Classifications

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  • A61K9/00—Medicinal preparations characterised by special physical form
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  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
  • C07D211/10—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms
  • C07D211/14—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms with hydrocarbon or substituted hydrocarbon radicals attached to the ring nitrogen atom
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  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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  • A61P3/00—Drugs for disorders of the metabolism
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
  • C07D211/10—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms
  • C07D211/16—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms with acylated ring nitrogen atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
  • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
  • C07D241/04—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
  • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
  • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
  • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
  • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
  • C07D295/182—Radicals derived from carboxylic acids
  • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• lipid nanoparticle compositions of the disclosure may include one or more cationic and/or ionizable amino lipids, phospholipids including polyunsaturated lipids, PEG lipids, structural lipids, and/or therapeutic and/or prophylactic agents in specific fractions.
• nucleic acids The effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids represents a continuing medical challenge.
• nucleic acids the delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of such species.
• Lipid-containing nanoparticle compositions, liposomes, and lipoplexes have proven effective as transport vehicles into cells and/or intracellular compartments for biologically active substances such as small molecule drugs, proteins, and nucleic acids.
• Such compositions generally include one or more "cationic" and/or amino (ionizable) lipids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), and/or lipids containing polyethylene glycol (PEG lipids).
• Cationic and/or ionizable lipids include, for example, amine- containing lipids that can be readily protonated. Though a variety of such lipid-containing nanoparticle compositions have been demonstrated, improvements in safety, efficacy, and specificity are still lacking.
• the disclosure provides a compound having the formula (I)
• t 1 or 2;
• Ai and A 2 are each independently selected from CH or N;
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• Ri, R 2 , R3, R4, and R5 are independently selected from the group consisting of C5 -2 o alkyl, C 5 - 20 alkenyl, -R"MR', -R*YR", -YR", and -R*OR";
• each M is independently selected from the group consisting of
• X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, -CH 2 -, -(CH 2 ) 2 -, -CHR-, -CHY-, -C(O)-, -C(0)0-, -OC(O)-, -C(0)-CH 2 -, -CH 2 -C(0)-,
• each Y is independently a C3-6 carbocycle
• each R* is independently selected from the group consisting of Ci_i 2 alkyl and C 2-12 alkenyl;
• each R is independently selected from the group consisting of C 1-3 alkyl and a C3-6 carbocycle; each R' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H; and
• each R" is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl,
• R 1 at least one of R 1; R 2 , R 3 , R 4 , and R 5 is -R"MR'.
• the compounds of formula (I) may include one or more of the following features when applicable.
• the compound is of any of formulae (Ial)-(Ia6):
• At least one of X 1 , X 2 , and X 3 is not -CH 2 -.
• X 1 is not -CH 2 -.
• at least one of X ⁇ X 2 , and X 3 is - C(O)-.
• X 3 is a bond while each of X 1 and X 2 is not a bond. In some embodiments, none of X 2 , and X 3 is a bond.
• Ri and R2 are the same.
• R 1; R2, and R3 are the same.
• R4 and R5 are the same.
• R 1; R 2 , R3, R4, and R5 are the same.
• At least one of R 1; R 2 , R3, R4, and R5 is -R"MR' . In some embodiments, at most one of R 1; R 2 , R3, R4, and R5 is -R"MR' . For example, at least one of R 1; R 2 , and R3 may be -R"MR', and/or at least one of R4 and R5 is -R"MR'. In certain embodiments,
• At least one M is -C(0)0-. In some embodiments, each M is -C(0)O. In some embodiments, at least one M is -OC(O)-. In some embodiments, each M is -OC(O)- In some embodiments, at least one R" is C3 alkyl. In certain embodiments, each R" is C3 alkyl. In some embodiments, at least one R" is C5 alkyl. In certain embodiments, each R" is C5 alkyl. In some embodiments, at least one R" is Ce alkyl. In certain embodiments, each R" is Ce alkyl. In some embodiments, at least one R" is C7 alkyl. In certain embodiments, each R" is C7 alkyl.
• At least one R' is C5 alkyl. In certain embodiments, each R' is C5 alkyl. In other embodiments, at least one R' is Ci alkyl. In certain embodiments, each R' is Ci alkyl. In some embodiments, at least one R' is C2 alkyl. In certain embodiments, each R' is C2 alkyl.
• R 1; R 2 , R3, R4, and R5 is C 12 alkyl. In certain embodiments, each of R 1; R 2 , R3, R4, and R5 are C 12 alkyl. [0013] In another aspect, the disclosure provides a compound having formula (II):
• Ai and A 2 are each independently selected from CH or N and at least one of Ai and A 2 is N;
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• Ri, R 2 , R3, R4, and R5 are independently selected from the group consisting of Ce-20 alkyl and Ce-20 alkenyl;
• Ri, R 2 , R3, R4, and R5 are the same, wherein Ri is not C 12 alkyl, Cig alkyl, or Cig alkenyl;
• R 1 only one of R 1; R 2 , R3, R4, and R5 is selected from Ce-20 alkenyl;
• R 1; R 2 , R3, R4, and R5 have a different number of carbon atoms than at least one other of R 1; R 2 , R3, R4, and R5;
• Ri are selected from Ce-2o alkenyl
• R4 and R5 are selected from Ce-2o alkyl
• Ri, R 2 , and R3 are selected from Ce-2o alkyl, and R4 and R5 are selected from Ce-2o alkenyl.
• the compounds of formula (II) may include one or more of the following features when applicable. [0015] In some embodiments, the compound is of formula (Ila):
• R 1; R2, R3, R4, and R5 are the same, and are not C 12 alkyl, C 18 alkyl, or C 18 alkenyl.
• Ri, R2, R3, R4, and R5 are the same and are C9 alkyl or Ci4 alkyl.
• R 1; R2, R3, R4, and R5 are selected from Ce-20 alkenyl.
• R 1; R 2 , R3, R4, and R5 have the same number of carbon atoms.
• R4 is selected from C5-2 0 alkenyl.
• R4 may be C 12 alkenyl or Ci 8 alkenyl.
• At least one of R 1; R 2 , R3, R4, and R5 have a different number of carbon atoms than at least one other of Ri, R2, R3, R4, and R5.
• R 1; R 2 , and R3 are selected from Ce-20 alkenyl, and R4 and R5 are selected from Ce-20 alkyl. In other embodiments, R 1; R 2 , and R3 are selected from Ce-20 alkyl, and R4 and R5 are selected from Ce-20 alkenyl. In some embodiments, R 1; R 2 , and R3 have the same number of carbon atoms, and/or R4 and R5 have the same number of carbon atoms. For example, R 1; R 2 , and R3, or R4 and R5, may have 6, 8, 9, 12, 14, or 18 carbon atoms. In some embodiments, R 1; R 2 , and R3, or R4 and R5, are C 18 alkenyl (e.g., linoleyl). In some
• R 1; R 2 , and R3, or R4 and R5, are alkyl groups including 6, 8, 9, 12, or 14 carbon atoms.
• Ri has a different number of carbon atoms than R 2 , R3, R4, and R5.
• R3 has a different number of carbon atoms than R 1; R 2 , R4, and R5.
• R4 has a different number of carbon atoms than R 1; R 2 , R3, and R5.
• the disclosure provides a compound according to formula (III):
• a 3 is CH or N
• a 4 is CH 2 or NH; and at least one of A 3 and A 4 is N or NH;
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• Ri, R-2, and R3 are independently selected from the group consisting of C5-20 alkyl, C5-20 alkenyl, -R"MR', -R*YR", -YR", and -R*OR";
• each M is independently selected
• X 1 and X 2 are independently selected from the group consisting of -CH 2 -,
• each Y is independently a C3-6 carbocycle
• each R* is independently selected from the group consisting of Ci-12 alkyl and C 2-12 alkenyl;
• each R is independently selected from the group consisting of C 1-3 alkyl and a C3-6 carbocycle;
• each R' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
• each R" is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
• the compounds of formula (III) may include one or more of the following features when applicable.
• the compound is of formula (Ilia):
• R 1; R2, and R3 are independently selected from the group consisting of C5-2 0 alkyl and C5-2 0 alkenyl. In some embodiments, R 1; R2, and R3 are the same. In certain embodiments, R 1; R 2 , and R3 are C 6 , C9, C 12 , or C 14 alkyl. In other embodiments, R 1; R 2 , and R3 are C 18 alkenyl. For example, R 1; R 2 , and R3 may be linoleyl.
• At least one of X 1 and X 2 is not -CH 2 -.
• X 1 is not -CH 2 -.
• at least one of X 1 and X 2 is -C(O)-.
• the disclosure provides a compound according to formula (lb):
• a 6 and A 7 are each independently selected from CH or N, wherein at least one of A 6 and A 7 is N;
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• X 4 and X 5 are independently selected from the group consisting of -CH 2 -,
• Ri, R2 , R3, R 4 , and R5 each are independently selected from the group consisting of C5-2 0 alkyl and C5-20 alkenyl, -R"MR', -R*YR", -YR", and -R*OR";
• each M is independently selected from the group consisting of
• each R is independently selected from the group consisting of C 1-3 alkyl and a C3-6 carbocycle;
• each R' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
• each R" is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
• the compounds of formula (lb) may include one or more of the following features when applicable.
• Ri and R2 are the same.
• R 1; R 2 , and R3 are the same.
• R4 and R5 are the same.
• R 1; R 2 , R3, R4, and R5 are the same.
• At least one of R 1; R 2 , R 3 , R4, and R5 is C9-12 alkyl. In certain embodiments, each of R 1; R 2 , R 3 , R4, and R5 independently is C9, C 12 or C 14 alkyl. In certain embodiments, each of R 1; R 2 , R 3 , R 4 , and R5 is C9 alkyl.
• Ae is N and A7 is N. In some embodiments, Ae is CH and A7 is N.
• X 4 is-CH 2 - and X 5 is -C(O)-. In some embodiments, X 4 and X 5 are -C(O)-.
• the compound has the formula (IV)
• the disclosure provides a compound having the formula (17-1):
• Ri a is -(CH2) n a Q a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 ) n aN(R a ) 2 , -C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)R a , and -N(R a )S(0) 2 R a and each na is independently selected from 1, 2, 3, 4, and 5;
• R 2a and R3 3 are each independently selected from the group consisting of C3 -2 4 alkyl, C3- 24 alkenyl, -R a *Y a R a ", -Y a R a ", and -R a *OR a ";
• each Y a is independently a C3-6 carbocycle
• each R a * is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• each R a is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H;
• each R a " is selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl;
• R 2a includes 7 or fewer carbon atoms.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n a is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• R3 3 includes 7 or fewer carbon atoms.
