Patent Application
20250127902
This disclosure describes, in one aspect a complex that includes a diblock polymer. The diblock polymer includes a first block and a second block. The first block includes an acrylate polymer. The second block includes an acrylamide polymer. The acrylamide polymer includes quaternary ammonium groups or amine cationizable groups. The complex further includes an oligonucleotide cargo.
In one or more embodiments, the acrylamide polymer is or includes amino ethyl acrylamide, dimethyl amine ethyl acrylamide, diethyl amine ethyl acrylamide, trimethyl amine ethyl acrylamide, or morpholino ethyl acrylamide.
In one or more embodiments, the acylate polymer is or includes a n-butyl acrylate polymer.
In one or more embodiments, the oligonucleotide cargo is an antisense oligonucleotide.
In one or more embodiments, the complex has an N/P ratio of 10 to 40.
In one or more embodiments, the complex includes or is a micelle.
In another aspect, the present disclosure describes a transfection composition that includes a plurality of the complexes of any preceding embodiment or aspect.
In one or more embodiments, the transfection composition includes a first plurality of complexes and a second plurality of complexes wherein the first plurality of complexes and the second plurality of complexes differ by at least by the identity of the second block of the diblock polymer.
In another aspect, the present disclosure describes a complex that includes a first diblock polymer and a second diblock polymer. The first diblock polymer includes a first block, the first block including an acrylate polymer. The first diblock polymer includes a second block, the second block including quaternary ammonium groups or amine cationizable groups. The second diblock polymer includes a first block, the first block including an acrylate polymer. The second diblock polymer includes a second block, the second block including quaternary ammonium groups or amine cationizable groups. The quaternary ammonium groups or amine cationizable groups of the second diblock polymer are different than the quaternary ammonium groups or amine cationizable groups of the first diblock polymer. The complex further includes an oligonucleotide cargo. In one or more embodiments, the complex is or includes a micelle.
In one or more embodiments, the first block of the first diblock polymer, the first block of the second diblock polymer, or both include or are an n-butyl-acrylate polymer.
In one or more embodiments, the acrylamide polymer of the first diblock polymer includes or is morpholino ethyl acrylamide and the acrylamide polymer of the second diblock polymer includes or is amino ethyl acrylamide, dimethyl amine ethyl acrylamide, diethyl amine ethyl acrylamide, or trimethyl amine ethyl acrylamide.
In one or more embodiments, the weight ratio or mole ratio of the first diblock polymer to the second diblock polymer is 0.8 parts or greater of the first diblock polymer for every 1 part of the second polymer. In one or more embodiments, the weight ratio or mole ratio of the first diblock polymer to the second diblock polymer is 0.1 to 0.3 parts of the first diblock polymer for every 1 part of the second polymer.
In another aspect, the present disclosure describes a transfection composition that includes a plurality of the complexes that include a first diblock polymer and a second diblock polymer as described herein.
In another aspect, the present disclosure describes a complex that includes a homopolymer. The homopolymer includes an acrylamide polymer. The acrylamide polymer includes quaternary ammonium groups or amine cationizable groups. The complex further includes an oligonucleotide cargo. In one or more embodiments, the oligonucleotide cargo has a length of 1000 bases or less. In one or more embodiments, the complex is or includes a polyplex. In one or more embodiments, the homopolymer is or includes amino ethyl acrylamide, dimethyl amine ethyl acrylamide, diethyl amine ethyl acrylamide, trimethyl amine ethyl acrylamide, or morpholino ethyl acrylamide.
In another aspect, the present disclosure describes a transfection composition that includes a plurality of the complexes that include a homopolymer.
In another aspect, the present disclosure describes a transfection method. The method includes contacting a cell with a transfection composition described herein.
The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Nucleic acid-based medicines have many important applications ranging from disease treatment to vaccine development. Antisense oligonucleotides (ASOs) are one type of pharmaceutical paradigm emerging as an innovative medicine for many diseases including cancer and spinal muscular atrophy. Some ASOs function by binding to RNA, such as mRNA, via Watson-Crick base pairing to modulate gene expression. For example, binding of an ASO to RNA can induce one or more mechanisms to prevent the translation of RNA into a protein, induce ribonuclease H mediated decay of RNA, and/or modulate the splicing of pre-mRNA. ASOs were one of the first genetic therapeutics on the market because new ASOs can be readily developed with knowledge of the targeted mRNA sequence. For example, MILASEN is an ASO therapy developed specifically for one patient suffering from Batten disease, a fatal genetic disease-causing loss of vision, seizures, and dementia. The patient's genome was sequenced to identify the exact mutation thought to cause the disease, and a specific ASO was developed (MILASEN) that improved the patient's health.
Although ASOs have the potential to change the way diseases are treated, the physiological environment makes ASO delivery challenging. To engage a target, the ASO may need to cross several cellular barriers including the cell membrane and the endosome. Due to the relatively large and anionic nature of many ASOs, crossing cellular barriers is challenging. Additionally, for in vivo applications, the ASO should avoid extracellular barriers such as nuclease degradation, renal clearance, protein sequestration, among others, to even arrive at the cellular barriers.
Delivery vehicles are commonly used to facilitate delivery of genetic payloads such as ASOs through the cell membrane. Delivery vehicles include viral vectors and lipid-based systems. Viral vectors are expensive and a potentially immunogenic gene delivery system. While more economically tractable than viral vector systems, lipid-base systems face stability issues.
Polymer-based delivery systems are being explored as genetic payload delivery systems. Polymer-based delivery systems may have improved stability over lipid-based delivery systems because polymers are large molecules that are less dynamic, whereas lipid-based systems are complex and involve dynamic assemblies of many lipids with semi-stable structures. Cationic polymers bind to anionic genetic material to form complexes such as polyplexes, which can facilitate delivery of many nucleic acid types (e.g., ASOs). Synthesis of cationic polymers is modular, easy to scale up, and inexpensive. Cationic polymers bind to oligonucleotides, such as ASOs, through interpolyelectrolyte complexation driven by entropy, forming complexes. While many cationic polymer structures and architectures have been studied, cationic polymers still have issues translating to clinical use. For example, serum instability of formulations is a challenge for nonviral formulations. Serum, a component of blood that includes many types of proteins, can cause aggregation, can cause burst release of payloads, and/or can bind nonspecifically to delivery systems, causing issues with both in vitro and in vivo delivery. Methods of improving serum stability include the incorporation of hydrophilic sheath layers such as polyethylene glycol, carbohydrates, or zwitterionic moieties. However, these methods of improving serum stability can decrease interactions of the cationic polymer with the oligonucleotide payload and/or the interactions between the complex and a cell, both of which can result in decreased efficacy and/or efficiency of such cationic polymer delivery systems.
In one aspect, the present disclosure describes a complex that includes a polymer and an oligonucleotide cargo. The polymer includes cationic groups, cationizable groups, or both. In another aspect, the present disclosure describes a composition that includes one or more of the complexes of the present disclosure. In yet another aspect, the present disclosure describes methods of using a complex and/or a composition containing the same. The complex and/or composition containing the same may be used as or in delivery systems for oligonucleotide cargo. The delivery system may be used for in vitro and/or in vivo delivery of an oligonucleotide cargo.
The complexes include an oligonucleotide cargo. As used herein, the terms “oligonucleotide” and “nucleic acid” are used interchangeably to refer to a polymer of two or more nucleotides. As use herein, the term “oligonucleotide cargo” refers to one or more oligonucleotides that are a part of a complex of the present disclosure. An oligonucleotide cargo may include a plurality of oligonucleotides. The plurality of oligonucleotides may include the oligonucleotide having the same sequence or two or more oligonucleotides of different sequences.
An oligonucleotide of an oligonucleotide cargo may be single stranded; double stranded; or include one or more portions that are single stranded and one or more portions that are double stranded. Oligonucleotides include nucleosides linked through internucleoside linkages, also called backbone linkages. Nucleosides include a pentose sugar (e.g., ribose or deoxyribose) and a nitrogenous base (nucleobase or base) covalently attached to the sugar. The canonical nucleobases found in DNA and/or RNA are adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The canonical sugars found in DNA and/or RNA are deoxyribose (DNA) and ribose (RNA). A conical nucleoside linkage (also termed backbone linkage) is a phosphodiester bond. Oligonucleotide cargos may include canonical sugars, canonical bases, canonical internucleoside linkages, non-canonical sugars, non-canonical bases, non-canonical internucleoside linkages, or any combination thereof. Examples of non-canonical sugars include 2′ modifications on a pentose or ribose such as 2′-O-methoxyethyl (2′O-MOE) and 2′ methoxy (2′O-Me); locked sugars (also known as locked nucleic acids); and sugar alternatives such as methylenemorpholine rings. Examples of noncanonical backbone linkages include phosphorothioate linkages and phosphorodiamidate linkages. In some cases, a non-canonical sugar can be combined with a non-canonical backbone linkage, such as, for example in a phosphorodiamidate morpholino oligomer.
Examples of oligonucleotide cargos include, but are not limited to, plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligonucleotides (ASOs), small interfering RNA (siRNA), micro-RNA (miRNA), guide RNA (gRNA), Cas9-gRNa complexes, aptamers, derivatives thereof, and combinations thereof.
In one or more embodiments, the oligonucleotide cargo includes an antisense oligonucleotide (ASO). An ASO is a single stranded oligonucleotide that is at least partially complementary to a target nucleic acid. ASOs are generally single stranded oligonucleotides; however, in the complexes of the present disclosure, an ASO may exists as single-stranded oligonucleotide, a double stranded oligonucleotide, or have one or more single stranded regions and one or more double stranded regions. ASOs may be linear and lack secondary structure. ASOs may include one or more non-canonical bases, one or more non-canonical sugars, one or more non-canonical internucleoside linkages, or any combination thereof.
An oligonucleotide cargo may have a variety of lengths. In one or more embodiments, the number of bases in an oligonucleotide cargo is 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 75 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 10000 or more, 20000 or more, 30000 or more, or 40000 or more. In one or more embodiments, the number of bases in an oligonucleotide cargo is 50000 or less, 40000 or less, 30000 or less, 20000 or less, 10000 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 75 or less, 50 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or 5 or less.
In one or more embodiments, the number of bases in an oligonucleotide cargo is 5 to 1000. In one or more embodiments, the oligonucleotide cargo is an ASO and the number of bases in the ASO is 5 to 1000, 5 to 75, 5 to 50, 5 to 30, 10 to 50, 10 to 40, 10 to 13, 15 to 50, 15 to 40, 15 to 30, 15 to 25, 20 to 50, 20 to 40, or 20 to 30.
In one or more embodiments, the oligonucleotide cargo is not self-replicating. In one or more embodiments, the oligonucleotide cargo is not methylated; that is, any cytosine present in the oligonucleotide sequence is not a methylated cytosine. In one or more embodiments, the oligonucleotide cargo does not encode a gene product.
