Intranuclear protein transduction through a nucleoside salvage pathway
Inventors
Assignees
US Department of Veterans Affairs
Publication Number
US-8956825-B2
Publication Date
2015-02-17
Expiration Date
2028-05-23
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Abstract
Provided herein are conjugate molecules containing a substrate for a nucleoside transport pathway linked to an active agent, wherein the conjugate can be transported into a cell or into the nucleus of a cell via a cellular nucleoside transport pathway. Further provided are methods of delivering a conjugate molecule to a target cell expressing a nucleoside transport pathway, wherein the conjugate contains a substrate for the nucleoside transport pathway linked to an active agent. Also provided are methods for screening for conjugates that are transported by nucleoside transport pathways. Further provided are methods of treating a patient having a disease or disorder affecting tissues expressing nucleoside transport pathways, in which a conjugate containing an agent effective in treating the disorder is administered to the patient. Also provided are methods of treating a patient having an autoimmune disorder involving administering to the patient a compound that inhibits a nucleoside transport pathway.
Core Innovation
The invention discloses conjugate molecules comprising a substrate capable of being transported by a nucleoside transport pathway linked to an active agent. These conjugates can be transported into cells or into the nucleus of cells via endogenous cellular nucleoside transport pathways, specifically through equilibrative nucleoside transporters such as ENT2 or concentrative nucleoside transporters. This delivery mechanism enables the active agent to enter target cells expressing nucleoside transport pathways.
The invention addresses the problem that polar or hydrophilic compounds typically diffuse poorly across lipid bilayers, limiting efficient intracellular delivery of biologically active molecules. While nucleoside transporters facilitate uptake of nucleosides and analogs, the mechanism for delivering therapeutic agents into the nucleus through these pathways was not established previously. The invention overcomes this challenge by utilizing substrates that can be transported via nucleoside salvage pathways, particularly ENT2, to enable intracellular and intranuclear delivery of active agents.
Claims Coverage
The patent contains multiple independent claims focusing on methods of screening conjugates for transport by the human equilibrative nucleoside transporter 2 (ENT2). Two main independent claims define methods using conjugates comprising substrates linked to active agents, and the transport by ENT2.
Method of screening conjugates with antibody substrates for ENT2 transport
A method to determine if a conjugate comprising a substrate that is an antibody or antibody fragment linked to an active agent is transported by human ENT2 by contacting cells expressing ENT2 with the conjugate and assessing intracellular transport.
Use of ENT2-insensitive transporter for screening with controlled expression
Use of cells either endogenously expressing or transfected with DNA encoding human ENT2 where transport of the conjugate is compared between ENT2-expressing and control cells to establish transport specificity.
Method of screening conjugates with polypeptide substrates excluding nucleosides
A method to assess transport of conjugates comprising a polypeptide substrate (excluding nucleosides or nucleoside analogs) linked to an active agent via ENT2 by contacting ENT2-expressing cells and determining transport.
Use of detectable labels in conjugates during screening
Conjugates may further comprise detectable labels (e.g., radioisotopes, fluorescent markers) to facilitate determination of transport into cells expressing ENT2.
Active agent composition in conjugates for ENT2 transport determination
The active agent linked to the substrate in the conjugate includes peptides, polypeptides, antibodies, or antibody fragments as defined in the independent screening claims.
The claims focus on screening methods for conjugates containing substrates, such as antibodies or polypeptides, linked to active agents that are transported into cells via the human ENT2 nucleoside transporter, including methods employing cells with endogenous or transfected ENT2 expression and use of labeling for detection.
Stated Advantages
The invention enables intracellular and intranuclear delivery of biological active agents through endogenous nucleoside transport pathways, overcoming poor diffusion of hydrophilic molecules across membranes.
It provides a novel molecular delivery vehicle based on nucleoside transporter substrates that can accomplish targeted delivery without toxicity, as exemplified by antibody 3E10 Fv.
The use of ENT2-mediated transport allows specific delivery to cells and nuclear localization, which is advantageous for regulation of gene expression and therapeutic applications involving intracellular targets.
Documented Applications
Delivering conjugates containing substrates transported via nucleoside transport pathways to cells expressing these transporters, for treatment of diseases or disorders.
Treatment of diseases involving skeletal muscle, including various muscular dystrophies and lysosomal muscle disorders, by transporting active agents such as deficient enzymes or proteins.
Treatment of glycogen storage disorders via transport into glycogen-storing cells including muscle and liver cells.
Treatment of vascular endothelium-related diseases such as inappropriate angiogenesis, atherosclerosis, and wound healing disorders.
Treatment of brain-related diseases including neurodegenerative diseases, mental illnesses, infectious diseases, inherited disorders, and lysosomal storage disorders.
Treatment of placental, thymic (including autoimmune diseases and thymus cancers), pancreatic, prostatic, renal, blood, and skin diseases by delivering active agents through targeted conjugates.
Delivering chemotherapeutic agents or tumor suppressor proteins to cancer cells expressing ENT2, useful for various cancers including leukemia, breast, colon, ovarian, lung, prostate, and others.
Delivery of nucleic acid therapeutics such as siRNA, antisense oligonucleotides, ribozymes, and aptamers into cells via ENT2-mediated transport for gene modulation therapies.
Modulation of nuclear receptor- or chromatin modification-associated diseases by delivery of proteins or nucleic acids affecting these pathways.
Pharmaceutical compositions comprising conjugates combined with agents that promote ENT2 expression, used to treat ENT2-deficient tissues, hypoxic tumors, ischemic tissues, diabetic ulcers, and other conditions involving hypoxia or ischemia.
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