Diblock copolymers and polynucleotide complexes thereof for delivery into cells
Inventors
Stayton, Patrick S. • Hoffman, Allan S. • Convertine, Anthony J. • Benoit, Danielle • Duvall, Craig L. • Johnson, Paul H. • Gall, Anna S.
Assignees
Genevant Sciences GmbH • WASHINGTON, University of • University of Washington
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Publication Number
US-9476063-B2
Publication Date
2016-10-25
Expiration Date
Abstract
Described herein are copolymers, and methods of making and utilizing such copolymers. Such copolymers have at least two blocks: a first block that has at least one unit that is hydrophilic at physiologic pH, and a second block that has hydrophobic groups. This second block further has at least one unit with a group that is anionic at about physiologic pH. The described copolymers are disruptive of a cellular membrane, including an extracellular membrane, an intracellular membrane, a vesicle, an organelle, an endosome, a liposome, or a red blood cell. Preferably, in certain instances, the copolymer disrupts the membrane and enters the intracellular environment. In specific examples, the copolymer is endosomolytic.
Core Innovation
The invention relates to diblock copolymers intended to form compositions with a therapeutic agent, including polynucleotides such as siRNA. The diblock copolymer includes a hydrophilic first block and a hydrophobic, substantially charge-neutral second block, and the second block contains anionic and/or cationic constitutional units that provide pH-responsive charge-state behavior tied to endosomal pH.
The anionic constituent groups are configured to become uncharged in endosomal pH, and the resulting pH-dependent charge-state change is described as producing endosomolytic behavior. This behavior is characterized as cellular membrane destabilizing activity associated with extracellular and intracellular membranes and vesicle, liposome, or red blood cell contexts.
Polynucleotides are associated with the hydrophilic block, including covalent or non-covalent association, with ionic complexation and cleavable covalent linkages such as disulfide. The copolymer structure is provided by chemical Formula I/II/III with substituent options and composition parameters controlling hydrophilicity, hydrophobicity, and charge state, and the document reports relationships among polymer composition, particle size/ζ-potential, hemolysis, internalization, and gene knockdown outcomes.
The document further describes that targeting moieties can be included, and it reports that nonspecific cytotoxicity is low in HeLa cells. In particular examples, pH-dependent hemolysis and internalization are shown to correlate with the hydrophobicity-enhancing moiety content and with charge ratio, and gene knockdown is described in terms of GAPDH knockdown.
Claims Coverage
Two independent composition claims are provided. Each claim centers on a diblock copolymer defined by chemical Formula I with constrained substituent/residue options and specified mole-fraction and molecular-weight ranges, together with a therapeutic agent, and the claims define additional inventive structure through residue identity options and constraints on molecular parameters and association possibilities in the dependent set.
Diblock copolymer composition defined by Formula I with controlled substituents and mole fractions
A copolymer comprising a diblock copolymer having chemical Formula I, with A0-A4, Y0-Y4, R1-R6, and hydrophilic/charge-state residues Q0-Q3 selected as specified, wherein mole fractions m and n satisfy m+n=1 and p+q+r=1, and wherein v and w are constrained to the stated kDa ranges, as part of a composition with a therapeutic agent.
Charge-state residue classes for Q1 and pH-responsive behavior
Q1 is defined as a residue which is hydrophilic at normal physiological pH, Q2 is defined as a residue which is positively charged at normal physiological pH but protonates at lower pH, and Q3 is defined as a residue which is negatively charged at normal physiological pH but undergoes protonation at lower pH, with Q0/Q1/Q2/Q3 selected within the listed options, as part of the composition.
Molecular-weight and mole-fraction constraints on the diblock copolymer
The diblock copolymer is constrained so that m is a mole fraction of 0 to less than 1.0, n is a mole fraction of greater than 0 to 1.0 with m+n=1, p is 0.1-0.9, q is 0.1-0.9, r is present up to 0.8, with p+q+r=1, and v is from 1 to 25 kDa while w is from 1 to 50 kDa.
Therapeutic agent in the composition, including specified siRNA in dependent claims
The composition comprises the Formula I diblock copolymer together with a therapeutic agent, with dependent refinement specifying that the therapeutic agent is siRNA in the dependent set associated with the independent composition claims.
Overall, the independent claims cover compositions combining a diblock copolymer defined by Formula I with specified residue/substituent options and composition constraints plus a therapeutic agent. The claim set further emphasizes residue identity options for hydrophilic and pH-responsive charge-state behavior and includes dependent refinements specifying therapeutic-agent identity and association mode.
Stated Advantages
Endosomolytic behavior described as cellular membrane destabilizing associated with extracellular and intracellular membranes and vesicle contexts.
pH-dependent hemolysis is reported, with the strongest endosomolytic response for BMA-rich variants.
Low nonspecific cytotoxicity is reported in HeLa cells.
Gene knockdown outcomes are described and correlated with polymer composition and charge ratio.
Documented Applications
Delivery/interaction with cells for intracellular gene knockdown, exemplified by GAPDH knockdown in HeLa cells.
Polynucleotide delivery compositions, including siRNA and other oligonucleotides/polynucleotides associated with the hydrophilic block.
Use of pH-dependent endosomal membrane destabilization/endosomolytic behavior to support intracellular delivery in cellular contexts.
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