The sequence of an oligonucleotide cargo depends at least in part upon the target nucleic acid. Targeting an oligonucleotide cargo to a target nucleic acid may be a multistep process. The process may include identifying the nucleic acid of interest, analyzing a transcript of the nucleic acid of interest, and identifying a particular region of the nucleic acid of interest to be targeted by the oligonucleotide. The nucleic acid of interest may be a pre-mRNA transcript, mRNA transcript, or a gene associated with a disease or disorder. Methods for designing, synthesizing, and screening oligonucleotide cargos are known.
A complex of the present disclosure includes a polymer. Polymers are polymerized from one or more monomers. A polymer polymerized from a particular monomer may be described as “monomer name” polymer. For example, a polymer polymerized from n-butyl acrylate monomers, may be described as an n-butyl acrylate polymer.
In one or more embodiments, a complex of the present disclosure includes a homopolymer. A homopolymer is a polymer polymerized from a single monomer.
In one or more embodiment, a complex of the present disclosure includes a block polymer. A block polymer is a polymer that includes two or more polymer segments (blocks) joined by a covalent linkage. A block copolymer may include two or more homopolymer segments; two or more copolymer segments; or one or more homopolymer segments and one or more copolymer segments that are joined by a covalent linkage.
In one or more embodiments, a complex includes a diblock polymer. A diblock polymer is a polymer that includes two homopolymer segments joined by a covalent linkage.
The homopolymers and block polymers of the present disclosure include cationic groups (also called cation groups, cationic moieties, cation moiety, or simply cations) or cationizable groups. A cationic group is a chemical functionality that has a positive charge. A cationizable group is a chemical functionality that when exposed to some pH values is protonated resulting in a positive charge. In some embodiments of the present disclosure, a cationizable group is an amine cationizable group that can be protonated to from a cation. In some embodiments of the present disclosure, a cationizable group is an amine cationizable group that has a pKa of 8.5 or less, 8.0 or less, 7.5 or less, 7.0 or less, 6.5 or less, 6.0 or less, or 5.5 or less. For example, an amine cationizable group may be protonated at physiological pH (e.g., 7.0-7.4) and/or at an acid pH, such as the pH observed in an endosome (e.g., 3 to 6.5). The cationic groups or cationizable groups may be a part of a monomer used to form a polymer. For example, a monomer may include a pendant cationic group or pendent cationizable group that is not chemically modified during polymerization.
In one or more embodiments, a cationic group includes a quaternary ammonium. In one or more embodiments a cationizable group is an amine cationizable group. An amine cationizable group includes a primary amine, a secondary amine, or a tertiary amine. Under certain conditions, an amine can be protonated forming an ammonium cation. For example, a primary amine can be protonated forming a primary ammonium cation, a secondary amine can be protonated forming a secondary ammonium cation, and a tertiary amine can be protonated forming a tertiary ammonium cation. In one or more embodiments, the cationic group includes an amine that is a part of a ring.
The cationizable groups of a polymer of the present disclosure may be described in their neutral form or in their cationic group form.
A cationic group or a cationizable group may be of the formula —NR1R2R3 where R1, R2, and R3 are each independently H, a lone pair of electrons, or alkyl. The alkyl may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons. In one or more embodiments, the alkyl is methyl, ethyl, isopropyl, n-propyl, iso-butyl, n-butyl, or sec-butyl. In one or more embodiments, R1, R2, and R3 are the same. In one or more embodiments, R1 and R2 are the same and R3 is different. In one or more embodiments, R1, R2, and R3 are different. In embodiments where R1, R2, and R3 are all alkyl, the group is a cationic group. An example of such a cationic group is trimethylammonium (—N(CH3)3+). Examples of cationizable groups include —NH2; dimethylamino (—N(CH3)2); and diethylamino (—N(CH2CH3)2). When exposed to certain pH values, the dimethylamino and diethylamino groups can be protonated to form dimethylammonium (—NH(CH3)2+) and diethylammonium (—NH(CH2CH3)2+) groups, respectively.
A cationizable group may be of the formula —NR4R5 where the nitrogen, R4 and R5 form a 5, 6, or 7 membered ring. Except for the nitrogen atom, the ring may be a hydrocarbon ring. The ring may be aromatic. In addition to the nitrogen atom of the formula, the ring may include one or more other heteroatoms such as O, N, or S. Examples of cationic groups that are rings include 1-imidazolyl and 2-morpholino.
The acid dissociation constant (pKa) of a polymer that includes cationic groups or cationizable may vary. The pKa of a polymer may affect the ability of the polymer to form complexes with an oligonucleotide cargo and/or the ability to release the oligonucleotide cargo from a complex. The pKa of a polymer may be determined, for example, via potentiometric titration. In one or more embodiments, the pKa of the polymer is 5.0 or greater, 5.5 or greater, 6.0 or greater, 6.1 or greater, 6.2 or greater, 6.3 or greater, 6.4 or greater, 6.5 or greater, 6.6 or greater, 6.7 or greater, 6.8 or greater, 6.9 or greater, 7.0 or greater, 7.2 or greater, or 7.5 or greater. In one or more embodiments, the pKa of the polymer is 8.0 or less, 7.5 or less, 7.2 or less, 7.0 or less, 6.9 or less, 6.8 or less, 6.7 or less, 6.6 or less, 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, or 5.5 or less. In one or more embodiments the pKa of the polymer is 5.0 to 8.0, 5.0 to 7.5, 5.0 to 7.2, 5.0 to 7.0, 5.0 to 6.9, 5.0 to 6.8, 5.0 to 6.7, 5.0 to 6.6, 5.0 to 6.5, 5.0 to 6.4, 5.0 to 6.3, 5.0 to 6.2, 5.0 to 6.1, 5.0 to 6.0, or 5.0 to 5.5. In one or more embodiments the pKa of the polymer is 5.5 to 8.0, 5.5 to 7.5, 5.5 to 7.2, 5.5 to 7.0, 5.5 to 6.9, 5.5 to 6.8, 5.5 to 6.7, 5.5 to 6.6, 5.5 to 6.5, 5.5 to 6.4, 5.5 to 6.3, 5.5 to 6.2, 5.5 to 6.1, or 5.5 to 6.0. In one or more embodiments the pKa of the polymer is 6.0 to 8.0, 6.0 to 7.5, 6.0 to 7.2, 6.0 to 7.0, 6.0 to 6.9, 6.0 to 6.8, 6.0 to 6.7, 6.0 to 6.6, 6.0 to 6.5, 6.0 to 6.4, 6.0 to 6.3, 6.0 to 6.2, or 6.0 to 6.1. In one or more embodiments the pKa of the polymer is 6.1 to 8.0, 6.1 to 7.5, 6.1 to 7.2, 6.1 to 7.0, 6.1 to 6.9, 6.1 to 6.8, 6.1 to 6.7, 6.1 to 6.6, 6.1 to 6.5, 6.1 to 6.4, 6.1 to 6.3, or 6.1 to 6.2. In one or more embodiments the pKa of the polymer is 6.2 to 8.0, 6.2 to 7.5, 6.2 to 7.2, 6.2 to 7.0, 6.2 to 6.9, 6.2 to 6.8, 6.2 to 6.7, 6.2 to 6.6, 6.2 to 6.5, 6.2 to 6.4, or 6.2 to 6.3. In one or more embodiments the pKa of the polymer is 6.3 to 8.0, 6.3 to 7.5, 6.3 to 7.2, 6.3 to 7.0, 6.3 to 6.9, 6.3 to 6.8, 6.3 to 6.7, 6.3 to 6.6, 6.3 to 6.5, or 6.3 to 6.4. In one or more embodiments the pKa of the polymer is 6.4 to 8.0, 6.4 to 7.5, 6.4 to 7.2, 6.4 to 7.0, 6.4 to 6.9, 6.4 to 6.8, 6.4 to 6.7, 6.4 to 6.6, or 6.4 to 6.5. In one or more embodiments the pKa of the polymer is 6.5 to 8.0, 6.5 to 7.5, 6.5 to 7.2, 6.5 to 7.0, 6.5 to 6.9, 6.5 to 6.8, 6.5 to 6.7, or 6.5 to 6.6. In one or more embodiments the pKa of the polymer is 6.6 to 8.0, 6.6 to 7.5, 6.6 to 7.2, 6.6 to 7.0, 6.6 to 6.9, 6.6 to 6.8, or 6.6 to 6.7. In one or more embodiments the pKa of the polymer is 6.7 to 8.0, 6.7 to 7.5, 6.7 to 7.2, 6.7 to 7.0, 6.7 to 6.9, or 6.7 to 6.8. In one or more embodiments the pKa of the polymer is 6.8 to 8.0, 6.8 to 7.5, 6.8 to 7.2, 6.8 to 7.0, or 6.8 to 6.9. In one or more embodiments the pKa of the polymer is 6.9 to 8.0, 6.9 to 7.5, 6.9 to 7.2, or 6.9 to 7.0. In one or more embodiments the pKa of the polymer is 7.0 to 8.0, 7.0 to 7.5, or 7.0 to 7.2. In one or more embodiments the pKa of the polymer is 7.2 to 8.0, 7.2 to 7.5, or 7.5 to 8.0.
In one or more embodiments, the complex includes an acrylamide polymer, the acrylamide polymer including the cationic groups and/or the cationizable groups. An acrylamide polymer includes the repeating acrylamide functionality —CH2CH(C(═O)NHR)— where R includes a cationic/cationizable group. R can include a linker that separates the cationic/cationizable group from the amide of the acrylamide functionality. The linker can be a hydrocarbon linker. The hydrocarbon linker can include —(CH2)n— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of acrylamide polymers include, but are not limited to, amino ethyl acrylamide (AEA; AEAm); dimethyl amine ethyl acrylamide (DMA; DMAm); diethyl amine ethyl acrylamide (DEA; DEAm); trimethyl amine ethyl acrylamide (TMA; TMAm); morpholino ethyl acrylamide (MEA; MEAm); trimethylammonium ethylamine acrylamide (TMAEA; TMAEAm); and (1-imidazolyl)propyl acrylamide (ImPAm).
An acrylamide polymer can be formed from acrylamide monomers. An acrylamide polymer can be formed by reacting a polymer having a carboxylic acid or an ester with a compound that includes an amine capable of forming an amide bond. For example, an acrylamide polymer can be formed by modifying an acrylate polymer. Acrylate monomers may be polymerized to form an acrylate polymer having the acrylate functionality repeating group —CH2CH(C(═O)ORX)— where RX is a labile ester functionality. A compound having an amine capable of reacting with the ester of the acrylate functionality can react with the ester to displace RX and form an amide bond thereby forming an acrylamide functionality. An acrylamide polymer formed this way may include residual acrylate functionalities that were not converted to acrylamide functionalities; that is, the polymer may include residual —CH2CH(C(═O)ORX)— groups. As used herein, the term “acrylamide polymer” refers to a polymer or polymer segment (block) that includes 80 mole percent (mol-%) or greater, 85 mol-% or greater, 90 mol-% or greater, 95 mol-% or greater, or 99 mol-% or greater acrylamide functionalities.
