Methods and compositions for nucleic acid and protein payload delivery
Inventors
WATSON, Andre Ronald • Foster, Christian
Assignees
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Publication Number
US-10975388-B2
Publication Date
2021-04-13
Expiration Date
Abstract
Provided are methods and compositions for delivering a nucleic acid, protein, and/or ribonucleoprotein payload to a cell. Also provided are delivery molecules that include a peptide targeting ligand conjugated to a protein or nucleic acid payload (e.g., an siRNA molecule), or conjugated to a charged polymer polypeptide domain (e.g., poly-arginine such as 9R or a poly-histidine such as 6H, and the like). The targeting ligand provides for (i) targeted binding to a cell surface protein, and (ii) engagement of a long endosomal recycling pathway. As such, when the targeting ligand engages the intended cell surface protein, the delivery molecule enters the cell (e.g., via endocytosis) but is preferentially directed away from the lysosomal degradation pathway.
Core Innovation
The document describes delivery molecules for intracellular delivery that include a peptide targeting ligand conjugated to a charged polymer polypeptide domain. The peptide targeting ligand comprises the amino acid sequence MIASQFLSALTLVLLIKESGA (SEQ ID NO:56) and is configured to provide targeted binding to a cell surface protein. The charged polymer polypeptide domain comprises the amino acid sequence RRRRRRRRR (9R) (SEQ ID NO:15).
The charged polymer polypeptide domain is condensed with a payload comprising a ribonucleoprotein complex. The intracellular delivery is biased toward a long endosomal recycling pathway and away from lysosomal degradation. The delivery molecules are discussed in the context of endocytosis and trafficking control to avoid lysosomal degradation.
The document also describes engineering of the peptide targeting ligand to enable conjugation, including cysteine addition and cysteine substitution or insertion at internal positions relative to a wild-type sequence. In illustrative examples, the peptide targeting ligand is shown as an engineered ligand such as exendin-4 with an S11C substitution, and cysteine-containing targeting ligands are used to support conjugation to the charged polymer polypeptide domain.
Claims Coverage
The partial content includes one independent claim. That independent claim defines a delivery molecule comprising a specified peptide targeting ligand, a charged polymer polypeptide domain conjugated to the targeting ligand, targeted binding to a cell surface protein, and condensation of the charged domain with a payload comprising a ribonucleoprotein complex. Dependent claims refine the targeting ligand by adding a cysteine residue and/or specifying internal cysteine substitution or insertion relative to a corresponding wild type sequence.
Peptide targeting ligand conjugated to charged polymer polypeptide domain for targeted binding
A delivery molecule comprising a peptide targeting ligand comprising the amino acid sequence MIASQFLSALTLVLLIKESGA (SEQ ID NO:56), the targeting ligand conjugated to a charged polymer polypeptide domain comprising the amino acid sequence RRRRRRRRR (9R) (SEQ ID NO:15), wherein the targeting ligand provides for targeted binding to a cell surface protein.
Condensation of charged polymer polypeptide domain with ribonucleoprotein payload
The charged polymer polypeptide domain is condensed with a payload comprising a ribonucleoprotein complex.
Cysteine residue in targeting ligand
The targeting ligand includes a cysteine residue.
Internal cysteine substitution or insertion in targeting ligand
The targeting ligand has a cysteine substitution or insertion at one or more internal amino acid positions compared to the corresponding wild type amino acid sequence.
Across the independent claim and its dependent refinements, the core coverage is a delivery molecule that combines a specified peptide targeting ligand (MIASQFLSALTLVLLIKESGA) conjugated to a charged polymer polypeptide domain (9R), targets a cell surface protein, and condenses the charged domain with a ribonucleoprotein complex payload, with additional targeting-ligand conjugation features specified via cysteine residues, including internal substitution or insertion relative to a wild type sequence.
Stated Advantages
Enhances activity, stability, distribution, or uptake for nucleic-acid payloads.
Supports targeted binding to a cell surface protein for targeted delivery.
Biases intracellular trafficking toward a long endosomal recycling pathway and away from lysosomal degradation.
Documented Applications
Gene editing in hematopoietic stem/progenitor cells.
Pancreatic indications.
Neural indications.
VWF-related editing via E-selectin targeting.
BCL11a disruption for fetal hemoglobin (HbF).
Beta-globin correction.
T-cell receptor (TCR) modification.
Intracellular delivery of payloads comprising ribonucleoprotein complex via endocytosis and trafficking biased toward long endosomal recycling and avoidance of lysosomal degradation.
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