• the disclosure provides a compound having the formula (17-1)
• Ria is -(CH 2 ) n aQ a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 ) n aN(R a ) 2 , - C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)R a , and -N(R a )S(0) 2 R a and each n a is independently selected from 1, 2, 3, 4, and 5;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• R 2a is selected from the group consisting of Cg -2 4 alkenyl
• R3a is selected from the group consisting of Cg -2 4 alkyl
• each R a is independently selected from the group consisting of C1-3 alkyl, C 2- 3 alkenyl, and H.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, ' is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• R3 3 is an alkyl including 9, 12, 14, or 18 carbon atoms.
• R 2a is Ci8 alkenyl (e.g., linoleyl).
• the disclosure provides a compound having the formula (17-1)
• Ria is -(CH 2 ) n aQ a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 ) n aN(R a ) 2 , - C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R)C(0)R a , and -N(R a )S(0) 2 R a and each n a is independently selected from 1, 2, 3, 4, and 5;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• R 2a is selected from the group consisting of Ci 3-2 o alkyl
• R3a is selected from the group consisting of Cg -2 o alkyl
• each R a is independently selected from the group consisting of C1-3 alkyl, C 2- 3 alkenyl, and H.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n a is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• R_2 a and R_3 A are the same.
• R_2 a and/or R 3a is CM alkyl.
• the disclosure provides a compound having the formula (17-1)
• Ri a is -(CH 2 ) n a Q a , where Q a is -OR a , R a is selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H, and n a is selected from 1, 2, 3, 4, and 5; and
• R2 a and R3 a are each independently selected from the group consisting of Cg-2o alkenyl, wherein
• R a is selected from the group consisting of C 1-3 alkyl and C2-3 alkenyl; or ii) Ri a is -(CH 2 )20H, and R2 a and R 3a each include one or fewer double bonds.
• R a is H. In other embodiments, R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n a is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• Ri a is -(CH 2 )20CH 3 . In other embodiments, Ri a is -(CH 2 )20H.
• R2 a is C 18 alkenyl (e.g., linoleyl).
• R 3a is Ci8 alkenyl (e.g., linoleyl).
• R2 a and R 3a are the same.
• the disclosure provides a compound of formula (17-1)
• Ria is -(CH 2 ) n aQ a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 ) n aN(R a ) 2 , - C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)R a , and -N(R a )S(0) 2 R a and each n a is independently selected from 1, 2, 3, 4, and 5;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• R 2a is selected from the group consisting of Cg-i 2 alkyl
• R3a is selected from the group consisting of Cg -2 o alkyl
• each R a is independently selected from the group consisting of C1-3 alkyl, C 2- 3 alkenyl, and H.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n a is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• Q a is -OR a and n a is selected from 2, 3, and 4.
• R 2a is C9 alkyl. In other embodiments, R 2a is C 12 alkyl.
• R 2a and R3 3 are the same.
• the disclosure provides a compound having the formula (19-1),
• Rib is selected from the group consisting of H, C1-5 alkyl, C2-5 alkenyl, -R b "M b R b ' , a C3-. carbocycle, -(CH 2 ) n Q b , and -(CH 2 ) n CHQ b R b , where Q b is selected from a heterocycle, -OR b , - 0(CH 2 ) n N(R b ) 2 , -C(0)OR b , -OC(0)R b , -CX b 3 , -CX b 2 H, -CX b 3 ⁇ 4, -CN, -N(R b ) 2 ,
• n is independently selected from 1, 2, 3, 4, and 5;
• R 2 b and R3b are independently selected from the group consisting of C 1-2 o alkyl, C 2-2 o alkenyl, -R b "MR b ' , -R b *YR b ", -YR b “, and -R b *OR b "; each M b is independently selected from the group consisting
• W is selected from the group consisting of -CH 2 -, -CHR b -, -C(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-;
• each X b is independently selected from the group consisting of F, CI, Br, and I;
• each Y b is independently a C 3 -6 carbocycle
• each R b * is independently selected from the group consisting of Ci-12 alkyl and Ci-12 alkenyl;
• each R b is independently selected from the group consisting of C1-3 alkyl, a C3-6 carbocycle, C2-3 alkenyl, and H;
• each R b ' is independently selected from the group consisting of Ci-12 alkyl, C 2-12 alkenyl, and H;
• each R b " is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
• W is not -CH 2 -. In particular such embodiments, W is -C(O)-.
• At least one of R2b and R3b is -R b "M b R b '. In certain embodiments, at least one M b is -C(0)0-. In some embodiments, at least one R b " is C5 alkyl. In certain embodiments, at least one R b ' is C 5 alkyl.
• R2b and/or R 3 b are selected from the group consisting of C 1-2 o alkyl.
• R2b and/or R 3 b may be alkyl groups including 9 or 12 carbon atoms.
• R2b and/or R 3 b are selected from the group consisting of C2-2 0 alkenyl.
• R2b and/or R 3 b may be alkenyl groups including 18 carbon atoms (e.g., linoleyl groups).
• R2b and R 3 b are the same.
• Rib is H, while in other embodiments, Rib is selected from C1-5 alkyl.
• Rib may be Ci alkyl.
• R3 ⁇ 4 is -(CH 2 ) n Q b
• Q b is a heterocycle such as a phenyl group.
• Q b may be a phenyl group with one or more substituents, as described herein.
• Rib is selected from the group consisting of Ce-20 alkyl
• R2b and R 3 b are independently selected from the group consisting of Ce-2o
• Rib is C 12 alkyl.
• R2b and/or R 3 b are C 18 alkenyl (e.g., linoleyl).
• R2b and are both linoleyl.
• the disclosure provides a compounds of formula (20-1):
• Ric is selected from the group consisting of a C3-6
• n c is independently selected from 1 , 2, 3, 4, and 5;
• R2c, R3c, and R4c are independently selected from the group consisting of C 1-2 o alkyl, C 2- 2 0 alkenyl, -R C "M C R C ', -R C *Y C R C ", -Y C R C “, and -R c *OR c ";
• each M c is independently selected from the group consisting of:
• each X c is independently selected from the group consisting of F, CI, Br, and I;
• each Y c is independently a C3-6 carbocycle
• each R c * is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl; each R c is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H;
• each R c ' is independently selected from the group consisting of C 1-12 alkyl, C2-12 alkenyl, and H;
• each R c " is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl,
• Ric is selected from the group consisting of a C3-6
• n c is independently selected from 1, 2, 3, 4, and 5;
• R 2c , R 3c , and R 4c is -R C "M C R C ' .
• Ri is selected from the group consisting of
• Ri c is independently selected from 1, 2, 3, 4, and 5.
• Ri c is independently selected from 1, 2, 3, 4, and 5.
• n c is 2.
• Q c is -C(0)OR c , where R c is, for example, H.
• At least one of R2 C , R 33 ⁇ 4 and R4 C is -R C "M C R C ' .
• R2 C , R 3 c and/or R4 C may be -R C "M C R C ' .
• at least one M c is -C(0)0-.
• each M c is -C(0)O.
• at least one R c " is C5 or C7 alkyl.
• each R c " is C5 alkyl.
• each R c " is C7 alkyl.
• at least one R c ' is C5, C 7 , or C9 alkyl.
• each R c ' is C5 alkyl. In other embodiments, each R c ' is C7 alkyl. In other embodiments, each R c ' is C9 alkyl. In some embodiments, R c ' is branched.
• R2 C , R 3C , and R 4c are selected from the group consisting of C5-20 alkyl. In certain embodiments, R2 C , R 3C , and R4 C are C 12 alkyl.
• R2 C is selected from the group consisting of C5-20 alkyl and C5-20 alkenyl.
• R2 C may be C 12 alkyl.
• R_3 C is selected from the group consisting of C5-20 alkyl and C5-20 alkenyl.
• R.3 C may be C 6 , C9, or C 12 alkyl.
• R4c is selected from the group consisting of C5-20 alkyl and C5-20 alkenyl.
• R 4c may be C 6 , C9, or C 12 alkyl.
• R.3 C and R4 C are the same.
• the disclosure provides a compound according to formula (20-1):
• Ri is selected from the group consisting of -(CH 2 ) ⁇ Q C , -(CH 2 ) ⁇ CHQ C R C , -CHQ C R C , and -CQ C (R C ) 2 , where Q c is selected from -OR c , -CN, and -N(R C ) 2 , and n c is selected from 1 , 2, 3, 4, and 5;
• R2c and R3 C are independently selected from the group consisting of Ce-20 alkyl and Ce-20 alkenyl;
• R4c is selected from the group consisting of C 13-2 0 alkyl and C5-2 0 alkenyl; and each R c is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H.
• R 3C is C 14 alkyl.
• R 4c is C 14 alkyl.
• R 3C is C 18 alkenyl.
• R 3C may be linoleyl.
• R4 C is C 18 alkenyl.
• R4 C may be linoleyl.
• R2 C is C 12 alkyl. In other embodiments, R2 C is C 14 alkyl. In some embodiments, R2 C is C 18 alkenyl. For example, R2 C may be linoleyl.
• R 3C and R4 C are the same.
• Ri c is -(CH 2 ) n c Q c .
• Q c is -OR c .
• Q c may be -OH.
• n c is 2 or 3.
• Ri is selected from the group consisting of -(CH 2 ) n c Q c , -(CH 2 ) ⁇ CHQ C R C , -CHQ C R C , and -CQ C (R c ) 2 , where Q c is selected from -OR c , -CN, and -N(R C ) 2 , and n c is selected from 1 , 2, 3, 4, and 5;
• R2c, R3c, and R4c are independently selected from the group consisting of C6 -2 o alkyl and C6- 2 o alkenyl;
• each R c is independently selected from the group consisting of C 1-3 alkyl, C 2- 3 alkenyl, and H;
• R 2c is selected from the group consisting of Ci-n alkyl and C 2- 5 alkenyl
• R3c is selected from the group consisting of Ci-n alkyl and C 2- 5 alkenyl
• R 2c is selected from the group consisting of Ci-n alkyl and C 2- 5 alkenyl.
• R 2c may be Ce or C9 alkyl.
• Ci-n alkyl and C 2- 5 alkenyl are selected from the group consisting of Ci-n alkyl and C 2- 5 alkenyl.
• C 2- 5 alkenyl may be Ce or C9 alkyl.
• R 2c is C 12 alkyl.