In one or more embodiments, a complex of the present disclosure includes a diblock polymer. A diblock polymer includes a first block and a second block. The first block includes a first homopolymer and the second block includes a second homopolymer. The first homopolymer may be a homopolymer that includes cationic groups or cationizable groups. In one or more embodiments, the first homopolymer (first block) includes an acrylamide polymer where the acrylamide polymer includes cationic groups or cationizable groups.
The second homopolymer (second block) of a diblock polymer may be a polymer that allows for the formation of micelles that include the diblock polymer (described herein). The second homopolymer may include a hydrophobic polymer. In one or more embodiments, the second homopolymer includes a hydrophobic group. The hydrophobic group may be alkyl. The alkyl may be linear, branched, or cyclic. The alkyl may include 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of hydrophobic alkyl groups include isopropyl, n-propyl, iso-butyl, n-butyl, and sec-butyl.
The second homopolymer may be an acrylate polymer or an acrylamide polymer. As such, the second homopolymer may include the repeating acrylamide functionality (—CH2CH(C(═O)NHRY)—) or the repeating acrylate functionality (—CH2CH(C(═O)ORY)—) where RY the is a hydrophobic group described herein. Examples of acrylate polymers include isopropyl acrylate (poly; n-butyl acrylate; sec-butyl; and combinations thereof acrylate polymers. Examples of acrylate polymers include isopropyl acrylamide; n-butyl acrylamide; sec-butyl; and combination thereof acrylamide polymers.
In embodiments where the complex includes a diblock polymer the diblock polymer may be of the formula [block 1]z-[block 2]q were z an q are each independently an integer from 1 to 1000.
The number-average molecular weight (Mn) of a polymer of a complex of the present disclosure may vary. Mn is calculated using the following equation:
where Mi is the mean molecular size of range i and xi is the number fraction of the total number of polymer chains that are within Mi range. Mn may be determined, for example, using size exclusion chromatography with a multi-angle light scattering detector.
In one or more embodiments, the Mn of the polymer is 5 kilodalton (kDa) or greater, 10 kDa or greater, 20 kDa or greater, 25 kDa or greater, 30 kDa or greater, 40 kDa or greater, 50 kDa or greater, or 75 kDa or greater. In one or more embodiments, the Mn of the polymer is 100 kDa or less, 75 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, or 10 kDa or less. In one or more embodiments, the Mn of the polymer is 5 kDa to 100 kDa, 5 kDa to 75 kDa, 5 kDa to 50 kDa, 5 kDa to 40 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, or 5 kDa to 10 kDa. In one or more embodiments, the Mn of the polymer is 10 kDa to 100 kDa, 10 kDa to 75 kDa, 10 kDa to 50 kDa, 10 kDa to 40 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, or 10 kDa to 20 kDa. In one or more embodiments, the Mn of the polymer is 20 kDa to 100 kDa, 20 kDa to 75 kDa, 20 kDa to 50 kDa, 20 kDa to 40 kDa, 20 kDa to 30 kDa, or 20 kDa to 25 kDa. In one or more embodiments, the Mn of the polymer is 25 kDa to 100 kDa, 25 kDa to 75 kDa, 25 kDa to 50 kDa, 25 kDa to 40 kDa, or 25 kDa to 30 kDa. In one or more embodiments, the Mn of the polymer is 30 kDa to 100 kDa, 30 kDa to 75 kDa, 30 kDa to 50 kDa, or 30 kDa to 40 kDa. In one or more embodiments, the Mn of the polymer is 40 kDa to 100 kDa, 40 kDa to 75 kDa, or 40 kDa to 50 kDa. In one or more embodiments, the Mn of the polymer is 50 kDa to 100 kDa or 50 kDa to 75 kDa. In one or more embodiments, the Mn of the polymer is 75 kDa to 100 kDa.
The weight-average molecular weight (Mw) of a polymer of the present disclosure may vary. Mw is calculated using the following equation:
where Mi is the mean molecular size of range i and wi is the weight fraction of the total number of polymer chains that are within Mi range. Mw may be determined using size exclusion chromatography with a multi-angle light scattering detector (see Example).
In one or more embodiments, the Mw of the polymer is 5 kilodalton (kDa) or greater, 10 kDa or greater, 20 kDa or greater, 25 kDa or greater, 30 kDa or greater, 40 kDa or greater, 50 kDa or greater, or 75 kDa or greater. In one or more embodiments, the Mw of the polymer is 100 kDa or less, 75 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, or 10 kDa or less. In one or more embodiments, the Mw of the polymer is 5 kDa to 100 kDa, 5 kDa to 75 kDa, 5 kDa to 50 kDa, 5 kDa to 40 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, or 5 kDa to 10 kDa. In one or more embodiments, the Mw of the polymer is 10 kDa to 100 kDa, 10 kDa to 75 kDa, 10 kDa to 50 kDa, 10 kDa to 40 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, or 10 kDa to 20 kDa. In one or more embodiments, the Mw of the polymer is 20 kDa to 100 kDa, 20 kDa to 75 kDa, 20 kDa to 50 kDa, 20 kDa to 40 kDa, 20 kDa to 30 kDa, or 20 kDa to 25 kDa. In one or more embodiments, the Mw of the polymer is 25 kDa to 100 kDa, 25 kDa to 75 kDa, 25 kDa to 50 kDa, 25 kDa to 40 kDa, or 25 kDa to 30 kDa. In one or more embodiments, the Mw of the polymer is 30 kDa to 100 kDa, 30 kDa to 75 kDa, 30 kDa to 50 kDa, or 30 kDa to 40 kDa. In one or more embodiments, the Mw of the polymer is 40 kDa to 100 kDa, 40 kDa to 75 kDa, or 40 kDa to 50 kDa. In one or more embodiments, the Mw of the polymer is 50 kDa to 100 kDa or 50 kDa to 75 kDa. In one or more embodiments, the Mw of the polymer is 75 kDa to 100 kDa.
The dispersity of the molecular weight of the polymer affect the characteristics of the polymer. The molecular weight dispersity may be quantified as the dispersity (ÐM). ÐM is the distribution of individual molecular masses of a polymer. ÐM is calculated as the quotient of the mass average molecular weight (Mw) divided by the number-average molecular weight (Mn). The Mw and Mn may be determined using various methods including, for example, viscometry, size exclusion chromatography, and mass spectrometry. Generally, a small ÐM is preferred. Although there is no desired lower limit, in practice the ÐM of the polymer may be 1.0 or greater, 1.1 or greater, 1.2 or greater, 1.3 or greater, 1.4 or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 or greater, 1.9 or greater, 2.0 or greater, 2.1 or greater, 2.2 or greater, 2.3 or greater. 2.3 or greater, or 2.4 or greater. In one or more embodiments, the ÐM of the polymer may be 2.5 or less, 2.2 or less, 2.0 or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, or 1.1 or less. In one or more embodiments, the ÐM for the polymer is 1.0 to 2.5, 1.0 to 2.2, 1.0 to 2.0, 1.0 to 1.8, 1.0 to 1.7, 1.0 to 1.6, 1.0 to 1.5, 1.0 to 1.4, 1.0 to 1.3, 1.0 to 1.2, or 1.0 to 1.1. In one or more embodiments, the ÐM for the polymer is 1.1 to 2.5, 1.1 to 2.2, 1.1 to 2.0, 1.1 to 1.8, 1.1 to 1.7, 1.1 to 1.6, 1.1 to 1.5, 1.1 to 1.4, 1.1 to 1.3, or 1.1 to 1.2. In one or more embodiments, the ÐM for the polymer is 1.2 to 2.5, 1.2 to 2.2, 1.2 to 2.0, 1.2 to 1.8, 1.2 to 1.7, 1.2 to 1.6, 1.2 to 1.5, 1.2 to 1.4, or 1.2 to 1.3. In one or more embodiments, the ÐM for the polymer is 1.3 to 2.5, 1.3 to 2.2, 1.3 to 2.0, 1.3 to 1.8, 1.3 to 1.7, 1.3 to 1.6, 1.3 to 1.5, or 1.3 to 1.4. In one or more embodiments, the ÐM for the polymer is 1.4 to 2.5, 1.4 to 2.2, 1.4 to 2.0, 1.4 to 1.8, 1.4 to 1.7, 1.4 to 1.6, or 1.4 to 1.5. In one or more embodiments, the ÐM for the polymer is 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, 1.5 to 1.8, 1.5 to 1.7, or 1.5 to 1.6. In one or more embodiments, the M for the polymer is 1.6 to 2.5, 1.6 to 2.2, 1.6 to 2.0, 1.6 to 1.8, or 1.6 to 1.7. In one or more embodiments, the ÐM for the polymer is 1.7 to 2.5, 1.7 to 2.2, 1.7 to 2.0, or 1.7 to 1.8. In one or more embodiments, the ÐM for the polymer is 1.8 to 2.5, 1.8 to 2.2, or 1.8 to 2.0. In one or more embodiments, the ÐM for the polymer is 2.0 to 2.5, 2.0 to 2.2, or 2.2 to 2.5.
A complex of the present disclosure includes a polymer (e.g., a homopolymer or diblock polymer) associated with an oligonucleotide cargo. The ratio of moles of the amine or ammonium containing cationic group or cationizable of the polymer to the moles of the negatively charged phosphorous containing groups (e.g., phosphate groups or phosphorothioate groups) of the oligonucleotide cargo (N/P) may vary. The N/P ratio of the polymer may affect the efficiency of the complex as a delivery system. As used herein, the N/P ratio refers to the N/P ratio of a single complex or the average N/P ratio of a plurality of complexes. It is understood that the N/P of a complex or a plurality of complexes is the predicted or calculated N/P of such complex or complexes. In one or more embodiments, the N/P ratio is 1 or greater, 3 or greater, 5 or greater, 6 or greater, 8 or greater, 10 or greater, 12 or greater, 14 or greater, 16 or greater, 18 or greater, 20 or greater, 25 or greater, or 30 or greater. In one or more embodiments, the N/P ratio is 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 18 or less, 16 or less, 14 or less, 12 or less, 10 or less, 8 or less, 6 or less, 5 or less, or 3 or less. In one or more embodiments, the N/P ratio is 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, or 1 to 3. In one or more embodiments the N/P ratio is 3 to 40, 3 to 35, 3 to 30, 3 to 25, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 12, 3 to 10, 3 to 8, 3 to 6, or 3 to 5. In one or more embodiments, the N/P ratio is 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 18, 5 to 16, 5 to 14, 5 to 12, 5 to 12, 5 to 10, 5 to 8, or 5 to 6. In one or more embodiments, the N/P ratio is 6 to 40, 6 to 36, 6 to 30, 6 to 26, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 12, 6 to 10, or 6 to 8. In one or more embodiments, the N/P ratio is 8 to 40, 8 to 36, 8 to 30, 8 to 26, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 12, or 8 to 10. In one or more embodiments, the N/P ratio is 10 to 40, 10 to 36, 10 to 30, 10 to 26, 10 to 20, 10 to 18, 10 to 16, 10 to 14, or 10 to 12. In one or more embodiments, the N/P ratio is 12 to 40, 12 to 36, 12 to 30, 12 to 26, 12 to 20, 12 to 18, 12 to 16, or 12 to 14. In one or more embodiments, the N/P ratio is 14 to 40, 14 to 36, 14 to 30, 14 to 26, 14 to 20, 14 to 18, or 14 to 16. In one or more embodiments, the N/P ratio is 16 to 40, 16 to 36, 16 to 30, 16 to 26, 16 to 20, or 16 to 18. In one or more embodiments, the N/P ratio is 18 to 40, 18 to 36, 18 to 30, 18 to 26, or 18 to 20. In one or more embodiments, the N/P ratio is 20 to 40, 20 to 36, 20 to 30, or 20 to 26. In one or more embodiments, the N/P ratio is 25 to 40, 25 to 36, 25 to 30, 30 to 36, or 35 to 40.
