies and therapeutic agents to the nucleus. Autoantibodies, such as those found in systemic lupus erythematosus (SLE), sometimes possess the unusual ability to penetrate live cells and localize to nuclei, but the mechanism for nuclear localization has been elusive. The patent identifies specific NLS sequences within the variable region of the 3E10 antibody and demonstrates that grafting such sequences can impart nuclear-penetrating properties to other molecules.
The invention encompasses compositions where the NLS is conjugated or linked to an active agent, and pharmaceutical compositions including these entities with a pharmaceutically acceptable carrier. Additionally, the patent provides methods for delivering these compositions to the nucleus of cells, screening compositions for desired intracellular activity, and treating diseases or conditions, including cancer, by facilitating nuclear entry of therapeutic or diagnostic agents.
Claims Coverage
The patent includes several independent claims that define the scope of the invention regarding NLS sequences, compositions containing such sequences, and methods of delivering said compositions to cell nuclei.
Nuclear localization signal comprising SEQ ID NOS: 50-52
The invention claims a composition containing a nuclear localization signal (NLS) comprising an amino acid sequence of any one of SEQ ID NOS: 50-52. The NLS is characterized by its ability to translocate from the cytoplasm to the nucleus. The composition explicitly excludes antibodies comprising an amino acid sequence 100% identical to SEQ ID NOS: 1, 2, 22, 23, 42, 54, 55, and 56.
Active agent cargo linked to the NLS
A key feature includes compositions wherein the NLS is linked, directly or indirectly, to an active agent cargo. The cargo is broadly defined to encompass proteins, peptides, carbohydrates, polysaccharides, nucleic acid molecules, inorganic molecules, organic molecules, and diagnostic agents.
Pharmaceutical composition containing the NLS and a carrier
The invention covers pharmaceutical compositions comprising the above NLS-containing composition and a pharmaceutically acceptable carrier. Such compositions may further include a therapeutic agent as an additional component.
Method of delivering composition to nucleus of a cell
The patent claims a method comprising contacting a cell with a composition containing the NLS, resulting in delivery of the composition to the nucleus of the cell.
The inventive features focus on the identification and use of specific NLS sequences to promote nuclear translocation, their application in delivering a range of cargos, their inclusion in pharmaceutical compositions, and methods enabling precise delivery of agents to the nucleus.
Stated Advantages
The disclosed NLS sequences enhance nuclear translocation of linked antibodies and cargos, enabling efficient delivery of diverse agents to the nucleus.
Grafting or linking the NLS to other proteins or antibodies imparts nuclear-penetrating capabilities, expanding therapeutic and diagnostic utility.
The approach facilitates delivery of therapeutic, diagnostic, and other active agents to intracellular targets, including the nucleus, which is typically challenging to access.
The method can increase the activity of the delivered agent on target cells or reduce the required dosage for therapeutic effect compared to using the agent alone.
Documented Applications
Delivery of therapeutic, diagnostic, or other active agents to the nucleus of cells for enhanced cellular activity or targeted treatment.
Treatment of diseases and disorders, including cancer, by administering compositions with NLS-mediated nuclear delivery alone or in combination with active agent cargo.
Screening and selecting compositions for desired cellular activities such as modulating cell growth, apoptosis, or cell signaling.
Use in pharmaceutical compositions for administration via enteral or parenteral routes, including systemic or local delivery.
Use in combination with standard cancer therapies such as chemotherapy, radiation therapy, or radiosensitizers to treat tumors, especially those resistant to conventional therapy.
Application in treatment of diseases involving nuclear events, such as genetic diseases, muscular dystrophies, lysosomal storage diseases, and traumatic injuries.
Use of NLS-modified antibodies or proteins as delivery vehicles for cargos such as proteins (e.g., p53), nucleic acids, or imaging agents in diagnostic and therapeutic strategies.
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