• R4 C is C 6 , C9, or C 12 alkyl.
• Ri c is -(CH 2 ) n cQ c .
• Q c is -OR c .
• R c is H.
• n c is 2 or 3.
• the disclosure provides a compound according to formula (20-1):
• Ri c is selected from the group consisting of -(CH 2 ) n cQ c , -(CH 2 ) n ' ; CHQ c R c , -CHQ C R C , and -CQ C (R C ) 2 , where Q c is selected from -OR c , -CN, and -N(R C ) 2 , and n c is selected from 1 , 2, 3, 4, and 5;
• R 2c is selected from the group consisting of H, Ci 2-2 o alkyl, and Ce-20 alkenyl;
• R3c and R4 C are C 12 alkyl
• each R c is independently selected from the group consisting of C 1-3 alkyl, C 2- 3 alkenyl, and H.
• R 2c is H. In other embodiments, R 2c is C 12 alkyl or alkenyl. In some embodiments, R 2c is C M alkyl. In other embodiments, R 2c is C 18 alkenyl. For example, R 2c may be linoleyl.
• Ri c is -(CH 2 ) n cQ c .
• Q c is -OR c .
• Q c may be OH.
• n c is 2, 3, or 4.
• the disclosure provides compounds of formula (21 -1):
• Rid and R 2 d are independently selected from the group consisting of H, C1-5 alkyl, and C 2- 5 alkenyl;
• n d is selected from 1, 2, 3, 4, and 5;
• k is selected from 0, 1 , 2, and 3;
• R 3 d and R4d are independently selected from the group consisting of C 1-2 o alkyl, C 2-2 o alkenyl,
• each M d is independently selected from the group consisting of -C(0)0-, -OC(O)-, -C(0)N(R d ')-, -N(R d ')C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(OR d ')0-, and -S(0) 2 -, or is absent;
• each R d ' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H; each Y d is independently a C3-6 carbocycle; each R * is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
• each R d ' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
• each R d " is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl,
• R 3d and R4d are bound to either i) the same carbon atom or ii) adjacent carbon atoms.
• R 3d and R4d are bound to the same carbon atom.
• R 3d and R4d may be bound to a carbon atom adjacent to C*.
• R 3d and R4d are not bound to a carbon atom adjacent to C*.
• R 3d and R4d are bound to adjacent carbon atoms. In certain embodiments, one or both of R 3d and R4d are bound to carbon atoms adjacent to C*.
• k is 0. In other embodiments, k is 1, 2, or 3.
• M d is absent. In other embodiments, M d is selected from the group consisting of
• M d is -C(0)0-.
• n d is 1, 2, or 3.
• Ri d and/or R 2d are selected from C1-5 alkyl. In certain embodiments, Ri d and/or R 2d are Ci alkyl.
• R 3d and/or R4d are selected from C2-20 alkenyl. In certain embodiments, R 3d and/or R4d are alkenyl groups including 17, 18, or 19 carbon atoms. For example, R 3d and/or R4d may be C 18 alkenyl groups (e.g., linoleyl).
• the disclosure features a nanoparticle composition including a lipid component comprising a compound as described above (e.g., a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1)).
• the lipid component of the nanoparticle composition includes a phospholipid.
• a phospholipid of a nanoparticle composition includes a phospholipid moiety and one or more fatty acid moieties, one or more of which may be unsaturated.
• a nanoparticle composition may include a lipid according to formula (V)
• R p represents a phospholipid moiety and Ri and R 2 represent unsaturated fatty acid moieties that may be the same or different.
• a phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
• a fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
• a phospholipid is selected from the group consisting of
• DLPC l,2-dilinoleoyl-sn-glycero-3-phosphocholine
• DMPC 1,2-dimyristoyl-sn-glycero-phosphocholine
• DOPC l,2-dioleoyl-sn-glycero-3-phosphocholine
• DPPC l,2-dipalmitoyl-sn-glycero-3-phosphocholine
• DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
• POPC l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
• OChemsPC l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
• the phospholipid is DOPE. In other embodiments, the phospholipid is DSPC.
• Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
• the lipid component of the nanoparticle composition includes a structural lipid.
• a structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.
• the structural lipid is cholesterol.
• the lipid component of the nanoparticle composition includes a PEG lipid.
• the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, and a PEG-modified dialkylglycerol.
• the nanoparticle composition includes a lipid component comprising a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1), a phospholipid (which may or may not be unsaturated), a PEG lipid, and a structural lipid.
• a lipid component comprising a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1), a phospholipid (which may or may not be unsaturated), a PEG lipid, and a structural lipid.
• the lipid component of the nanoparticle composition includes about 30 mol % to about 60 mol % compound of one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21- I), about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid.
• the lipid component of the nanoparticle composition includes about 30 mol % to about 45 mol % compound of one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1), about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid.
• the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % compound of one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1), about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid.
• the lipid component includes about 50 mol % said compound, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In other embodiments, the lipid component includes about 40 mol % said compound, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In some of these embodiments, the phospholipid is DOPE, while in other embodiments the phospholipid is DSPC. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the PEG lipid is PEG-DMG. In any of the above, the total content of the lipid component may not exceed 100%.
• the nanoparticle composition includes more than one phospholipid, PEG lipid, structural lipid, or other lipid. In certain embodiments, the
• nanoparticle composition further includes a cationic and/or ionizable lipid such as an amino- lipid.
• a cationic and/or ionizable lipid is selected from the group consisting of 3-(didodecylamino)-Nl,Nl,4-tridodecyl-l-piperazineethanamine (KL10),
• DODMA l,2-dioleyloxy-N,N-dimethylaminopropane
• the nanoparticle composition includes a therapeutic and/or prophylactic agent.
• the therapeutic and/or prophylactic agent may be selected from the group consisting of a protein, a small molecule drug, a cytotoxic agent, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or a nucleic acid (such as a deoxyribonucleic acid or a ribonucleic acid).
• the therapeutic and/or prophylactic agent is a ribonucleic acid (RNA).
• RNA may be selected from the group consisting of a small interfering RN A (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsR A), a small hairpin RNA (shRNA), a messenger RNA (niRNA), and mixtures thereof
• the therapeutic and/or prophylactic agent is a messenger RNA (mRNA).
• RNA of a nanoparticle composition may be naturally or non-naturally occurring and may include one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and/or a 5' cap structure.
• the nanoparticle composition includes more than one therapeutic and/or prophylactic agent, such as one or more RNAs.
• the therapeutic and/or prophylactic agents may be of the same or different types (e.g., two mRNAs, two siRNAs, one mRNA and one siRNA, one mRNA and one small molecule drug, etc.).
• the encapsulation efficiency of a therapeutic and/or prophylactic agent of a nanoparticle composition is at least 50%. In certain embodiments, the encapsulation efficiency is at least 80%. In certain embodiments, the encapsulation efficiency is greater than 90%.
• the wt wt ratio of the lipid component to a therapeutic and/or prophylactic agent in the nanoparticle composition is from about 10: 1 to about 60: 1. In certain embodiments, the wt/wt ratio is about 20: 1.
• the N:P ratio of the nanoparticle composition is from about 2: 1 to about 30:1. In certain embodiments, the N:P ratio is from about 2: 1 to about 8: 1. In certain embodiments, the N:P ratio is from about 5: 1 to about 8: 1. For example, the N:P ratio may be about 5.0:1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1.
• the mean size of a nanoparticle composition is from about 40 nm to about 150 nm. In certain embodiments, the mean size is from about 70 nm to about 100 nm. In one embodiment, the mean size may be about 80 to about 100 nm. In certain embodiments, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.
• the polydispersity index of the nanoparticle composition is from about 0 to about
• the polydispersity index is from about 0.10 to about 0.20.
• the nanoparticle composition has a zeta potential of about - 10 mV to about +20 mV.
• the cell uptake of the compound or nanoparticle composition is LDLR-independent.
• the cell uptake of the compound or nanoparticle composition is LDLR-dependent.
• the cell uptake of the compound or nanoparticle composition is apoE-independent.
• the cell uptake of the compound or nanoparticle composition is apoE-dependent. In some embodiments, the cell uptake of the compound or nanoparticle composition is LDLR- apoE-interaction independent. In some embodiments, the cell uptake of the compound or nanoparticle composition is LDLR-apoE-interaction dependent.
• the production of the polypeptide is higher in mammalian hepatocytes than cells from a different tissue (e.g., spleen or kidney).
• the production of the polypeptide occurs substantively in mammalian hepatocytes (e.g., little or no production of the polypeptide in other cells, e.g., spleen cells or renal cells).
• the nanoparticle composition includes one or more other components including, but not limited to, one or more pharmaceutically acceptable excipients, small hydrophobic molecules, therapeutic and/or prophylactic agents, carbohydrates, polymers, permeability enhancing molecules, buffers, and surface altering agents.
• the disclosure features a pharmaceutical composition comprising a nanoparticle composition according to the preceding aspects and a
• the pharmaceutical composition is refrigerated or frozen for storage and/or shipment (e.g., being stored at a temperature of 4 °C or lower, such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C).
• a temperature of 4 °C or lower such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C,
• the pharmaceutical composition is a solution that is refrigerated for storage and/or shipment at, for example, about -20° C, -30 °C, - 40 °C, -50 °C, -60 °C, -70 °C, or -80 °C.
• the disclosure provides a method of delivering a therapeutic and/or prophylactic agent (e.g., an mRNA) to a cell (e.g., a mammalian cell).
• a therapeutic and/or prophylactic agent e.g., an mRNA
• This method includes the step of administering to a subject (e.g., a mammal, such as a human) a nanoparticle composition including (i) a lipid component including a phospholipid (such as a polyunsaturated lipid), a PEG lipid, a structural lipid, and a compound according to one of formulae (I), (Ial)- (Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1) and (ii) a therapeutic and/or prophylactic agent, in which administering involves contacting the cell with the nanoparticle composition, whereby the therapeutic and/or prophy
• the disclosure provides a method of producing a polypeptide of interest in a cell (e.g., a mammalian cell).
• the method includes the step of contacting the cell with a nanoparticle composition including (i) a lipid component including a phospholipid (such as a polyunsaturated lipid), a PEG lipid, a structural lipid, and a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1) and (ii) an mRNA encoding the polypeptide of interest, whereby the mRNA is capable of being translated in the cell to produce the polypeptide.
• a lipid component including a phospholipid (such as a polyunsaturated lipid), a PEG lipid, a structural lipid, and a compound according to one of formulae (I), (Ial)-
• the disclosure provides a method of treating a disease or disorder in a mammal (e.g., a human) in need thereof.