A complex of the present disclosure generally includes a plurality of polymers associated with a cargo in a particular molecular configuration. The type of molecular configuration may be dependent at least in part on the type and/or identity of polymers included in the complex. For example, a complex that includes homopolymers may be a relatively unorganized complex of a plurality of homopolymer chains and a plurality of polynucleotide chains (oligonucleotide cargo), termed a polyplex. In a polyplex, the oligonucleotide associates with (e.g., binds to) the polymer via electrostatic interactions between the negatively charged oligonucleotide backbone and the cationic groups.
In one or more embodiments, a complex that includes a diblock polymer may include or be a micelleplex. A micelleplex includes a micelle and an oligonucleotide cargo non-covalently bound to the micelle. The cargo may be non-covalently bound to the exterior of the micelle. A micelle is supramolecular assembly of a plurality of amphipathic molecules organized such that the hydrophobic (or lipophilic) portion of the molecules are packed together forming a hydrophobic core and the hydrophilic portion of the molecules are displayed exterior to the hydrophobic core. For example, the hydrophobic block of a plurality of diblock polymers of the present disclosure may assemble together to form a hydrophobic core of a micelle while the block that includes the cationic groups or cationizable groups is displayed on the surface of the micelle. The cationic groups displayed on the micelle are able to associate with (e.g., bind to) the oligonucleotide cargo via electrostatic interactions between the negatively charged oligonucleotide backbone and the cationic groups to form a micelleplex.
The size of a complex (e.g., a micelleplex or polyplex) may affect its efficacy and/or efficiency as an oligonucleotide delivery system. Complex size can be measured, for example, using dynamic light scattering such as described in Example 1 and Example 2. In one or more embodiments, a complex has a size of, or a plurality of complexes have an average size of 0.05 nm or greater, 0.5 nm or greater, 1 nm or greater, 10 nm or greater, 20 nm or greater, 30 nm or greater, 40 nm or greater, 50 nm or greater, 75 nm or greater, 100 nm or greater, or 200 nm or greater. In one or more embodiments a complex has a size of, or a plurality of complexes have an average size of 500 nm or less, 200 nm or less, 100 nm or less, 75 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, 1 nm or less, or 0.5 nm or less.
The molecular configuration of the complex may affect its efficacy and/or efficiency as an oligonucleotide delivery system. Additionally, the configuration of the cells to which the complex is delivered may affect the complexes efficacy and/or efficiency as a delivery system. For example, in one or more embodiments, polyplexes may be able to more effectively delivery an oligonucleotide cargo to cells that are adhered to a surface as compared to micelleplexes. In one or more embodiments, micelleplexes may be able to more effectively delivery an oligonucleotide cargo to cells that free in solution (not adhered to a surface) as compared to polyplexes.
Nucleic acid delivery (e.g., oligonucleotide cargo delivery) is often assessed via in vitro cultures with cell monolayers. One method to enhance cellular interactions with delivery systems is to mediate aggregation, where complexes settling down on cells faster may lead to higher interactions with those cells. For example, this trend was observed when CRISPR/Cas9 ribonucleoprotein delivery using polymers in different media (without serum) was previously explored. Specifically, when CRISPR/Cas9 ribonucleoprotein polymer-complexes were formulated in PBS compared to water, some formulations had faster aggregation and settling kinetics onto the cells, which resulted in more efficient gene editing. However, there may be a limit to this phenomenon because large sizes of aggregates may affect internalization pathways and settling onto adhered cells is not a factor in the case of in vivo studies. To increase the cell interaction with complexes independent of the settling kinetics, cells can also be transfected while lifted or in suspension (for cell types that are cultured via suspension or adherent methods). Adhered cell lines can easily be exposed to transfection formulations after detachment and prior to plating. While suspension cells may be more challenging to transfect, the solution mixing can cause increased delivery system-cell interactions. Suspension cell growth methods are often used in bioreactors (for production of biopharmaceuticals) due to scale-up practicality and adherent cells can be cultured on microbeads that promote scaled culture growth in bioreactors.
A series of 10 polymers were synthesized (see Example 1, Polymer Synthesis). A homopolymer of pentafluoro phenyl acrylate (PFPA) as well as a diblock of PFPA and a hydrophobic block of n-butyl acrylate (nBA) were synthesized to yield similar molecular weight PFPA regions. Aminolysis reactions were performed on these two scaffolds to replace the PFPA moiety with one of five different cationic groups or cationizable groups (AEA, DMA, DEA, TMA, MEA) yielding a total of 10 polymers: 5 diblock and 5 linear variants, respectively (
Each of the 10 linear polymers and micelles were complexed with a model ASO that knocks down deGFP production and tested in vitro to determine the delivery efficacy of each formulation. Cells were transfected either while adhered or through simultaneous addition of cells and complexes (using a lifted cell procedure) so that identical adhered cell lines could be used in both conditions (see Example 1, Transfection Study). A green fluorescent protein (GFP) knockdown assay was used to determine delivery efficacy. In the GFP knockdown assay, human embryonic kidney (HEK) cells modified to consistently express destabilized GFP (deGFP) were transfected with a polyplex or micelleplex that included an ASO that binds to the deGFP mRNA and knocks down production of the deGFP. The extent of GFP knockdown was measured using flow cytometry by quantifying the decrease in the geometric mean GFP fluorescence intensity of the cells, as compared to the untreated control. Additionally, polyplexes and micelleplexes including \a scrambled or mismatch (MM) ASO sequence were included to screen for unrelated knockdown effects deriving from the protocol and/or toxicity.
Many of the complexes exhibited good knockdown efficacy in the presence of serum (10% fetal bovine serum (FBS)) and also maintained high cell viability especially compared to the commercial controls, LIPOFECTAMINE (L2K) and Jet PEI (
Micelles performed better than their linear analogues at both N/Ps studied. In each grouping of architecture and N/P there is a trend in cationic group/cationizable group performance that is similar. The primary amine, AEA, displays the highest levels of deGFP knockdown, followed by DMA, DEA, TMA, and lastly MEA. A similar trend was observed in previous studies when comparing primary, secondary, tertiary substituted amines for pDNA delivery, where less substituted amines performed better likely due to increased endosomal escape. However, these studies did not use post-polymerization methods to control for differences in length and are not comparing cations in a micelle architecture.
Two procedures were compared for transfection, the adhered cell and the simultaneous addition of cell and complex conditions (see Example 1, Transfection Study). Polyplex perform better when added to adhered cells compared to when cells are simultaneously added with the polyplexes. This effect may be due to linear polymers being more prone to aggregation in the presence of serum and thus have faster settling kinetics leading to an increase in cell-polyplex interaction when compared to micelleplexes (
The internalization of the ASO, or lack thereof, may explain the trend seen in transfection efficiency. HEK deGFP cells were transfected in serum using a Cy3 tagged ASO (see Example 1, Cy3 ASO Internalization Study). Interestingly, the nonpackaged ASO, or ‘ASO only’, was found to be present in the full population of cells (
When comparing cationic/cationizable groups (excluding MEA), groups such as TMA with high internalization showed lower gene knockdown performance. This may be explained by the minimal release of ASO or minimal endosomal escape, supporting the hypothesis that internalization may not be the main factor producing high knockdown, but rather something more complex. It is important to note that Cy3 is a large hydrophobic molecule that could change the ASO properties significantly. However, it is thought that the Cy3 assay can give important insight into internalization using these delivery vehicles. When comparing adhered versus simultaneous addition probed by the Cy3 internalization assay, various trends were observed (
Hence, micelleplexes generally have better knockdown and internalization after five hours under simultaneous addition compared to the adhered cell addition condition, and vice versa for polyplexes. This supports the hypothesis that micelles (micelleplexes) perform better under the simultaneous addition condition and linear polymers (polyplexes) perform better under adhered cell conditions due to increased cell-complex interactions and internalization.
A dye exclusion binding study with QUANT-IT OLIGREEN reagent was used to compare the relative strengths of binding between the linear polymers or micelles and the ASO payload (see Example 1, Binding Study;
Tighter binding can be important for getting the ASO into the cell, however tighter binding can also be detrimental to delivery since the cargo must be released or unpackaged to perform its function. In this case, the ASO needs to be released from the vehicle (polymer) to bind to mRNA and cause knockdown. Adding a small amount of serum (10% FBS) to the culture media decreased the ASO-polymer binding. Interestingly, some of the cationic/cationizable groups that had stronger relative binding affinities (when no serum was present) also seemed to have increased release when exposed to FBS (such as AEA, DMA, and DEA) (
Correlating these results, the delivery efficacy of polymers with different cationic/cationizable groups seems to be closely correlated to their binding strength with the ASO, thus influencing its intracellular transport. However, once in the cell, proteins at even higher concentrations (>10% serum) may help unpackage the ASO from the polymer or micelle so that the ASO can then bind to mRNA knocking down deGFP production.
It was hypothesized that (1) polyplexes have higher aggregation causing them to settle down faster and interact with adhered cells (compared to cells that were simultaneously added) leading to increased delivery efficacy in adhered cells and (2) micelleplexes were more colloidally stable and interact with cells in the solution that were added simultaneously with micelleplexes, again leading to increased ASO delivery. To further understand the impact of solution properties, the micelleplex and linear polyplex hydrodynamic radii was measured in serum via dynamic light scattering (DLS) (see Example 1, Dynamic Light Scattering;
A complex of the present disclosure may include more than one type of polymer. A type of polymer is the composition of the polymer, for example, including the cationic or cationizable groups. In one or more embodiments, a complex of the present disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of polymers. For example, a complex of the present disclosure may include a first polymer (e.g., homopolymer or diblock polymer) having a first cationic or cationizable group and a second polymer (e.g., homopolymer of diblock polymer) having a second cationic or cationizable group.
In one or more embodiments, a complex of the present disclosure includes a first polymer (e.g., a first diblock polymer) having a morpholino ethyl acrylamide block and a second polymer (e.g., a second diblock polymer) having an amino ethyl acrylamide, dimethyl amine ethyl acrylamide, diethyl amine ethyl acrylamide, or trimethyl amine ethyl acrylamide block.
