Intranuclear protein transduction through a nucleoside salvage pathway
Inventors
Assignees
US Department of Veterans Affairs
Publication Number
US-10702543-B2
Publication Date
2020-07-07
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 provides conjugate molecules comprising a substrate capable of being transported by 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 the cellular nucleoside transport pathway. The invention also includes methods of delivering such conjugates to target cells expressing nucleoside transport pathways, methods for screening conjugates for transport via these pathways, and methods of treating diseases affecting tissues that express nucleoside transport pathways by administering such conjugates. Additionally, methods of treating autoimmune disorders by administering compounds that inhibit nucleoside transport pathways are provided.
The problem being solved is the inefficient delivery of biologically active molecules into cells and their nuclei. Many small hydrophilic molecules diffuse poorly across lipid bilayers, and while transporter proteins exist for nucleosides and their analogs, protein transduction through such pathways had not been demonstrated. The invention addresses the need for effective molecular delivery vehicles that utilize endogenous transport mechanisms to deliver active agents into cells, particularly into the nucleus, overcoming barriers to cellular and nuclear uptake of therapeutic molecules.
Claims Coverage
The patent includes one independent claim focused on a method of contacting an equilibrative nucleoside transporter (ENT)-2 conjugate to hypoxic tissue, detailing its components and therapeutic strategy.
Method of targeting ENT2 conjugates to hypoxic tissue
A method comprising administering (a) a cDNA encoding human ENT2 or any fragment thereof having the same therapeutic transport activity as full-length ENT2, followed by (b) administering a conjugate comprising an antibody capable of being transported by ENT2 linked to an active agent, where the conjugate is transported by ENT2, and the antibody has the same binding specificity as an antibody produced by the hybridoma with ATCC accession number PTA 2439.
Inclusion of antibody or antigen-binding fragment
The conjugate includes an antibody or an antigen-binding fragment thereof capable of transport via ENT2.
Antibody or fragment binding nucleic acid
The antibody or antigen-binding fragment in the conjugate binds nucleic acid.
Active agent selection including specified proteins and compounds
The active agent linked to the antibody may be selected from hamartin (TSC1), tuberin (TSC2), or almitrine.
Active agent selection comprising chemotherapeutic drugs and biologicals
The active agent may be selected from topotecan, NSC 644221, PX-478, YC-1, 17-AAG, or bevacizumab.
The claims cover methods of delivering ENT2-transported antibody conjugates to hypoxic tissues using DNA encoding ENT2, specifying antibody characteristics and linking to active agents including proteins and therapeutic compounds, thus covering targeted delivery methods and therapeutic compositions involving ENT2-mediated transport.
Stated Advantages
3E10 Fv penetrates living cells with specific nuclear localization and absence of toxicity.
3E10 Fv can deliver therapeutic cargo proteins successfully in vitro and in vivo.
Utilization of the nucleoside salvage pathway, specifically ENT2, enables novel protein transduction.
The invention provides a delivery method distinct from endosome-limited cell-penetrating peptides, potentially increasing therapeutic efficacy.
Inhibition of nucleoside transporters may limit tissue damage by cytotoxic autoantibodies in autoimmune diseases.
Documented Applications
Delivery of therapeutic proteins, nucleic acids, small molecules, and antibodies into various cell types including muscle, brain, vascular endothelium, thymus, placenta, pancreas, prostate, kidney, blood, skin, and cancer cells.
Treatment of skeletal muscle disorders including muscular dystrophies and lysosomal storage diseases by delivering deficient enzymes or proteins such as dystrophin.
Treatment of glycogen storage diseases by delivering relevant enzymes to glycogen-storing cells.
Treatment of vascular endothelium disorders including inappropriate or deficient angiogenesis, restenosis, and atherosclerosis.
Treatment of neurodegenerative diseases, autoimmune disorders, cancers (including ovarian, colon, breast, leukemia, lung, prostate), and other diseases involving cells expressing nucleoside transporters.
Delivery of nucleic acid therapeutics such as siRNAs, antisense oligonucleotides, ribozymes, and aptamers to targeted cells.
Use in molecular therapy to deliver therapeutic proteins, hormones, cytokines, enzymes, and expression constructs to affected tissues.
Treatment of hypoxic tissues or ENT2-deficient tissues by administering agents that promote ENT2 expression or activity along with the conjugates.
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