• the method includes the step of administering to the mammal a therapeutically effective amount of a nanoparticle composition including (i) a lipid component including a phospholipid (such as a polyunsaturated lipid), a PEG lipid, a structural lipid, and a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1) and (ii) a therapeutic and/or prophylactic agent (e.g., an mRNA).
• a lipid component including a phospholipid (such as a polyunsaturated lipid), a PEG lipid, a structural lipid, and a compound according to one of formulae (I), (Ial)-(Ia6),
• the disease or disorder is characterized by dysfunctional or aberrant protein or polypeptide activity.
• the disease or disorder is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.
• the disclosure provides a method of delivering (e.g., specifically delivering) a therapeutic and/or prophylactic agent to a mammalian organ (e.g., a liver, spleen, lung, or femur).
• a mammalian organ e.g., a liver, spleen, lung, or femur.
• This method includes the step of administering to a subject (e.g., a mammal) a nanoparticle composition including (i) a lipid component including a phospholipid, a PEG lipid, a structural lipid, and a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1) and (ii) a therapeutic and/or prophylactic agent (e.g., an mRNA), in which administering involves contacting the cell with the nanoparticle composition, whereby the therapeutic and/or prophylactic agent is delivered to the organ such as liver.
• a subject e.g., a mammal
• a nanoparticle composition including (i) a lipid component including a phospholipid, a PEG lipid, a structural lipid, and a compound according to one of formulae (I), (Ial
• the disclosure features a method for the enhanced delivery of a therapeutic and/or prophylactic agent (e.g., an mRNA) to a target tissue (e.g., a liver, spleen, lung, or femur).
• a therapeutic and/or prophylactic agent e.g., an mRNA
• a target tissue e.g., a liver, spleen, lung, or femur.
• This method includes administering to a subject (e.g., a mammal) a nanoparticle composition, the composition including (i) a lipid component including a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1), a phospholipid, a structural lipid, and a PEG lipid; and (ii) a therapeutic and/or prophylactic agent, the administering including contacting the target tissue with the nanoparticle composition, whereby the therapeutic and/or prophylactic agent is delivered to the target tissue.
• a lipid component including a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1), a phospholipid, a structural
• the delivery is enhanced as compared to a reference composition which comprises a reference lipid instead of a compound of one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21- I).
• the disclosure features a method of lowering
• the immunogenicity comprising introducing the nanoparticle composition of the disclosure into cells, wherein the nanoparticle composition reduces the induction of the cellular immune response of the cells to the nanoparticle composition, as compared to the induction of the cellular immune response in cells induced by a reference composition which comprises a reference lipid instead of a compound of one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1).
• the cellular immune response is an innate immune response, an adaptive immune response, or both.
• a cell contacted in a method is in a mammal.
• a mammal may be, for example, a rodent, non- human primate, or a human.
• the mammal is a human.
• the mammal is LDLR-deficient, or apoE-deficient, or both.
• the mammal is not LDLR-deficient.
• the mammal is not apoE-deficient.
• the mammal is neither LDLR-deficient nor apoE- deficient.
• the mammal has an abnormal LDLR-apoE interaction.
• the mammal has a normal LDLR-apoE interaction.
• a therapeutic and/or prophylactic agent may be an mRNA.
• the therapeutic and/or prophylactic agent may be specifically delivered to a target tissue of interest (e.g., a mammalian liver, spleen, lung, or femur).
• a polypeptide of interest may be specifically produced in a target cell or tissue of interest (e.g., a hepatocyte, a mammalian liver, spleen, lung, or femur), e.g., the production of polypeptide is substantively higher in the target cell or tissue than in a non-target cell/tissue.
• a target cell or tissue of interest e.g., a hepatocyte, a mammalian liver, spleen, lung, or femur
• the nanoparticle composition is administered
• a dose of about 0.001 mg/kg to about 10 mg/kg of therapeutic and/or prophylactic agent is administered to a mammal in certain embodiments.
• the delivery (e.g., delivery efficiency) of the therapeutic and/or prophylactic agent to the mammalian cell is LDLR- independent. In some embodiments, the delivery of the therapeutic and/or prophylactic agent to the mammalian cell is LDLR-dependent. In some embodiments, the delivery of the therapeutic and/or prophylactic agent to the mammalian cell is apoE-independent. In some embodiments, the delivery of the therapeutic and/or prophylactic agent to the mammalian cell is apoE- dependent. In some embodiments, the delivery of the therapeutic and/or prophylactic agent to the mammalian cell is LDLR-apoE-interaction independent. In some embodiments, the delivery of the therapeutic and/or prophylactic agent to the mammalian cell is LDLR-apoE-interaction dependent.
• the production (e.g., yield) of the polypeptide of interest in the mammalian cell is LDLR-independent. In some embodiments, the production of the polypeptide of interest in the mammalian cell is LDLR- dependent. In some embodiments, the production of the polypeptide of interest in the mammalian cell is apoE-independent. In some embodiments, the production of the polypeptide of interest in the mammalian cell is apoE-dependent. In some embodiments, the production of the polypeptide of interest in the mammalian cell is LDLR-apoE-interaction independent. In some embodiments, the production of the polypeptide of interest in the mammalian cell is LDLR-apoE-interaction dependent.
• one or more nanoparticle compositions each including one or more therapeutic and/or prophylactic agents may be used in combination.
• one or more nanoparticle compositions each including one or more therapeutic and/or prophylactic agents may be simultaneously contacted with a cell or delivered to a mammalian cell or organ.
• the one or more nanoparticle compositions are contacted with a cell or delivered to a mammalian cell or organ at different times.
• one or more additional therapeutic and/or prophylactic agents or compounds may be used in combination with a nanoparticle composition including a therapeutic and/or prophylactic agent.
• an additional therapeutic and/or prophylactic agent or compound may be administered at or near the same time as a nanoparticle composition (e.g., within one hour).
• an additional therapeutic and/or prophylactic agent or compound may be administered before or after (e.g., one or more hours before or after) a nanoparticle composition as a pretreatment or post-treatment therapy.
• an additional therapeutic and/or prophylactic agent or compound is selected from the group consisting of an anti -inflammatory compound, a steroid (e.g., a corticosteroid), a statin, an estradiol, a BTK inhibitor, an S 1P1 agonist, a glucocorticoid receptor modulator (GRM), or an anti -histamine.
• an additional therapeutic and/or prophylactic agent or compound is selected from the group consisting of dexamethasone, methotrexate, acetaminophen, an HI receptor blocker, or an H2 receptor blocker.
• the patent or application file contains at least one drawing executed in color.
• Figure 1 is a pair of graphs comparing luciferase expression levels in mice (whole body) after administration of nanoparticle compositions containing compounds of the disclosure over time.
• Figure 2 is a graph summarizing luciferase expression levels at 3 h after administration of nanoparticle compositions containing compounds of the disclosure. Total light flux values were acquired via body luminescent imaging (BLI) 3 h after administration.
• the numbers 1-12 refer to the compositions containing Compounds 42-52 and MC3 respectively.
• Figure 3 is a graph summarizing luciferase expression levels at 6 hr after administration of nanoparticle compositions containing compounds of the disclosure. Total light flux values were acquired via BLI 6 h after administration.
• the numbers 1-12 refer to the compositions containing Compounds 42-52 and MC3 respectively.
• Figure 4 is graph summarizing luciferase expression levels at 24 h after administration of nanoparticle compositions containing compounds of the disclosure. Total light flux values were acquired via BLI 24 h after administration.
• the numbers 1-12 refer to the compositions containing Compounds 42-52 and MC3 respectively.
• Figure 5 is graph summarizing expression levels of luciferase in mouse liver 6 hours after administration of nanoparticle compositions including compounds of the disclosure.
• the numbers 1-12 refer to the compositions containing Compounds 42-52 and MC3 respectively.
• Figure 6 is a graph summarizing expression levels of luciferase in mouse lungs 6 hours after administration of nanoparticle compositions including compounds of the disclosure.
• the numbers 1-12 refer to the compositions containing Compounds 42-52 and MC3 respectively.
• Figure 7 is a graph summarizing expression levels of luciferase in mouse spleen 6 hours after administration of nanoparticle compositions including compounds of the disclosure.
• the numbers 1-12 refer to the compositions containing Compounds 42-52 and MC3 respectively.
• Figure 8 is a pair of graphs illustrating hEPO expression levels in rats dosed with compounds of the disclosure as compared to with KL22, showing that KL22 and its chain length derivatives (previously showing improved protein expression in mice), do not express hEPO in rats.
• PBS phosphate buffered saline
• Graph A compares the hEPO concentration after administration of nanoparticle compositions containing Compound 23,
• Graph B illustrates the results of a dose response study using Compound 20, KL22, and MC3 at dose of 0.2, 0.5 and 1 mpk. KL22 and Compound 23 were shown to be toxic at the 2 mg/kg dose.
• the numbers 1-5 refer to compositions containing the following: 1 : Compound 23, 2: Compound 11, 3: KL22; 4: MC3; 5: PBS.
• the numbers 1-9 refer to compositions containing the following: 1 : Compound 20, 0.2 mpk; 2: Compound 20, 0.5 mpk; 3: Compound 20, 1 mpk; 4: KL22, 0.2 mpk; 5: KL22, 0.5 mpk; 6: KL22, 1 mpk; 7: MC3, 0.2 mpk; 8: MC3, 0.5 mpk; 9: MC3, 1 mpk; 10: PBS.
• Figure 9 is a pair of graphs illustrating performance of Compound 11 as an LDLr independent lipid.
• Graph A is a bar graph showing expression of luciferase induced by administration of nanoparticle compositions including Compound 11 at dosages of 0.05 mpk, 0.25 mpk, and 0.5 mpk to LDLR -/- knockout and wild-type mice.
• Graph B shows LDL-c levels in LDLR knockout mice after administration of a control mRNA, i.e., non-translating Factor IX ("NT-FIX") and various compositions comprising mRNAs encoding the LDL receptor in mice, with KL22 at 0.5 mpk, or with Compound 11 at 0.5 and 1 mpk. LDL-c levels in mice were found to drop with nanoparticle composition containing Compound 11.
• NT-FIX non-translating Factor IX
• Figure 10 is a graph showing hEPO levels in non-human primates up to -50 h after administration of a nanoparticle composition containing Compound 4, compared to a composition containing MC3.