In one or more embodiments, a complex of the present disclosure includes a first polymer having a first cation or cationizable group and a second polymer having a second cation or cationizable group. In such embodiments, the weight ratio or mole ratio of the first polymer to the second polymer can vary. It is understood that the weight ratio or mole ratio is the predicted or calculated weight ratio or mole ratio determined from the amount of the polymers present during complex or micelle formation. For example, a complex or micelle formed from a mixture that included 5 g of a first polymer and 5 g of a second polymer is said to have weight ratio of 1 part first polymer to 1 part second polymer. In one or more embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 0.1 parts or greater of the first polymer for every 1 part of the second polymer; 0.2 parts or greater of the first polymer for every 1 part of the second polymer; 0.3 parts or greater of the first polymer for every 1 part of the second polymer; 0.4 parts or greater of the first polymer for every 1 part of the second polymer; 0.5 parts or greater of the first polymer for every 1 part of the second polymer; 0.6 parts or greater of the first polymer for every 1 part of the second polymer; 0.7 parts or greater of the first polymer for every 1 part of the second polymer; 0.8 parts or greater of the first polymer for every 1 part of the second polymer; or 0.9 parts or greater of the first polymer for every 1 part of the second polymer. In one or more embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 1 part or less of the first polymer for every 1 part of the second polymer; 0.9 parts or less of the first polymer for every 1 part of the second polymer; 0.8 parts or less of the first polymer for every 1 part of the second polymer; 0.7 parts or less of the first polymer for every 1 part of the second polymer; 0.6 parts or less of the first polymer for every 1 part of the second polymer; 0.5 parts or less of the first polymer for every 1 part of the second polymer; 0.4 parts or less of the first polymer for every 1 part of the second polymer; 0.3 parts or less of the first polymer for every 1 part of the second polymer; or 0.2 parts or less of the first polymer for every 1 part of the second polymer.
In embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 0.8 parts to 1 part of the first polymer for every 1 part of the second polymer. In embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 0.1 parts to 0.5 part of the first polymer for every 1 part of the second polymer. In embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 0.1 parts to 0.3 part of the first polymer for every 1 part of the second polymer. In embodiments, the weight ratio or mole ratio of the first polymer to the second polymer is 0.2 parts of the first polymer for every 1 part of the second polymer. A complex having two or more different polymers (e.g., a first diblock polymer and a second diblock polymer) has a total polymer content. The total polymer content is the sum of the number of moles or weight of the polymers. In one or more embodiments, the total polymer content a complex having two or more different polymers includes 5 mol-% or weight-% or greater, 10 mol-% or weight-% or greater, 20 mol-% or weight-% or greater, 30 mol-% or weight-% or greater, 40 mol-% or weight-% or greater, 50 mol-% or weight-% or greater, 60 mol-% or weight-% or greater, 70 mol-% or weight-% or greater, or 80 mol-% or weight-% or greater of the first polymer. In one or more embodiments, the total polymer content a complex having two or more different polymers includes 90 mol-% or weight-% or less, 80 mol-% or weight-% or less, 70 mol-% or weight-% or less, 60 mol-% or weight-% or less, 50 mol-% or weight-% or less, 40 mol-% or weight-% or less, 30 mol-% or weight-% or less, 20 mol-% or weight-% or less, or 10 mol-% or weight-% or less of the first polymer. In one or more embodiments, a complex having two or more polymers has a total polymer content that includes 30 mol-% or weight-% to 70 mol-% of weight-%, 40 mol-% or weight-% to 60 mole-% or weight-%, or 50 mol-% or weigh-% of the first polymer. In one or more embodiments, a complex having two or more polymers has a total polymer content that includes 5 mol-% or weight-% to 40 mol-% of weight-%, 10 mol-% or weight-% to 30 mole-% or weight-%, or 20 mol-% or weigh-% of the first polymer. It is understood that the mole-% or weight-% of the polymers in the total polymer content is the predicted of calculated amount determined from the amount of the polymers present during complex formation. For example, a complex formed from a mixture that included 5 g of a first polymer and 5 g of a second polymer is said to have a total polymer content that has 50 weight-% first polymer and 50 weight-% second polymer.
In another aspect, the present disclosure describes a composition that includes one or more complexes of the present disclosure. The composition may be a delivery system composition. In one or more embodiments, the delivery system is a transfection composition. A transfection composition is a composition that includes one or more components for transfection. Transfection is the in vivo or in vitro process of introducing an exogenous oligonucleotide (e.g., an oligonucleotide cargo) into a cell.
A transfection composition includes a plurality of complexes of the present disclosure. The plurality of complexes may include one complex species or two or more complex species. It is understood that a “complex species” refers to a complex that includes a specific oligonucleotide cargo and a specific polymer or polymers (e.g., a complex that includes a first polymer and a second polymer). A first complex species may differ from a second complex species by the oligonucleotide cargo, the polymer or polymers, or both.
In one or more embodiments, a transfection composition includes a first complex and a second complex. The amount of each complex in a transfection composition may vary. In one or more embodiments, the weight ratio or mole ratio of the first complex to the second complex is 0.1 parts or greater of the first complex for every 1 part of the second complex; 0.2 parts or greater of the first complex for every 1 part of the second complex; 0.3 parts or greater of the first complex for every 1 part of the second complex; 0.4 parts or greater of the first complex for every 1 part of the second complex; 0.5 parts or greater of the first complex for every 1 part of the second complex; 0.6 parts or greater of the first complex for every 1 part of the second complex; 0.7 parts or greater of the first complex for every 1 part of the second complex; 0.8 parts or greater of the first complex for every 1 part of the second complex; or 0.9 parts or greater of the first complex for every 1 part of the second complex. In one or more embodiments, the weight ratio or mole ratio of the first complex to the second complex is 1 part or less of the first complex for every 1 part of the second complex; 0.9 parts or less of the first complex for every 1 part of the second complex; 0.8 parts or less of the first complex for every 1 part of the second complex; 0.7 parts or less of the first complex for every 1 part of the second complex; 0.6 parts or less of the first complex for every 1 part of the second complex; 0.5 parts or less of the first complex for every 1 part of the second complex; 0.4 parts or less of the first complex for every 1 part of the second complex; 0.3 parts or less of the first complex for every 1 part of the second complex; or 0.2 parts or less of the first complex for every 1 part of the second complex.
The transfection complex may further include a transfection medium. The transfection medium may be any suitable medium for contact with cells. In one or more embodiments, the medium comprises water. In one or more embodiments, the medium includes salts and or agents or compounds that promote cell health and/or growth. In one or more embodiments, the medium is a cell growth medium, such as, for example, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), RPMI 1640 Medium, OPTIMEM I, or any combination thereof. In one or more embodiments, the medium includes proteins. In other embodiments, the medium does not include proteins. In one or more embodiments, the medium is serum free. In other embodiments, the medium includes serum.
The pH of the transfection composition may vary. In one or more embodiments, the pH of the transfection composition is 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, or 8 or greater. In one or more embodiments, the pH of the transfection composition is 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less. In one or more embodiments, the pH of the transfection composition is 4 to 8, 5 to 8, 6 to 8, or 7 to 8. In another aspect, the present disclosure describes a method. The method includes forming a complex of the present disclosure. The method used to form the complex may depend at least in part on the polymer or polymers used to form the complex.
In one or more embodiments, the method includes mixing a polymer or polymers (e.g., one or more different types of polymers) with an oligonucleotide cargo to create a first mixture. The method may further include incubating the first mixture for a first period of time to form a second mixture comprising the complex. In one or more embodiments, the second mixture is a transfection composition that can be used to treat cells. For example, this method may be used to form a complex that includes a homopolymer. This method may be used to form a polyplex.
In one or more embodiments, the method includes mixing a polymer or polymers in a pre-first mixture. The method may further include incubating the pre-first mixture for period of time to form a first mixture that include micelles. Incubating the pre-first mixture allows at least a portion of the polymers to organize into one or more micelles. In one or more embodiments, the method includes incubating the first mixture with an oligonucleotide to from a second mixture, the second mixture including a plurality of complexes. Incubating the first mixture with the oligonucleotide allows at least a portion of the oligonucleotide to associate with the micelles to form micelleplexes. In one or more embodiments, the second mixture is a transfection composition that can be used to treat cells. For example, this method may be used to form a complex that includes a diblock polymer or two or more diblock polymers.
The pre-first mixture includes a carrier. The carrier may include water. The carrier may further include one or more additional components. For example, the carrier may include a buffering agent and/or a salt. Examples of buffering agents include 3-(N-morpholino)propanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid; and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
The pre-first mixture may have one or more properties that may facilitate micelle formation. For example, the pre-first mixture may have a pH of 4 to 9, such as 6.5 to 7.5 or 7. The pre-first mixture may have an ionic strength of 10 mM to 1000 mM, such as 50 mM to 200 mM or 100 mM.
In one or more embodiments, the method may further include agitating the pre-first mixture after incubation. For example, ultrasonication may be used to agitate the pre-first mixture. In one or more embodiments, the method may further include incubating the agitated pre-first mixture at an elevated temperature.
The absolute concentration of the one or more polymers and the absolute concentration of the oligonucleotide in the first mixtures of the methods presented herein may vary depending at least in part on the desired N/P ratio, the identity and properties of the polymer or polymers, the identity and properties of the oligonucleotide, the number of cells to be treated, other factors, or any combination thereof.
In one or more embodiments, the first mixture includes water. In one or more embodiments, the first mixture includes an aqueous solution of water and an acid. The acid is used to adjust the pH to a value of 1 to 5. A low pH may be beneficial to complex as a low pH may increase the probability that the cationizable groups of the polymers are ionized (e.g., protonated). Protonation of the cationizable amines gives the cationizable amines a positive charge which may increase electrostatic interactions between the polymer and any negatively charged backbone groups of an oligonucleotide. Any suitable acid or buffer may be included. Examples of acids include, but are not limited to, citric acid, acetic acid, hydrochloric acid, formic acid, lactic acid, uric acid, malic acid, tartaric acid, sulfuric acid, and the like. Examples of buffer include sodium acetate buffer, chloroacetate buffer, and the like.
The length of first incubation period of time may affect the transfection efficiency of a complex. In one or more embodiments, the first period of time is 1 minute or greater, 15 minutes or greater, 20 minutes or greater, 25 minutes or greater, 30 minutes or greater, 40 min or greater, 1 hour or greater, 2 hours or greater, 4 hours or greater, or 5 hours or greater. In one or more embodiments, the first period of time is 5 hours or less, 4 hours or less, 2 hours or less, 1 hour or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, or 15 minutes or less. In one or more embodiments, the first period of time is 15 min to 1 hour, 15 minutes to 40 minutes, 15 minutes to 35 minutes, 15 minutes to 30 minutes, 15 minutes to 25 minutes, or 15 minutes to 20 minutes. In one or more embodiments, the first period of time is 20 minutes to 1 hour, 20 minutes to 40 minutes, 20 minutes to 35 minutes, 20 minutes to 30 minutes, or 20 minutes to 25 minutes. In one or more embodiments, the first period of time is 25 minutes to 1 hour, 25 minutes to 40 minutes, 25 minutes to 35 minutes, or 25 minutes to 30 minutes. In one or more embodiments, the first period of time is 30 minutes to 1 hour, 30 minutes to 40 minutes or 30 minutes to 35 minutes. In one or more embodiments, the first period of time is 25 minutes to 35 minutes.
