Ligands to enhance cellular uptake of biomolecules
OwnerPaul O.P. Ts`o, Robert Duff +3inventors
InventorsPaul O.P. Ts`o, Robert Duff, Yuanzhong Zhou, Scott Deamond, Clinton Roby
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Abstract
The present invention relates to the design and synthesis of homogeneous A-L-P constructs, which contain a hepatic ligand to direct an oligomer or “payload” to a hepatocyte intracellularly via a receptor-mediated, ligand-directed pathway.
Claims71
A construct comprising a homogeneous conjugate of formula A-L-P, wherein
- A represents a hepatic ligand that specifically binds to a hepatic receptor, thereby facilitating the entrance of said conjugate into cells having said receptor;
- L represents a bifunctional linker that is covalently linked to A in a regiospecific manner to form A-L;
- A-L is covalently linked to P in a regiospecific manner to form A-L-P;
- P represents a biologically stable oligomer, wherein P is released from the conjugate following hydrolysis or reduction of at least one specific biochemical linkage, and contains internucleotide linkages resistant to enzymatic hydrolysis or biodegradation upon release from the conjugate.
A purified ligand-linker construct comprising a liver ligand covalently linked to a bifunctional linker to form the A-L construct.
A method for synthesizing conjugates comprising a Conjugation Method 1, wherein
- a) a 2′-O-Me-nucleotide phosphodiester linkage is incorporated to the 5′-end of the oligonucleotide or oligonucleotide analogs;
- b) the 5′-end of the oligonucleotide or oligonucleotide analog is enzymatically phosphorylated using PNK and ATP;
- c) the 5′-phosphate group of the oligonucleotide or oligonucleotide analog is modified to introduce a disulfide linkage to form 5′-disulfide-modified oligonucleotide or oligonucleotide analog;
- d) the 5′-disulfide group of the 5′-disulfide-modified oligonucleotide or oligonucleotide analog is reduced to a thiol group to form a thiol-modified oligonucleotide;
- and
- f) one reactive group of the heterobifunctional linker is covalently conjugated to a ligand and a second group of the heterobifunctional linker is covalently conjugated to said thiol-modified oligonucleotide or oligonucleotide analogs to form the A-L-P conjugate.
A method for synthesizing conjugates comprising a Conjugation Method 2 wherein
- a) a ligand is modified with a bifunctional linker to form an A-L construct;
- b) said A-L construct is purified to greater than 95% homogeneity and unreacted linker is removed;
- c) the oligonucleotide or oligonucleotide analog is modified to form a thiol-modified oligomer;
- d) said thiol-modified oligomer is purified under degassed conditions to remove unreacted reagent and impurities;
- e) a conjugation reaction using a purified A-L construct and a purified thiol-oligomer in a two-component conjugation reaction is executed under degassed conditions;
- wherein said conjugation can be performed by using either excess amounts of said ligand scaffold or said thiol-modified oligomer to form purified A-L-P conjugates;
- and the A-L-P conjugate is purified.
A method for radiolabeling an oligonucleotide-containing or oligonucleotide analog-containing conjugate, comprising radiolabeling an A-LP conjugate, wherein
- a) a tri-nucleotide tracer unit, 5′-T-3′-ps-3′-T-ps-T-5′ is added to the 3′-end of an oligonucleotide or an oligonucleotide analog during solid-phase synthesis;
- b) said tracer unit undergoes enzymatic phosphorylation using PNK and ATP to form a modified tracer unit;
- and
- c) said modified tracer unit is chemically modified with an amine of the radioactive phosphate group of the A-L-P conjugate to prevent cellular enzymatic degradation.
A method for the synthesis of oligonucleotide-containing conjugates wherein
- a) a bifunctional linker terminating in a disulfide moiety is incorporated onto an oligonucleotide or an oligonucleotide analog during solid-phase synthesis to form a disulfide-modified oligomer;
- b) said disulfide-modified oligomer is purified;
- c) the disulfide moiety of said disulfide-modified oligomer is reduced to a thiol group to form a thiol-modified oligomer;
- d) said thiol-modified oligomer is purified under degassed conditions;
- e) a conjugation reaction using a purified A-L and a purified thiol-oligomer is executed under degassed conditions to form an A-L-P conjugate;
- and
- f) the synthesized A-L-P conjugate is purified.
A method for the synthesis of a radiolabeled conjugate comprising the radiolabel of an A-L-P conjugate containing an oligonucleotide or an s oligonucleotide analog;
- wherein
- a) a disulfide linker is incorporated into the 5′-end and a trinucleotide tracer unit, 5′-T-3′-ps-3′-T-ps-T-5′, is incorporated at the 3′-end of an oligonucleotide analog during solid-phase synthesis;
- b) the disulfide- and tracer-containing oligomer is purified;
- c) the disulfide is reduced to a thiol group to form a thiol-modified oligomer;
- d) said thiol-modified oligomer is purified using size exclusion chromatography under degassed conditions to remove unreacted reagent and impurities;
- e) a purified A-L is conjugated to a purified thiol-oligomer under degassed conditions to form an A-L-P conjugate;
- f) the tracer unit is enzymatically phosphorylated to incorporate a radiolabeled phosphate into the A-L-P conjugate using PNK and radiolabeled ATP;
- and
- g) the radioactive phosphate group of the ATP conjugate is chemically modified with an amine to protect it from a cellular enzymatic degradation.
Dependencies are inferred from the claim text.