• the Compound 4 LNP demonstrated 3-fold expression of hEPO compared to MC3, establishing Compound 4 as an LDLr independent lipid that translates to higher species.
• Figure 11 is a pair of images comparing the results of a mouse liver
• IHC immunohistochemistry
• GFP green fluorescent protein
• Graph A shows CD-I mouse liver cells after administration of GFP mRNA in a MC3 LNP, 6h after intravenous administration at a dose of 0.5 mpk.
• GFP mRNA Protein expression from the MC3 LNP composition was observed in both hepatocytes and Kupffer cells.
• Graph B shows LDLR knockout mouse liver cells after administration of GFP mRNA in a Compound 4 LNP, 8h after intravenous administration at a dose of 0.5 mpk.
• the LNP containing Compound 4 appears to show less protein expression in Kupffer cells.
• FIG. 12 is a set of graphs illustrating hEPO expression levels in CD-I mice dosed with compounds of the disclosure, compared to MC3.
• PBS is used as control.
• Graph A shows the hEPO concentration 3 h after administration of the nanoparticle compositions.
• Graph B shows the hEPO concentration 6 h after administration of the nanoparticle compositions.
• Graph C shows the hEPO concentration 24 h after administration of the nanoparticle compositions.
• A-C numbers 1-14 refer to compositions containing the following: 1 : Compound 73, 2: Compound 80, 3: Compound 70; 4: Compound 81; 5: Compound 69; 6:
• Figure 13 is a graph showing hEPO levels (pg/mL) in CD-I mice up to -25 h after administration of a nanoparticle composition containing compounds of the disclosure, compared to a composition containing MC3.
• Numbers 1-13 refer to compositions containing the following: 1 : Compound 73, 2: Compound 80, 3: Compound 70; 4: Compound 81 ; 5:
• Figure 14 is a pair of graphs showing percentages of activated B-cells in the spleens of CD-I mice dosed with compounds of the disclosure, compared to MC3, and compared to mice not having received any treatment (naive test subject). PBS is used as control.
• Graph A shows the percentage of CD 19+ cells.
• Graph B shows the percentage of CD 19+ CD69+ CD86+ cells.
• Numbers 1-13 refer to compositions containing the following: 1 :
• Figure 15 is a graph summarizing luciferase expression levels at 6 h after administration of nanoparticle compositions containing compounds of the disclosure to CD-I mice at a dose of 0.5 mpk. Total light flux values were acquired via body luminescent imaging (BLI) 6 h after administration.
• the numbers 1-7 and 10 refer to the compositions containing Compounds 4-10 and MC3 respectively.
• Figure 16 is a graph showing GFP levels in the livers of LDLR knockout mice at 30 min to 24 h after intravenous administration of an eGFP RNA in a lipid composition containing Compound 4.
• the liver GFP levels were determined via IHC.
• the square markers represent the number of GFP positive cells following administration of a dose of 0.1 mpk of the composition.
• the circular markers represent the number of GFP positive cells following administration of a dose of 0.5 mpk of the composition.
• Figure 17 is a pair of graphs showing the ApoE dependence of luciferase ("Luc") expression following administration of a composition containing Luc mRNA and Compound 4 to mice at a dose 0.5 mpk.
• the expression following administration of a composition containing Luc mRNA and MC3 is presented for comparison.
• Graph A shows the total flux in the liver 6h after administration.
• the % change in Luc expression in livers of ApoE knockout vs. wild-type mice i.e., (WT mean expression-KO mean expression)AVT mean expression]* 100%
• Graph B shows the total flux in the spleen 6h after administration.
• the % change in expression in spleens of ApoE knockout vs. wild-type mice was 4.34 % for Compound 4 and 72.2 % for MC3.
• Numbers 1-4 refer to the following: 1 :
• composition containing Compound 4, administered to ApoE knockout mice 2: Composition containing Compound 4, administered to wild-type mice; 3: Composition containing MC3, administered to ApoE knockout mice; 4: Composition containing MC3, administered to wild- type mice.
• Figure 18 is a pair of graphs showing the effect of a composition containing
• Compound 4 on liver enzymes The composition was administered to rats at 0.1 mpk and 1 mpk. The effects of MC3 are shown for comparison. PBS is used as a control.
• Graph A shows the effect on aspartate aminotransferase (AST).
• Graph B shows the effect on alanine
• ALT aminotransferase
• Figure 19 is a set of graphs showing immune cell activation by a composition containing Compound 4.
• the effects of MC3 are shown for comparison.
• PBS phosphate buffered saline
• Compositions were administered to rats at 0.1 mpk or 1 mpk.
• Graph A shows the effect on activation of neutrophil.
• Graph B shows the effect on activation of lymphocytes.
• Graph C shows the effect on activation of monocytes. Numbers 1-5 in Graphs A- C refer to the following: 1 : Compound 4, 0.1 mpk; 2: Compound 4; 1 mpk; 3: MC3, 0.1 mpk 4: MC3, 1 mpk; 5: PBS.
• Figure 20 is a graph showing the expression of Stefin A Quadruple Mutant-Tracy
• SQT SQT protein in mouse liver determined via FLAG IHC at different time points following intravenous administration of various nanoparticle compositions comprising SQT mRNA and lipids disclosed herein.
• Numbers 1-11 in the figure refer to the following: 1 : 0 h, PBS; 2: 0 h, Compound 4; 3: 0.5 h, Compound 4; 4: 4 h, Compound 4; 5: 8 h, Compound 4; 6: 24 h, Compound 4; 7: O h, MC3; 8: 0.5 h, MC3; 9: 4 h, MC3; 10: 8 h, MC3; 11 : 24 h, MC3.
• the disclosure relates to novel lipids and lipid nanoparticle compositions including a novel lipid.
• the disclosure also provides methods of delivering a therapeutic and/or prophylactic agent to a mammalian cell, specifically delivering a therapeutic and/or prophylactic agent to a mammalian organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof.
• a method of producing a polypeptide of interest in a cell involves contacting a nanoparticle composition comprising an mRNA with a mammalian cell, whereby the mRNA may be translated to produce the polypeptide of interest.
• a method of delivering a therapeutic and/or prophylactic agent to a mammalian cell or organ may involve administration of a nanoparticle composition including the therapeutic and/or prophylactic agent to a subject, in which the administration involves contacting the cell or organ with the composition, whereby the therapeutic and/or prophylactic agent is delivered to the cell or organ.
• the present disclosure provides lipids including a central piperazine moiety.
• the lipids described herein may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents to mammalian cells or organs.
• the lipids described herein have little or no immunogenicity.
• the lipid compound of any of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1) has a lower immunogenicity as compared to a reference lipid (e.g. , MC3, KC2, or DLinDMA).
• a reference lipid e.g. , MC3, KC2, or DLinDMA
• a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.
• a reference lipid e.g., MC3, KC2, or DLinDMA
• Lipids may be compounds of formula (I),
• t is 1 or 2; Ai and A 2 are each independently selected from CH or N;
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• Ri, R 2 , R3, R4, and R5 are independently selected from the group consisting of C5 -2 o alkyl, C 5-20 alkenyl, -R"MR', -R*YR", -YR", and -R*OR";
• each M is independently selected from the group consisting of
• X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, -CH 2 -, -(CH 2 ) 2 -, -CHR-, -CHY-, -C(O)-, -C(0)0-, -OC(O)-, -C(0)-CH 2 -, -CH 2 -C(0)-,
• each Y is independently a C3-6 carbocycle
• each R* is independently selected from the group consisting of Ci_i 2 alkyl and C 2-12 alkenyl;
• each R is independently selected from the group consisting of C 1-3 alkyl and a C3-6 carbocycle;
• each R' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
• each R" is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl
• R 1 at least one of R 1; R 2 , R 3 , R 4 , and R 5 is -R"MR'.
• the compound is of any of formulae (Ial)-(Ia6):
• the compounds of Formula (I) or any of (Ial)-(Ia6) include one or more of the following features when applicable.
• ring A is [00166] In some embodiments, ring A is
• ring A is
• ring A is
• ring A is
• ring A is
• Z is CH 2
• Z is absent.
• At least one of Ai and A 2 is N
• each of Ai and A 2 is N.
• each of Ai and A 2 is CH.
• Ai is N and A 2 is CH.
• Ai is CH and A 2 is N.
• at least one of X 1 , X 2 , and X 3 is not -CH 2 -.
• X 1 is not -CH 2 -.
• at least X 2 , and X 3 is -C(O)-.
• X 2 is -C(O)-, -C(0)0-, -OC(O)-, -C(0)-CH 2 -,
• X 3 is -C(O)-, -C(0)0-, -OC(O)-, -C(0)-CH 2 -,
• X 3 is -CH 2 -.
• X 3 is a bond or -(CH 2 ) 2 -.
• Ri and R 2 are the same. In certain embodiments, Ri, R 2 , and R3 are the same. In some embodiments, R4 and R5 are the same. In certain embodiments, Ri, R 2 , R3, R4, and R5 are the same.
• At least one of R 1; R 2 , R3, R4, and R5 is -R"MR' . In some embodiments, at most one of R 1; R 2 , R3, R4, and R5 is -R"MR' . For example, at least one of R 1; R 2 , and R 3 may be -R"MR', and/or at least one of R 4 and R 5 is -R"MR'. In certain embodiments,
• At least one M is -C(0)O. In some embodiments, each M is -C(0)O. In some embodiments, at least one M is -OC(O)-. In some embodiments, each M is -OC(O)-. In some embodiments, at least one M is -0C(0)O. In some embodiments, each M is -0C(0)O. In some embodiments, at least one R" is C3 alkyl. In certain embodiments, each R" is C3 alkyl. In some embodiments, at least one R" is C5 alkyl. In certain embodiments, each R" is C5 alkyl. In some embodiments, at least one R" is e alkyl. In certain embodiments, each R" is e alkyl. In certain embodiments, each R" is e alkyl. In certain embodiments, each R" is e alkyl. In certain embodiments, each R" is e alkyl.
• At least one R" is C7 alkyl. In certain embodiments, each R" is C7 alkyl. In some embodiments, at least one R' is C5 alkyl. In certain embodiments, each R' is C5 alkyl. In other embodiments, at least one R' is Ci alkyl. In certain embodiments, each R' is Ci alkyl. In some embodiments, at least one R' is C 2 alkyl. In certain embodiments, each R' is C 2 alkyl.
• At least one of R 1; R 2 , R3, R4, and R5 is C 12 alkyl. In certain embodiments, each of R 1; R 2 , R3, R4, and R5 are C 12 alkyl.