In one or more embodiments, the method further includes diluting the second mixture with a dilution medium to create a third mixture. In one or more embodiments, the third mixture is a transfection composition that can be used to treat cells. In such embodiments, the dilution medium is a transfection medium.
The second mixture may be diluted with an amount of a medium such that the concentration of the complex in the third mixture is 99% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less 0.1% or less, 0.01% or less, 0.001% or less, 0.0001% or less of the concentration of the complex in the second mixture. In one or more embodiments, the second mixture may be diluted with an amount of a medium such that the concentration of the complex in the third mixture is 0.0001% or greater, 0.001% or greater, 0.01% or greater, 0.1% or greater, 1% or greater, 5% or greater, 10% or greater, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or 90% or greater of the concentration of the complex in the second mixture. In one or more embodiments, the second mixture may be diluted with an amount of a medium such that the concentration of the complex in the third mixture is 10% to 90%, 20% to 80%, or 40% to 60% of the concentration of the complex in the second mixture.
The length of the second period of time may affect the efficiency of transfection. In one or more embodiments, the second period of time is 1 minute or greater, 15 minutes or greater, 20 minutes or greater, 25 minutes or greater, or 30 minutes or greater, 40 minutes or greater, 1 hour or greater, 2 hours or greater, 4 hours or greater, or 5 hours or greater. In one or more embodiments, the second period of time is 5 hours or less, 4 hours or less, 2 hours or less, 1 hour or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, or 15 minutes or less. In one or more embodiments, the second period of time is 15 minutes to 1 hour, 15 minutes to 40 minutes, 15 minutes to 35 minutes, 15 minutes to 30 minutes, 15 minutes to 25 minutes, or 15 minutes to 20 minutes. In one or more embodiments, the second period of time is 20 minutes to 1 hour, 20 minutes to 40 minutes, 20 minutes to 35 minutes, 20 minutes to 30 minutes, or 20 minutes to 25 minutes. In one or more embodiments, the second period of time is 25 minutes to 1 hour, 25 minutes to 40 minutes, 25 minutes to 35 minutes, or 25 minutes to 30 minutes. In one or more embodiments, the second period of time is 30 minutes to 1 hour, 30 minutes to 40 minutes or 30 minutes to 35 minutes. In one or more embodiments, the second period of time is 25 minutes to 35 minutes.
In another aspect, the present disclosure describes a method of transfecting a cell with a complex of the present disclosure. In some such embodiments, the complex is in a transfection composition of the present disclosure. The complex and/or transfection composition may include any components as disclosed herein and may be formed according to any method disclosed herein. The method includes contacting a complex and/or a transfection composition with a cell to form a transfection mixture. In one or more embodiments, the transfection composition is the third mixture following the second incubation time from the method of forming the complex described herein.
The technique of contacting a complex and/or transfection composition with a cell may depend at least in part on if the cell is a part of an in vitro or in vivo system. In one or more embodiments where the cell is a part of an in vitro system, contacting the cell with the complex of a transfection composition may include placing the transfection composition on top of an adhered cell or in a mixture that includes non-adhered cells. In one or more embodiments where the cell is a part of an in vivo system, contacting a complex of a transfection composition with a cell may include administering the transfection composition to a subject. Administration may be, for example, topical, enteral, or parenteral.
The pH of the complex or transfection composition used to treat the cells may affect the efficiency of transfection. For example, a pH that is too high or too low may kill the cells. In one or more embodiments, the pH of the complex or transfection composition is between 5 and 8. In one or more embodiments, the pH of complex or transfection composition is between 7 and 8.
In one or more embodiments, the method further incudes incubating the transfection mixture for a transfection time. For example, in embodiments where the cell is a part of an in vitro system, the transfection composition may be in contact with the cell for a transfection time. In one or more embodiments, the transfection time is 1 minute or greater, 15 minutes or greater, 20 minutes of greater, 25 minutes or greater, or 30 minutes or greater, 40 minutes or greater, 1 hour or greater, 5 hours or greater, 12 hours or greater, 24 hours or greater, 48 hours or greater, 64 hours or greater, or 96 hours or greater. In one or more embodiments, the transfection time of time is 96 hours or less, 64 hours or less, 48 hours or less, 24 hours or less, 12 hours or less, 5 hours or less, 1 hour or less, 40 minutes of less, 35 minutes or less, 30 minutes or less, 25 minutes or less, or 20 minutes or less. In one or more embodiments, the transfection time of time is 15 minutes to 40 minutes, 15 minutes to 35 minutes, 15 minutes to 30 minutes, 15 minutes to 25 minutes, or 15 minutes to 20 minutes. In one or more embodiments, transfection time is 20 minutes to 40 minutes, 20 minutes to 35 minutes, 20 minutes to 30 minutes, or 20 minutes to 25 minutes. In one or more embodiments, the transfection time is 25 minutes to 40 minutes, 25 minutes to 35 minutes, or 25 minutes to 30 minutes. In one or more embodiments, the transfection time is 30 minutes to 40 minutes or 30 minutes to 35 minutes. In one or more embodiments, the transfection time is 25 minutes to 35 minutes.
In one or more embodiments, the method further includes quenching the transfection mixture. For example, in one or more embodiments where the cell is a part of an in vitro system, the method further includes quenching the transfection mixture. Quenching the transfection mixture decreases the likelihood of transfection. Quenching the transfection mixture may increases cell survival during and/or after the transfection process. In one or more embodiments, quenching the transfection mixture includes adding serum-containing media to the transfection mixture to create a quenched mixture. The serum in the serum-containing media disrupts the ability of the transfection complex to be transfected into a cell.
Mixed and blended micelleplex formulations were explored. Mixed micelleplexes were compositions that included two micelleplex species differing by the identity of the diblock polymer. A blended micelleplex was a single micelleplex that included two different diblock polymers, each diblock polymer having a different cationic/cationizable group. It was hypothesized the mixed and blended formulations of polymers could influence and improve properties involved in nucleic acid delivery and offer a new facile method to study and rapidly optimize polymeric formulations in a manner similar to lipids. To this end, three diblock polymers were synthesized via post polymerization modifications of a pentafluoro phenyl acrylate and n-butyl acrylate (PFPA-nBA) diblock polymer to include 3 different amine types (M, D, and A in
Once synthesized, the diblocks were formulated into micelles using direct dissolution in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH=7). They were stirred for 1 week, followed by ultrasonication for 2 hours (see Example 2, Homomicelle Formation and Blended Diblock Formation). Two different versions of micelles were formulated (1) homo-micelles, formed from one type of diblock (M100, D100, and A100) and (2) blended diblock micelles, where 2 different diblocks were blended into one micelle (BldR′% R″% where R′% and R″% indicate the amount of the diblock containing cationizable moieties R′ or R″, where R′ and R″ are different and are independently M, D, or A) (
All of the micelles were of similar size of about 50-60 nm radius and low polydispersity (
Micelleplexes were formulated at N/Ps of 15 for each homomicelle formulations (A100, D100, and M100) and N/P of 30 for blended diblock formulations (BldA50M50 and BldD50M50) and mixed micelle formulations (MixA50+M50 and MixD50+M50). These N/Ps were chosen based on the high performance of N/P 15 of micelle formulations in previous studies. Additionally, the N/P of each cationizable moiety was 15 leading to a total N/P of 30, as each cationizable moiety was in a 50:50 mixture in the mixed micelle and blended diblock system. When the micelles were complexed with ASO, it was observed that they formed unimodal micelleplex populations that mimic the size of the uncomplexed micelles (
The binding of micelles to the ASO in PBS was studied (
The micelleplex systems were studied for their delivery of ASOs in vitro. A GFP knockdown assay was used to study delivery efficacy (see Example 2, Transfection). Briefly, HEK cells that have been modified to continually produce destabilized GFP (deGFP) were lifted using trypsin and then added to micelleplexes containing an ASO that would knockdown deGFP mRNA and prevent it from being translated into the protein. These transfections were all completed in conditions containing serum. The cells and micelleplex formulations were added to plates allowing the cells to adhere. 72 hours after transfection, these cells were analyzed via flow cytometry to measure GFP fluorescence and corresponding gene knockdown. It has been observed that colloidally stable micelleplexes interact more when cells are temporarily suspended in solution and yield better transfection efficacy compared to cells that were pre-adhered and then transfected (
A100 and D100 were chosen because they were the best-performing micelles in previous studies. Additionally, the moiety M was also explored as it was hypothesized that it could improve transfection. For example, it was hypothesized that the incorporation of a moiety with a lower pKa would only be protonated under acidic conditions such as endosomal pH and not in extracellular conditions therefore potentially improving endosomal escape without detrimental interactions with the cell membrane, which often causes of toxicity.
A100, D100, M100 and 50:50 formulations of M with either D or A as either blended (BldA50M50 or BldD50M50) or mixed (MixA50+M50 or MixD50+M50) micelles were first studied (
Coformulating M with D increased transfection, but both mixing or blending of M with A did not increase transfection compared to transfections of D100 and A100, respectively (
The shifts in pKa may alter the micelles' ability to escape the endosome via the proton sponge effect. To test the hypothesis that mixing or blending M with D causes more endosomal escape then mixing or blending M with A, the same transfection conditions with an additional preincubation step with Baf-A1, a small molecule known to inhibit the endosomal proton pumps that are necessary for the proton sponge effect (
Due to the ability of M to modulate D's performance in the blended and mixed systems, these formulations were studied at different ratios of D and M to enable optimization of performance. The corresponding D100 and M100 homomicelles at N/P formulation ratios that correlate to the amount of D and M present in the mixed and blended systems were also studied. There was little difference in the micelleplex size and dispersity for the blended diblock systems and the micelle mixed systems (
Binding was studied using the OLIGGREEN assay to observe if changing the ratio of D and M would affect binding in either PBS (the media used for micelleplex formulation) or after addition of DMEM with 10% FBS (the media used for transfection) (see Example 2, Binding Study). It was hypothesized that because all N/Ps were well above 1 that formulations including D (D100, BldD20M80 MixD20+M80, BldD80M20, and MixD20+M80) would bind similarly (as previously observed when studying only the 50:50 mixtures,
Transfection of the D and M mixed and blended systems with ASO payload was further examined to determine optimal ratios of D:M. It was hypothesized that increasing the amount of M in the system (whether as a mixed micelle or blended diblock) would improve transfection efficacy because there would be a greater change in pKa of the D moieties when more hydrophobic M is added. Additionally, the hydrophobic M moiety itself could improve delivery as increasing hydrophobicity often improves nucleic acid delivery through a variety of different mechanisms including modulation of nucleic acid binding/release, internalization, membrane-interactions, etc. Furthermore, hydrophobicity can cause differences in cooperative protonation/deprotonation, a factor in delivery of oligonucleotide cargos. One of the mixed systems with higher M incorporation, BldD20M80 N/P=30, had a much higher GFP knockdown compared to the D100 micelle at the corresponding N/P (D100 N/P=6) (
Transfection with and without Baf-A1 was examined to understand if this trend was due to changes in endosomal escape through the proton sponge hypothesis since Baf-A1 blocks proton pumps in the endosome. BldD20M80 had the most decrease in knockdown when Baf-A1 was added versus when it wasn't compared to MixD20+M80 and D100, indicating that BldD20M80 is using the proton sponge effect more than MixD20+M80 and D100, possibly explaining its high performance (
Mixing micelles and blending diblocks systems can lead to improved transfection efficiency over homomicelle systems. This approach provides an easy and modular formulation method for optimized delivery into the cells. M could improve transfection efficiency when mixed or blended with D, but did not improve efficacy when mixed or blended with A. This may be due to the static charge density of the chains containing A within the cellular and endosomal pH range. Micelles including D were much more affected by addition of Baf-A1 indicating said formulations utilize a proton sponge mechanism of endosomal escape (compared to micelle formulations including A that were much lesser affected). M is able to increase endosomal escape using the proton sponge when D is in the system. However, A is not as reliant on endosomal escape via proton sponge effect. Therefore, no improvements in transfection were observed when M is mixed or blended with A. Additionally, the ratios of D and M were tuned within blended diblock and mixed micelle formulations. It was found that a ratio of 80:20 M:D as a blended diblock (BldD20M80) yielded optimal ASO transfection in this system.