• the compound is selected from the group consisting of:
• a lipid has the formula (II)
• Ai and A2 are each independently selected from CH or N and at least one of Ai and A2 is
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• Ri, R2, R3, R4, and R5 are independently selected from the group consisting of C6-20 alkyl and C6-20 alkenyl;
• Ri, R2, R3, R4, and R5 are the same, wherein Ri is not C 12 alkyl, Cig alkyl, or Cig alkenyl;
• Ri, R2, R3, R4, and R5 is selected from Ce-20 alkenyl
• R2 and R3 are selected from Ce-2o alkenyl, and R4 and R5 are selected from C 6- alkyl; or
• Ri, R2, and R3 are selected from Ce-20 alkyl, and R4 and R5 are selected from Ce-20 alkenyl.
• the compound is of formula (Ila):
• the compounds of Formula (II) or (Ila) include one or more of the following features when applicable.
• Z is CH 2 .
• Z is absent.
• At least one of Ai and A 2 is N.
• each of Ai and A 2 is N.
• each of Ai and A 2 is CH.
• Ai is N and A 2 is CH.
• Ai is CH and A 2 is N.
• R 1; R2, R3, R4, and R5 are the same, and are not C 12 alkyl
• Ci8 alkyl or C 18 alkenyl.
• Ri, R2, R3, R4, and R5 are the same and are C9 alkyl or C 14 alkyl.
• R 1; R2, R3, R 4 , and R5 are selected from C6-2 0 alkenyl.
• R 1; R 2 , R3, R4, and R5 have the same number of carbon atoms.
• R4 is selected from C5-2 0 alkenyl.
• R4 may be C 12 alkenyl or C 18 alkenyl.
• At least one of R 1; R 2 , R3, R4, and R5 have a different number of carbon atoms than at least one other of R 1; R 2 , R3, R4, and R5.
• R 1; R 2 , and R3 are selected from C6-2 0 alkenyl, and R 4 and R5 are selected from C6-2 0 alkyl. In other embodiments, R 1; R 2 , and R3 are selected from C6-2 0 alkyl, and R 4 and R5 are selected from C6-2 0 alkenyl. In some embodiments, R 1; R 2 , and R3 have the same number of carbon atoms, and/or R4 and R5 have the same number of carbon atoms. For example, R 1; R2, and R3, or R 4 and R5, may have 6, 8, 9, 12, 14, or 18 carbon atoms.
• Ri, R2, and R3, or R4 and R5, are C 18 alkenyl (e.g., linoleyl).
• R 1; R2, and R3, or R4 and R5, are alkyl groups including 6, 8, 9, 12, or 14 carbon atoms.
• Ri has a different number of carbon atoms than R 2 , R3, R4, and R5.
• R3 has a different number of carbon atoms than R 1; R 2 , R4, and R5.
• R4 has a different number of carbon atoms than R 1; R 2 , R3, and R5.
• the compound is selected from the group consisting of:
• the compound has the formula (III)
• a 3 is CH or N
• a 4 is CH 2 or NH; and at least one of A 3 and A 4 is N or NH;
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• Ri, R 2 , and R3 are independently selected from the group consisting of C5 -2 o alkyl, C5 -2 o alkenyl, -R"MR', -R*YR", -YR", and -R*OR";
• each M is independently selected
• X 1 and X 2 are independently selected from the group consisting of -CH 2 -,
• each Y is independently a C3-6 carbocycle
• each R* is independently selected from the group consisting of Ci_i 2 alkyl and C 2-12 alkenyl;
• each R is independently selected from the group consisting of C 1-3 alkyl and a C3-6 carbocycle;
• each R' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
• each R" is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
• the compounds of Formula (III) or (Ilia) include one or more of the following features when applicable.
• Z is CH 2.
• Z is absent.
• At least one of A 3 and A 4 is N or NH.
• A3 is N and A4 is NH.
• a 3 is N and A 4 is CH 2 .
• A3 is CH and A4 is NH.
• At least one of X 1 and X 2 is not -CH 2 -.
• X 1 is not -CH 2 -.
• at least one of X 1 and X 2 is - C(O)-.
• X 2 is -C(O)-, -C(0)0-, -OC(O)-, -C(0)-CH 2 -, -CH 2 -C(0)-
• R 1; R 2 , and R3 are independently selected from the group consisting of C5 -2 o alkyl and C5 -2 o alkenyl. In some embodiments, R 1; R 2 , and R3 are the same. In certain embodiments, R 1; R 2 , and R3 are C 6 , C9, C 12 , or C 14 alkyl. In other embodiments, R 1; R 2 , and R3 are C 18 alkenyl. For example, Ri, R 2 , and R3 may be linoleyl.
• the compound is selected from the group consisting of:
• a 6 and A 7 are each independently selected from CH or N, wherein at least one of A 6 and A 7 is N;
• Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
• X 4 and X 5 are independently selected from the group consisting of -CH 2 -,
• Ri, R 2i R3, R4, and R5 each are independently selected from the group consisting of C5 -2 o alkyl, C 5-20 alkenyl, -R"MR', -R*YR", -YR", and -R*OR";
• each M is independently selected from the group consisting of
• each Y is independently a C3-6 carbocycle
• each R* is independently selected from the group consisting of Ci_i 2 alkyl and C 2-12 alkenyl;
• each R is independently selected from the group consisting of C 1-3 alkyl and a C3-6 carbocycle; each R' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H; and
• each R" is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
• R 1; R 2i R3, R4, and R5 each are independently selected from the group consisting of Ce-20 alkyl and Ce-20 alkenyl.
• Ri and R2 are the same.
• R 1; R 2 , and R3 are the same.
• R4 and R5 are the same.
• Ri, R 2 , R3, R4, and R5 are the same.
• At least one of Ri, R2, R3, R4, and R5 is C9-12 alkyl.
• each of R 1; R 2 , R 3 , R4, and R5 independently is C9, C 12 or C 14 alkyl.
• each of R 1; R 2 , R 3 , R4, and R5 is C9 alkyl.
• a 6 is N and A 7 is N. In some embodiments, A 6 is CH and
• a 7 is N.
• X 4 is-CH 2 - and X 5 is -C(O)-. In some embodiments, X 4 and X 5 are -C(O)-.
• At least one of X 4 and X 5 is not -CH 2 -, e.g., at least one of X 4 and X 5 is -C(O)-.
• at least one of Ri, R 2 , R 3 , R 4 , and R 5 is -R"MR'.
• At least one of R 1; R 2 , R 3 , R4, and R5 is not -R"MR'.
• the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-phenyl)-2-aminoethyl
• the compound has the formula (IV)
• the disclosure provides a compound having the formula (17-1)
• Ri a is -(CH 2 ) n a Q a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 ) n aN(R a ) 2 , -C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)R a , and -N(R a )S(0) 2 R a and each n a is independently selected from 1, 2, 3, 4, and 5;
• R 2a and R3 3 are each independently selected from the group consisting of C3 -2 4 alkyl, C3- 24 alkenyl, -R a *Y a R a ", -Y a R a ", and -R a *OR a ";
• each Y a is independently a C3-6 carbocycle
• each R a * is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• each R a is independently selected from the group consisting of C 1-3 alkyl, C 2 -3 alkenyl, and H;
• each R a " is selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl;
• R 2a includes 7 or fewer carbon atoms.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• R3 a includes 7 or fewer carbon atoms.
• the compound is selected from the group consisting of:
• the disclosure provides a compound having the formula (17-1)
• Ria is -(CH 2 ) n aQ a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 yN(R a ) 2 , - C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)R a , and -N(R a )S(0) 2 R a and each n a is independently selected from 1, 2, 3, 4, and 5;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• R 2a is selected from the group consisting of Cg -2 4 alkenyl
• R3a is selected from the group consisting of Cg -2 4 alkyl
• each R is independently selected from the group consisting of C1-3 alkyl, C 2- 3 alkenyl, and H.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n a is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• R3 3 is an alkyl including 9, 12, 14, or 18 carbon atoms.
• R 2a is C ig alkenyl (e.g., linoleyl).
• the compound is selected from the group consisting of:
• the disclosure provides a compound having the formula (17-1)
• Ria is -(CH 2 ) n aQ a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 yN(R a ) 2 , - C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)R a , and -N(R a )S(0) 2 R a and each n a is independently selected from 1, 2, 3, 4, and 5;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• R 2a is selected from the group consisting of Ci3 -2 o alkyl
• R3a is selected from the group consisting of Cg -2 o alkyl
• each R a is independently selected from the group consisting of C1-3 alkyl, C 2- 3 alkenyl, and H.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n a is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• R 2a and R3 3 are the same.
• R 2a and/or R 3a is C14 alkyl.
• the compound is (Compound 17-7).
• the disclosure provides a compound having the formula (17-1)
• Ria is -(CH2)n a Q a , where Q a is -OR a , R a is selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H, and n a is selected from 1, 2, 3, 4, and 5; and
• R 2a and R 33 are each independently selected from the group consisting of Cg-2o alkenyl, wherein
• R a is selected from the group consisting of C 1-3 alkyl and C2-3 alkenyl; or iv) Ri a is -(CH 2 )20H, and R2 a and R 33 each include one or fewer double bonds.
• R a is H. In other embodiments, R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n a is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• Ri a is -(CH 2 )20CH 3 . In other embodiments, Ri a is -(CH 2 ) 2 OH.
• R2 a is C 18 alkenyl (e.g., linoleyl).
• R 3a is Ci8 alkenyl (e.g., linoleyl).
• R2 a and R 3a are the same.
• the compound is selected from the group consisting of
• Ri is -(CH 2 ) n aQ a , where Q a is selected from a heterocycle, -OR a , -0(CH 2 ) n aN(R a ) 2 , - C(0)OR a , -OC(0)R a , -CX a 3 , -CX a 2 H, -CX a H 2 , -CN, -N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)R a , and -N(R a )S(0) 2 R a and each n a is independently selected from 1, 2, 3, 4, and 5;
• each X a is independently selected from the group consisting of F, CI, Br, and I;
• R 2a is selected from the group consisting of Cg-i 2 alkyl
• R3a is selected from the group consisting of Cg -2 o alkyl
• each R a is independently selected from the group consisting of C1-3 alkyl, C 2- 3 alkenyl, and H.
• Q a is -OR a .
• R a is H.
• R a is -CH 3 .
• n a is 1. In other embodiments, n a is 2. In other embodiments, n is 3. In other embodiments, n a is 4. In some embodiments, n a is 5.