In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. The term “consisting of” means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of” indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.
In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.
As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.
In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.
As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.
As used herein, the terms “formed from” and “polymerized from” are open ended, unless expressly stated, and may include other components that may not be expressly described relative to the subject that is formed from or polymerized from the stated components. For example, a polymer formed from or polymerized from a quinoline-containing monomer may include capping groups or other groups not expressly mentioned.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
N-Boc-ethylenediamine; N,N-dimethylethylenediamine; N,N-diethylethylenediamine, (2-aminoethyl)trimethylammonium chloride hydrochloride; 4-(2-aminoethyl)morpholine; and trifluoroacetic acid were purchased from Sigma Aldrich (St. Louis, MO). Human embryonic kidney cells that express destabilized green fluorescent protein (deGFP HEK cells) were procured. deGFP knockdown (+T+T+GCCGGTGGTGCAGAT+A+A+A (SEQ ID NO: 1)) and deGFP knockdown mismatched sequence ASOs (+G+G+AGTACACTATATCGG+T+G+G (SEQ ID NO: 2)) were purchased from Integrated DNA Technologies (Coralville, IA). ASO modifications included phosphorothioated backbones. Additionally, the +indicates a locked nucleic acid. Dulbecco's Modified Eagle Medium (DMEM), high glucose, GLUTAMAX FBS, trypsin-EDTA, penicillin-streptomycin, PBS, optimum, OLIGOFECTAMINE, LIPOFECTAMINE 2000, QUANT-IT OliGreen ssDNA Reagent, and calcein violet stain were from Invitrogen Life Technologies ThermoFisher Scientific (Carlsbad, CA). JetPEI was purchased through Polyplus (Illkirch-Graffenstaden, France). CCK-8 kit was purchased from Dojindo Molecular Technologies (Rockville, MD).
The synthesis of the cationic linear and diblock polymers from poly(pentafluorophenyl acrylate) (PPFPA) followed the procedure outlined in Santa Chalarca, C. F.; Dalal, R. J.; Chapa, A.; Hanson, M. G.; Reineke, T. M. Cation Bulk and PKa Modulate Diblock Polymer Micelle Binding to PDNA. ACS Macro Lett. 2022, 588-594. doi.org/10.1021/acsmacrolett.2c00015.). Briefly, the diblock or homopolymer with pendant PFPA was mixed with the ethylamine diamine-functionalized derivative of each cationic moiety and 2-hydroxyethyl acrylate (HEA) in 1,4-dioxane, or N,N-Dimethyl formamide (DMF) for TMA and MEA. The solution was stirred under nitrogen for 18 hours at 40° C. Dialysis in saline, followed by water, was used for purification.
PPFPA was synthesized as described in Santa Chalarca, C. F.; Dalal, R. J.; Chapa, A.; Hanson, M. G.; Reineke, T. M. Cation Bulk and PKa Modulate Diblock Polymer Micelle Binding to PDNA. ACS Macro Lett. 2022, 588-594. doi.org/10.1021/acsmacrolett.2c00015. Aminolysis and Michael addition of the PPFPA yielded the five different linear polymers following the general procedure described in
L-TMA Synthesis: To a 4 mL scintillation vial, the PFPA polymer (1 eq. per PFPA repeat unit, Mn=40K, 71.4 mg) dissolved in anhydrous DMF (2.1 mL) was added. Next, HEA (0.15 eq. per PFPA repeat unit, 25 eq. per end group, 5.1 μL) was run through a basic alumina plug column and added. Triethylamine (2 eq. per PFPA repeat unit, 83.6 μL) was added at the same time as (2-aminoethyl)trimethylammonium chloride hydrochloride (2 eq. per PFPA repeat unit, 105 mg) was added. The vial was sealed with a septum, put under N2 and heated at 40° C. for 18 hours. Next an 19F NMR was taken (
L-AEA-Boc Synthesis: To a 20 mL scintillation vial, the PFPA polymer (1 eq. per PFPA repeat unit, Mn=40K, 119.0 mg) dissolved in anhydrous 1,4 dioxane (3.6 mL) was added. Next, HEA (0.15 eq. per PFPA repeat unit, 25 eq. per end group, 5.1 μL) was run through a basic alumina plug column and added. Next the N-Boc-ethylenediamine (2 eq. per PFPA repeat unit, 158 μL) was added. The vial was sealed with a septum, put under N2 and heated at 40° C. for 18 hours. Next an 19F NMR was taken (
L-AEA Synthesis: To a 4 mL scintillation vial, the L-AEA-Boc polymer (10.9 mg) and trifluoroacetic acid (TFA) (0.4 mL) were added. This was stirred at room temperature for 2 hours. Next, the TFA was evaporated off using a rotary evaporator. Next, 5 mL of milliQ water was added to the resulting L-AEA polymer which was then transferred to a dialysis membrane (RC MWCO 12-15 kDa). This was dialyzed against 100 mM NaCl for 2 changes/2 days and then MilliQ water for 3 changes/3 days. The solution was then freeze dried yielding the L-AEA polymer. The 1H NMR of L-AEA is shown in
L-DEA Synthesis: To a 20 mL scintillation vial, the PFPA polymer (1 eq. per PFPA repeat unit, Mn=40K, 119.0 mg) dissolved in anhydrous 1,4 dioxane (3.6 mL) was added. Next, HEA (0.15 eq. per PFPA repeat unit, 25 eq. per end group, 5.1 μL) was run through a basic alumina plug column and added. Next the N,N-diethylenediamine (2 eq. per PFPA repeat unit, 141.7 μL) was added. The vial was sealed with a septum, put under N2 and heated at 40° C. for 18 hours. Next an 19F NMR was taken (
L-DMA Synthesis: To a 4 mL scintillation vial, the PFPA polymer (1 eq. per PFPA repeat unit, Mn=40K, 23.81 mg) was dissolved in anhydrous 1,4 dioxane (0.71 mL) was added. Next, HEA (0.15 eq. per PFPA repeat unit, 25 eq. per end group, 5.1 μL) was run through a basic alumina plug column and added. Next the N,N-dimethylenediamine (2 eq. per PFPA repeat unit, 21.8 μL) was added. The vial was sealed with a septum, put under N2 and heated at 40° C. for 18 hours. Next an 19F NMR was taken (
L-MEA Synthesis: To a 20 mL scintillation vial, the PFPA polymer (1 eq. per PFPA repeat unit, Mn=40K, 119.0 mg) dissolved in anhydrous DMF (3.6 mL) was added. Next, HEA (0.15 eq. per PFPA repeat unit, 25 eq. per end group, 5.1 μL) was run through a basic alumina plug column and added. Next the 4-(2-aminoethyl)morpholine (2 eq. per PFPA repeat unit, 131.2 μL) was added. The vial was sealed with a septum, put under N2 and heated at 40° C. for 18 hours. Next an 19F NMR was taken (
The formulation of the cationic micelles followed the procedure outlined in Santa Chalarca, C. F.; Dalal, R. J.; Chapa, A.; Hanson, M. G.; Reineke, T. M. Cation Bulk and PKa Modulate Diblock Polymer Micelle Binding to PDNA. ACS Macro Lett. 2022, 588-594. doi.org/10.1021/acsmacrolett.2c00015.). Briefly, diblock polymers were dissolved in MOPS (10 mM, ionic strength=100 mM, pH=7.0) at a 1 mg/mL concentration and stirred for 7 days. Next, the solutions were ultrasonicated for 2 hours, annealed at 40° C. overnight, and ultrasonicated again for 2 hours. Micelle size and dispersity were determined via dynamic light scattering (DLS) on a Zetasizer Nano ZS (Malvern Instruments Limited, Worcestershire, UK, Model: ZEN3600).
Complexes were formed by adding 50 μL of micelle or linear polymer solution including JETPEI at the corresponding N/P concentration in PBS to 50 μL of ASO (0.5 μg/well, 50 μL of ASO stock solution per well) in PBS. For OLIGFECTAMINE, the ASO was added to OLIGFECTAMINE and prepared in Optimem as outlined in the recommended procedure for this product. This was pipette mixed 3 times and then allowed to sit and complex for 30 minutes.
For the adhered cell condition, 200 μL of DMEM with 10% FBS containing 1% penicillin-streptomycin was added to the above solutions, and pipette mixed 3 times. This was added to adhered cells (plated 24 hours before at 50,000 cells per well in a 24 well plate). LIPOFECTAMINE 2000 was formulated with ASO payloads according to the commercial procedure, but scaled down to accommodate 0.5 μg of ASO/well.
For the lifted cell addition procedure, 200 μL of DMEM with 10% FBS and 1% penicillin-streptomycin was formulated with 50,000 cells and the above solutions were added and pipette mixed 3 times. This solution was added to an empty 24 well plate. Lipofectamine 2000 formulated with the commercial procedure with ASO (scaled down to 0.5 μg of ASO/well) was added to 200 μL of DMEM with 10% FBS and 1% penicillin-streptomycin with 50,000 cells that had just been added to the plate rather than adhered cells. For untreated cells, only 200 μL of DMEM+10% FBS and 1% penicillin-streptomycin and cells to each well.
24 hours after transfection, the media was replaced with 1 mL of DMEM containing 10% FBS and 1% penicillin-streptomycin. 72 hours after transfection, a CCK-8 assay was run following the commercial procedure to measure viability followed by flow cytometry to measure deGFP knockdown in live cells.
Knockdown was measured through flow cytometry by gating for cells and single cells using the forward and side scattering detectors. Cells were then gated by live cells using a calcein violet stain. The mean fluorescence of GFP in the live cells was used to analyze the GFP knockdown. The decrease in GFP fluorescence compared to untreated cells was normalized to untreated cells to get a percent knockdown value with 100% knockdown meaning no GFP fluorescence was observed.