• Q a is -OR a and n a is selected from 2, 3, and 4.
• R 2a is C9 alkyl. In other embodiments, R 2a is C 12 alkyl.
• R 2a and R3 3 are the same.
• the compound is selected from the group consisting of:
• the disclosure provides a compound having the formula (19-1),
• Ri b is selected from the group consisting of H, C1-5 alkyl, C2-5 alkenyl, -R b "M b R b ', a C3-6 carbocycle, -(CH 2 ) n Q b , and -(CH 2 ) n CHQ b R b , where Q b is selected from a heterocycle, -OR b , -0(CH 2 ) n N(R b ) 2 , -C(0)OR b , -OC(0)R b , -CX b 3 , -CX b 2 H, -CX b H 2 , -CN,
• n is independently selected from 1, 2, 3, 4, and 5;
• R 2 b and are independently selected from the group consisting of C 1-2 o alkyl, C 2-2 o alkenyl, -R b "MR b ', -R b *YR b ", -YR b “, and -R b *OR b ";
• each M b is independently selected from the group consisting of:
• W is selected from the group consisting of -CH 2 -, -CHR b -, -C(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-;
• each X b is independently selected from the group consisting of F, CI, Br, and I;
• each Y b is independently a C3-6 carbocycle
• each R b * is independently selected from the group consisting of Ci_i 2 alkyl and Ci_i 2 alkenyl;
• each R b is independently selected from the group consisting of C1-3 alkyl, a C3-6 carbocycle, C 2- 3 alkenyl, and H;
• each R b ' is independently selected from the group consisting of Ci_i 2 alkyl, C 2-12 alkenyl, and H;
• each R b " is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
• W is not -CH 2 -, e.g., W is -C(O)-.
• R 2b and R 3b is -R b "M b R b '.
• M b is -C(0)0-.
• at least one R b " is C 5 alkyl.
• at least one R b ' is C 5 alkyl.
• I1 ⁇ 2, and/or R3 ⁇ 4 are selected from the group consisting of
• R2b and/or R3 ⁇ 4 may be alkyl groups including 9 or 12 carbon atoms.
• R2b and/or R3 ⁇ 4 are selected from the group consisting of C2-20 alkenyl.
• R2b and/or R3 ⁇ 4 may be alkenyl groups including 18 carbon atoms (e.g., linoleyl groups).
• R2b and R3 ⁇ 4 are the same.
• Rib is H, while in other embodiments, Rib is selected from
• Rib may be Ci alkyl.
• Rib is -(CH 2 ) n Q b
• Q b is a heterocycle such as a phenyl group.
• Q b may be a phenyl group with one or more substituents, as described herein.
• the compound is selected from the group consisting of: (Compound 19-1),
• lipids are compounds of formula (19-11)
• Rib is selected from the group consisting of Ce-20 alkyl
• R2b and R 3 b are independently selected from the group consisting of Ce-20 alkenyl.
• Rib is C 12 alkyl.
• R2b and/or R 3 b are C 18 alkenyl (e.g., linoleyl).
• R2b and are both linoleyl.
• the compound is 19-6).
• lipids may be compounds of formula (20-1),
• Ric is selected from the group consisting of a C3-6
• n c is independently selected from 1 , 2, 3, 4, and 5;
• R2c, R3c, and R4c are independently selected from the group consisting of C 1-2 o alkyl, C 2- 20 alkenyl, -R C "M C R C ' , -R C *Y C R C ", -Y C R C “, and -R c *OR c ";
• each M c is independently selected from the group consisting of:
• each X c is independently selected from the group consisting of F, CI, Br, and I;
• each Y c is independently a C 3 -6 carbocycle
• each R c * is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
• each R c is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H;
• each R c ' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
• each R c " is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl,
• Ric is selected from the group consisting of a C 3 -6
• R 2c , R 3c , and R 4c is -R C "M C R C ' .
• Ri c is selected from the group consisting of
• Ri c is -(CH 2 ) n cQ c .
• n c is 2.
• Q c is -C(0)OR c , where R c is, for example, H.
• R 2c , R 3c , and Ric is -R C "M C R C ' .
• R 2c , R 3c , and/or R 4c may be -R C "M C R C '.
• at least one M c is -C(0)O.
• each M c is -C(0)0-.
• at least one R c " is C5 or C7 alkyl.
• each R c " is C5 alkyl.
• each R c " is C7 alkyl.
• at least one R c ' is C5, C 7 , or C 9 alkyl.
• each R c ' is C5 alkyl. In other embodiments, each R c ' is C7 alkyl. In other embodiments, each R c ' is C 9 alkyl. In some embodiments, R c ' is branched.
• R 2c , R 3c , and R 4c are selected from the group consisting of
• R_2 C is selected from the group consisting of C5-20 alkyl and
• R_2 C may be C 12 alkyl.
• R_3 C is selected from the group consisting of C5-20 alkyl and
• R_3 C may be C 6 , C9, or C 12 alkyl.
• R4 C is selected from the group consisting of C5-20 alkyl and
• R4 C may be C 6 , C9, or C 12 alkyl.
• R_3 C and R4 C are the same.
• the compound is selected from the group consisting of:
• the lipid is a compound according to formula (20-1)
• Ri c is selected from the group consisting of -(CH 2 ) n cQ c , -(CH 2 ) n ' ; CHQ c R c , -CHQ C R C , and -CQ C (R C ) 2 , where Q c is selected from -OR c , -CN, and -N(R C ) 2 , and n c is selected from 1, 2, 3, 4, and 5;
• R 2c and R3 C are independently selected from the group consisting of C6 -2 o alkyl and C6 -2 o alkenyl;
• R4c is selected from the group consisting of Ci3 -2 o alkyl and C5 -2 o alkenyl; and each R c is independently selected from the group consisting of C 1-3 alkyl, C 2- 3 alkenyl, and H.
• R 3c is C 14 alkyl.
• R4 C is C 14 alkyl.
• R 3c is C 18 alkenyl.
• R3 C may be linoleyl.
• R4 C is C 18 alkenyl.
• R4 C may be linoleyl.
• R 2c is C 12 alkyl. In other embodiments, R 2c is C 14 alkyl. In some embodiments, R 2c is C 18 alkenyl. For example, R 2c may be linoleyl.
• R 3c and R4 C are the same.
• Ri c is -(CH 2 ) n c Q c .
• Q c is -OR c .
• Q c may be -OH.
• n c is 2 or 3.
• the compound is selected from the group consisting of:
• the lipid is a compound having formula (20-1)
• Ri c is selected from the group consisting of -(CH 2 ) n ' ; CHQ c R c , -CHQ C R C , and -CQ C (R c ) 2 , where Q c is selected from -OR c , -CN, and -N(R C ) 2 , and n c is selected from 1 , 2, 3, 4, and 5;
• R2c, R3c, and R4c are independently selected from the group consisting of C6-20 alkyl and Ce-20 alkenyl;
• each R c is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H;
• R2c is selected from the group consisting of Ci-n alkyl and C2-5 alkenyl
• R 3c is selected from the group consisting of Ci-n alkyl and C2-5 alkenyl.
• R2 C is selected from the group consisting of Ci-n alkyl and
• R2 C may be Ce or C9 alkyl.
• R.3 C is selected from the group consisting of Ci-n alkyl and
• R.3 C may be e or C9 alkyl.
• R_3 C is C 12 alkyl.
• R2 C is C 12 alkyl.
• R4 C is C 6 , C9, or C 12 alkyl.
• Ri c is -(CH 2 ) n c Q c .
• Q c is -OR c .
• R c is H.
• n c is 2 or 3.
• the compound is selected from the group consisting of:
• the lipid is a compound according to formula (20-1) l c
• R LC is selected from the group consisting of -(CH 2 ) D CQ C , -(CH 2 ) N ' ; CHQ C R C , -CHQ C R C , and -CQ C (R C ) 2 , where Q C is selected from -OR C , -CN, and -N(R C ) 2 , and n c is selected from 1 , 2, 3, 4, and 5;
• R2c is selected from the group consisting of H, C12-20 alkyl, and C6-20 alkenyl
• R3c and R4 C are C 12 alkyl
• each R C is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H. [00295] In some embodiments, R_2 C is H. In other embodiments, R_2 C is C 12 alkyl or alkenyl. In some embodiments, R_2 C is C 14 alkyl. In other embodiments, R_2 C is C 18 alkenyl. For example, R2 C may be linoleyl.
• Ri c is -(CH 2 ) n c Q c .
• Q c is -OR c .
• Q c may be OH.
• n c is 2, 3, or 4.
• the compound is selected from the group consisting of:
• lipids may be compounds of formula (21-1),
• Rid and !1 ⁇ 2 are independently selected from the group consisting of H, C1-5 alkyl, and C 2- 5 alkenyl;
• n d is selected from 1, 2, 3, 4, and 5;
• k is selected from 0, 1 , 2, and 3;
• R 3 d and R4d are independently selected from the group consisting of C 1-2 o alkyl, C2-2 0 alkenyl, -R d "M d R d ', -R d *Y d R d ", -Y d R d ", and -R d *OR d ";
• each M d is independently selected from the group consisting of:
• each R d ' is independently selected from the group consisting of C 1-12 alkyl, C2-12 alkenyl, and H; each Y d is independently a C3-6 carbocycle;
• each R d * is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
• each R d ' is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
• each R d " is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl,
• R 3 d and R4d are bound to either i) the same carbon atom or ii) adjacent carbon atoms.
• Ru and R4d are bound to the same carbon atom.
• Ru and R4d may be bound to a carbon atom adjacent to C*.
• Ru and R4d are not bound to a carbon atom adj acent to C*.
• R ⁇ and R4d are bound to adjacent carbon atoms. In certain embodiments, one or both of Ru and R4d are bound to carbon atoms adjacent to C*.
• k is 0. In other embodiments, k is 1 , 2, or 3.
• M d is absent. In other embodiments, M d is selected from the group consisting of
• M d is -C(0)0-.
• n d is 1, 2, or 3.
• Rid and/or R 2 d are selected from C1-5 alkyl.
• Rid and/or R2d are Ci alkyl. [0008] In certain embodiments, and/or are selected from C2-20 alkenyl. In certain embodiments, R.3d and/or are alkenyl groups including 17, 18, or 19 carbon atoms. For example, ⁇ 1 ⁇ 2 and/or may be C 18 alkenyl groups (e.g., linoleyl).
• the compound is selected from the group consisting of:
• alkyl or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted.
• C 1-2 4 alkyl means an optionally substituted linear or branched, saturated hydrocarbon including 1-24 carbon atoms.