Cells were transfected using the same methods outlined under the transfection study, however a 5′ Cy3 labeled ASO was used. The same molarity of Cy3 ASO as ASO was used therefore, 0.547 μg Cy3 ASO per well was used to account for the added molecular weight of the Cy3 in the Cy3 ASO. 24 hours after transfection, the cells were trypsinized, washed with PBS, incubated in cell scrub for 10 min, and analyzed via flow cytometry to measure Cy3 fluorescence of live cells using a similar method described in the Transfection Study section.
The complexes were formed by adding 50 μL micelle or linear polymer diluted at the corresponding N/P concentration to 50 μL ASO (1 μg ASO per well and 50 μL ASO stock per well). These were allowed to complex for 30 min. Next, 100 μL of QUANT-IT OLIGREEN (200x diluted from stock) was added and allowed to complex for 5 min. After, 66.6 μL of the complex QUANT-IT OLIGREEN mixture was transferred to another well and 133.3 μL of either PBS or FLUOROBRITE with 10% FBS. QUANT-IT OLIGREEN fluorescence intensity was measured on a Synergy H1 Hybrid Reader with excitation at 485 nm and emission at 528 nm (BioTek, Winooski, VT).
Working solutions of linear polymers, micelles, and ASO were filtered with a 0.2 μm hydrophilic polypropylene (GHP) filter. Complexes were formed by adding 33.3 μL micelle/polymer (at corresponding N/P ratio) to 33.3 μL ASO (0.6 μg/uL ASO). These were allowed to complex for 30 min. Then, 133.3 μL unfiltered DMEM with 10% FBS and 1% penicillin-streptomycin was added to each and let complex for 1 min. Note, there was not a difference in the DLS traces of filtered and unfiltered DMEM with 10% FBS and 1% penicillin-streptomycin, so unfiltered media was used to preserve FBS contents and avoid changes in concentration and/or shear-related denaturing. Complexes were then analyzed via DLS on a Wyatt DynaPro III instrument (Wyatt Technology, Santa Barbara, CA).
ASO concentration in the supernatant was compared after centrifugation to measure the amount of ASO that was in an aggregated state and “settled down”. To measure this, complexes were formed by adding linear polymers and micelles (100 μL) to Cy3-ASO (100 μL) (0.5 μg Cy3 ASO per centrifuged tube) at each designated N/P ratio. This was allowed to complex for 30 min. Next, 400 μL of FLUOROBRITE with 10% FBS was added to each complex solution. This was allowed to aggregate for 30 min. The vials were then centrifuged (15,700 rcf, 2 min). The supernatant was sampled (100 μL) and Cy3 fluorescence was measured on a Synergy H1 Hybrid Reader (Excitation: 488, Emission: 570). To normalize with respect to the ‘non-settled’ control, the vials were then vortexed for 2 min. 100 μL was sampled again from the solution and Cy3 fluorescence was measured on the plate reader. The relative amount of ASO in the supernatant was calculated as the ‘centrifuged fluorescence’ divided by the ‘vortexed fluorescence’ of each vial.
deGFP HEK cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), high glucose, GLUTAMAX Supplement (with high glucose, GLUTAMAX, and phenol Red, without sodium pyruvate) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin. Cells were kept in a 5% CO2 incubator at 37° C.
PFPA-nBa diblock copolymers were synthesized via our previously described in Example 1; N,N-dimethylethylenediamine, N-Boc-ethylenediamine, 4-(2-aminoethyl)morpholine; and trifluoroacetic acid were purchased from Sigma Aldrich (St. Louis, MO). GFP knockdown ASO (+T+T+GCCGGTGGTGCAGAT+A+A+A(SEQ ID NO: 1)) and GFP knockdown MM ASO (+G+G+AGTACACTATATCGG+T+G+G(SEQ ID NO: 2)) (+indicates a locked nucleic acid) were purchased from Integrated DNA Technologies (Coralville, IA). All ASOs had phosphorothioated backbones. Modified HEK cells that continually produce destabilized green fluorescent protein (deGFP HEK cells) were. CCK-8 kit was purchased from Dojindo Molecular Technologies (Rockville, MD). Dulbecco's Modified Eagle Medium (DMEM) high glucose GLUTAMAX, Fetal bovine serum, penicillin-streptomycin, trypsin-EDTA, phosphate-buffered saline, LIPOFECTAMINE 2000, OLIGOFECTAMINE, QUANT-IT OLIGREEN ssDNA Reagent, and calcein violet stain were from Invitrogen Life Technologies ThermoFisher Scientific (Carlsbad, CA). JETPEI was purchased through Polyplus (Illkirch-Graffenstaden, France). Bafilomycin-A1 was purchased from Cayman Chemical Company (Ann Arbor, MI).
The synthetic steps followed a similar procedure to Santa Chalarca, C. F.; Dalal, R. J.; Chapa, A.; Hanson, M. G.; Reineke, T. M. Cation Bulk and PKa Modulate Diblock Polymer Micelle Binding to PDNA. ACS Macro Lett. 2022, 588-594. doi.org/10.1021/acsmacrolett.2c00015. A PFPA-nBA diblock polymer was synthesized via RAFT chain extension of a PnBA macro-CTA. The PFPA-nBA diblock polymer was modified using either N-Boc-ethylenediamine(A-Boc protected), N,N-dimethylethylenediamine (D), or 4-(2-aminoethyl)morpholine (M) for aminolysis and HEA for thiocarboate cleavage and subsequent Michael addition in either 1,2-dioxane or DMF by stirring at 40° C. for 18 hours. The resulting solution was purified via dialysis in saline solution followed by Millipore water to yield the diblock polymers A-Boc protected, D, and M after freeze drying. The A-Boc protected polymer was deprotected by mixing with TFA for 2 hours followed by dialysis in saline solution followed by water to yield the A polymer after freeze drying.
The micelle formulation steps followed the general procedure outlined in Santa Chalarca, C. F.; Dalal, R. J.; Chapa, A.; Hanson, M. G.; Reineke, T. M. Cation Bulk and PKa Modulate Diblock Polymer Micelle Binding to PDNA. ACS Macro Lett. 2022, 588-594. doi.org/10.1021/acsmacrolett.2c00015. Polymers were directly dissolved in MOPS (20 mM, pH=7.0 100 ionic strength adjusted with NaCl) at 1 mg/mL in a 20 mL glass vial. This was stirred at room temperature for 7 days. After 7 days, the solutions were ultrasonicated for 2 hours.
Micelle size and dispersity were determined via DLS at 1 mg/mL concentrations on a Zetasizer Nano ZS (Malvern Instruments Limited, Worcestershire, UK, Model: ZEN3600).
The micelleplexes were formed mimicking the transfection procedure by adding 50 μL micelle solution diluted at the corresponding N/P concentration to 50 μL ASO solution (0.25 μg ASO per well and 50 μL ASO stock per well). These were both diluted in PBS. These were allowed to complex for 30 min. Complexes were then analyzed via DLS on a Wyatt DynaPro III instrument (Wyatt Technology, Santa Barbara, CA). For studies in DMEM with 10% FBS, 200 μL of DMEM with 10% FBS and 1% penstrep was added. Complexes were then analyzed again via DLS on a Wyatt DynaPro III instrument.
The micelleplexes were formed mimicking the transfection process. 50 μL micelle solution diluted at the corresponding N/P concentration was added to 50 μL ASO solution (0.25 μg ASO per well and 50 μL ASO stock per well). These were all diluted in PBS. These were allowed to complex for 30 min. Next, 0.5 μL of QUANT-IT OLIGREEN stock was added and allowed to complex for 5 min. Fluorescence intensity of the QUANT-IT OLIGREEN was measured on a Synergy H1 Hybrid Reader (excitation=485 nm, emission=528 nm) (BioTek, Winooski, VT). Next, if studied in DMEM with 10% FBS, 200 μL of DMEM with 10% FBS and 1% penstrep was added followed by chloroacetate buffer (if studied and at the concentration desired). Fluorescence intensity of the QUANT-IT OLIGREEN was measured again on the Synergy H1 Hybrid Reader (excitation=485 nm, emission=528 nm.
The two different diblocks were mixed in the powder form. This mixture was then directly dissolved in MOPS (20 mM, pH=7.0, 100 ionic strength adjusted with NaCl) at 1 mg/mL in a 20 mL glass vial. This was stirred at room temperature for 7 days. After 7 days, the solutions were ultrasonicated for 2 hours. DLS was used to analyze the size and dispersity of the micelles using a Zetasizer Nano ZS (Malvern Instruments Limited, Worcestershire, UK, Model: ZEN3600).
In a centrifuge tube, 50 μL of micelle solutions (concentration depended on N/P studied) made from either pre-formed homomicelles or blended diblock micelles or by mixing two solutions of homomicelles to form a mixed micelle was added to 50 μL of ASO solutions (5 ug/mL ASO so that each well contained 0.25 ug ASO (note: Example 1 studies were done at 0.5 ug ASO per well 22), pipette mixed, and then allowed to complex for 30 min to form the micelleplex solutions. Next, 200 μL of DMEM with 10% FBS and 1% penicillin-streptomycin containing 50,000 deGFP HEK cells was added to the 100 μL of micelleplex solution, chloroacetate (at designated concentration if studied) pipette mixed and added to the well in a 24 well plate. LIPOFECTMINE 2000 was formulated similarly to the commercial protocol however it was scaled down to 0.25 ug ASO per well. Additionally, 200 μL of DMEM with 10% FBS and 1% penicillin-streptomycin with 50,000 cells was added to the lipofectamine-ASO solution and this was mixed and added to the plate instead of adding it to adhered cells. For untreated cells, 200 μL of DMEM+10% FBS and 1% penicillin-streptomycin with 50,000 cells was added to 100 uL PBS (instead of micelleplex solution), mixed and added to each well. These cell and micelleplex mixtures were allowed to sit and settle for 30 minutes before being put in an 5% CO2, 37° C. incubator.
24 hours after introducing the cells to the micelleplexes, the media was changed out for 1 mL of DMEM containing 10% FBS and 1% penicillin-streptomycin. 72 hours after transfection, cells were analyzed via the CCK-8 viability assay following the commercial protocol. Additionally, the cells were analyzed via flow cytometry to measure GFP fluorescence. The forward and side scattering detectors were used to gate for cells and single cells and calcein violet staining was used to gate for live cells. GFP knockdown was analyzed using the mean fluorescence of GFP in the live, single cells. The decrease in GFP fluorescence was normalized to untreated cells to get a percent knockdown value where 100% knockdown meant no GFP fluorescence was observed.
Transfection conditions were identical to previous method; however, cells were preincubated with 200 nM Bafilomycin-A1 in DMEM containing 10% FBS and 1% penicillin-streptomycin for 30 minutes prior to transfection. After the pre-incubation step, cells were counted and transfected as described above.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims the benefit of U.S. Provisional Patent Application No. 63/544,654, filed Oct. 18, 2023, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63544654 | Oct 2023 | US |