• An alkyl group described herein refers to both unsubstituted and substituted alkyl group unless otherwise specified.
• alkenyl or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted.
• C2-24 alkenyl means an optionally substituted linear or branched hydrocarbon including 2 to 24 carbon atoms and at least one carbon-carbon double bond.
• An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds.
• C 18 alkenyl may include one or more double bonds.
• a C 18 alkenyl group including two double bonds may be a linoleyl group.
• An alkenyl group described herein refers to both unsubstituted and substituted unless otherwise specified.
• the term "carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, or twelve membered rings.
• the notation "C3-6 carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g. , cycloalkyl or aryl groups).
• carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups.
• Carbocycles described herein refers to both unsubstituted and substituted carbocycles unless otherwise specified.
• the term "cycloalkyl" as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.
• heterocycle or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom.
• Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, or twelve membered rings.
• Heterocycles may include one or more double or triple bonds and may be non- aromatic or aromatic.
• heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. Heterocycles may be optionally substituted.
• a “biodegradable group” is a group that may facilitate faster metabolism of a lipid in a mammalian entity.
• a biodegradable group may be selected from the group consisting of, but is not limited to, -C(0)0-, -OC(O)-, -C(0)N(R')-, -N(R')C(0)-,
• an "aryl group” is a carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a "heteroaryl group” is a heterocyclic group including one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl.
• Aryl and heteroaryl groups may be optionally substituted.
• each M, M b , M c , or M d can be independently selected from the non-limiting group consisting of phenyl, oxazole, and thiazole.
• each M, M b , M c , or M d can be independently selected from the list of biodegradable groups above.
• Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified.
• a sulfonic acid e.g., -OR
• an acetal e.g.,-C(OR)2R"
• each OR are alkoxy groups that can be the same or different and R"" is an alkyl or alkenyl group
• a phosphate e.g., P(0)4 3"
• a thiol e.g., -SH
• a sulfoxide e.g., -S(O)R
• a sulfinic acid e.g., -S(O)OH
• sulfonyl e.g., -S(0) 2 OH
• a thial e.g., -C(S)H
• a sulfate e.g., S(0) 4 2"
• a sulfonyl e.g., -S(0) 2 -
• an amide e.g., -C(0)NR 2 , or -N(R)C(0)R
• an azido e.g., -N 3
• a nitro e.g., -N0 2
• a cyano e.g., -CN
• an isocyano e.g., -NC
• an acyloxy e.g.,-OC(0)R
• an amino e.g., -NR 2 , -NRH, or -NH 2
• carbamoyl e.g., -OC(0)NR 2 , -OC(0)NRH, or -OC(0)
• R is an alkyl or alkenyl group, as defined herein.
• the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein.
• a C5 -2 o alkyl group may be further substituted with one, two, three, four, five, six, or more substituents as described herein.
• a lipid may have a positive or partial positive charge at physiological pH.
• Such lipids may be referred to as cationic or ionizable (amino)lipids.
• Lipids may be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.
• Nanoparticle compositions comprising a lipid component comprising a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1) as described herein.
• the largest dimension of a nanoparticle composition is 1 ⁇ or shorter (e.g., 1 ⁇ , 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter), e.g. , when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method.
• DLS dynamic light scattering
• Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes.
• LNPs lipid nanoparticles
• nanoparticle compositions are vesicles including one or more lipid bilayers.
• a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments.
• Lipid bilayers may be functionalized and/or crosslinked to one another.
• Lipid bilayers may include one or more ligands, proteins, or channels.
• Nanoparticle compositions comprise a lipid component including at least one lipid, such as a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1), as described herein.
• a nanoparticle composition may include a lipid component including one of Compounds 1 through 88, Compounds 17-1 through 17-13, Compounds 19-1 through 19-6, Compounds 20-1 through 20-25 and Compounds 21-1 through 21-6.
• Nanoparticle compositions may also include a variety of other components.
• the lipid component of a nanoparticle composition may include one or more other lipids in addition to a lipid according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1).
• Cationic/ionizable lipids may include one or more other lipids in addition to a lipid according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), (20-1) and (21-1).
• a nanoparticle composition may include one or more cationic and/or ionizable lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH) in addition to a lipid according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19- I), (19-11), (20-1) and (21-1).
• Cationic and/or ionizable lipids may be selected from the non-limiting group consisting of
• DLin-MC3-DMA 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane
• DLin-KC2-DMA 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane
• DODMA l,2-dioleyloxy-N,N-dimethylaminopropane
• a cationic lipid may also be a lipid including a cyclic amine group.
• Additional cationic and/or ionizable lipids that are suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, and WO2015011633, the entire contents of each of which are hereby incorporated by reference in their entireties.
• the lipid component of a nanoparticle composition may include one or more
• PEG or PEG-modified lipids Such species may be alternately referred to as PEGylated lipids.
• a PEG lipid is a lipid modified with polyethylene glycol.
• a PEG lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
• a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG- DSPE lipid.
• the lipid component of a nanoparticle composition may include one or more structural lipids.
• Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.
• the structural lipid is cholesterol.
• the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.
• the lipid component of a nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids.
• Phospholipids may assemble into one or more lipid bilayers.
• phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
• a phospholipid may be a lipid according to formula (V)
• R p represents a phospholipid moiety and Ri and R 2 represent fatty acid moieties with or without unsaturation that may be the same or different.
• a phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
• a fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
• Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
• a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
• alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
• an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide.
• Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
• Phospholipids useful in the compositions and methods described herein may be selected from the non-limiting group consisting of l,2-distearoyl-s «-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-OT-glycero-3-phosphoethanolamine (DOPE),
• DLPC l,2-dilinoleoyl-sn-glycero-3-phosphocholine
• DMPC 1,2-dimyristoyl-sn-glycero-phosphocholine
• DOPC l,2-dioleoyl-sn-glycero-3-phosphocholine
• DPPC l,2-dipalmitoyl-sn-glycero-3-phosphocholine
• POPC l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
• OChemsPC l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
• a nanoparticle composition includes DSPC.
• a nanoparticle composition includes DOPE.
• a nanoparticle composition includes both DSPC and DOPE.
• a nanoparticle composition that includes one or more lipids described herein may further include one or more adjuvants, e.g., Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.
• GLA Glucopyranosyl Lipid Adjuvant
• CpG oligodeoxynucleotides e.g., Class A or B
• poly(I:C) poly(I:C)
• aluminum hydroxide e.g., aluminum hydroxide, and Pam3CSK4.
• Nanoparticle compositions may include one or more therapeutic and/or prophylactic agents.
• the disclosure features methods of delivering a therapeutic and/or prophylactic agent to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof comprising administering to a mammal and/or contacting a mammalian cell with a nanoparticle composition including a therapeutic and/or prophylactic agent.
• Therapeutic and/or prophylactic agents include biologically active substances and are alternately referred to as "active agents.”
• a therapeutic and/or prophylactic agent may be a substance that, once delivered to a cell or organ, brings about a desirable change in the cell, organ, or other bodily tissue or system. Such species may be useful in the treatment of one or more diseases, disorders, or conditions.
• a therapeutic and/or prophylactic agent is a small molecule drug useful in the treatment of a particular disease, disorder, or condition.
• drugs useful in the nanoparticle compositions described herein include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensive agents (e.g., clonidine
• a therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent.
• a cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells.
• Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof.
• taxol cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine,
• Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium.
• iodine e.g., iodine 125 or iodine 131
• strontium 89 phosphorous
• palladium cesium
• iridium phosphate
• cobalt yttrium 90
• samarium 153 samarium 153
• praseodymium praseodymium
• Vaccines include compounds and preparations that are capable of providing immunity against one or more conditions related to infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis and can include mRNAs encoding infectious disease derived antigens and/or epitopes.
• Vaccines also include compounds and preparations that direct an immune response against cancer cells and can include mRNAs encoding tumor cell derived antigens, epitopes, and/or neoepitopes.
• Compounds eliciting immune responses may include vaccines, corticosteroids (e.g., dexamethasone), and other species.
• a vaccine and/or a compound capable of eliciting an immune response is administered intramuscularly via a composition including a compound according to one of formulae (I), (Ial)-(Ia6), (lb), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19- II), (20-1) and (21-1).
• therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU),
• antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil, dacarbazine
• alkylating agents e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU)
• cyclophosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).
• anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
• antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
• anti-mitotic agents e.g.
• a therapeutic and/or prophylactic agent is a protein.
• Therapeutic proteins useful in the nanoparticles of the disclosure include, but are not limited to, gentamycin, amikacin, insulin, erythropoietin (EPO), granulocyte-colony stimulating factor (G- CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), Factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, Hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.
• gentamycin amikacin
• insulin erythropoietin
• G- CSF granulocyte-colony stimulating factor
• GM-CSF granulocyte-macrophage colony stimulating factor
• LHRH luteinizing hormone-releasing hormone
• a therapeutic and/or prophylactic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid).
• nucleic acid e.g., ribonucleic acid or deoxyribonucleic acid.
• polynucleotide in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
• Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
• a therapeutic and/or prophylactic agent is an RNA.
• RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.
• the RNA is an mRNA.
• a therapeutic and/or prophylactic agent is an mRNA.
• An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide.
• a polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity.
• a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.
• a therapeutic and/or prophylactic agent is an siRNA.
• An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest.
• an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA.
• An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest.
• the siRNA may be an immunomodulatory siRNA.
• a therapeutic and/or prophylactic agent is an shRNA or a vector or plasmid encoding the same.
• An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.
• Nucleic acids and polynucleotides useful in or suitable for the compounds and methods of the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5 ' -terminus of the first region (e.g., a 5 -UTR), a second flanking region located at the 3 ' -terminus of the first region (e.g., a 3 -UTR), at least one 5 ' -cap region, and a 3 ' -stabilizing region.
• a first region of linked nucleosides encoding a polypeptide of interest
• a first flanking region located at the 5 ' -terminus of the first region e.g., a 5 -UTR
• a second flanking region located at the 3 ' -terminus of the first region e.g., a 3 -UTR
• a nucleic acid or polynucleotide further includes a poly -A region or a Kozak sequence (e.g., in the 5 ' -UTR).
• polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide.
• a polynucleotide or nucleic acid e.g., an mRNA
• any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside).
• the 3 ' -stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2 ' -0-methyl nucleoside and/or the coding region, 5 ' -UTR, 3 ' -UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5 -methoxy uridine), a 1 -substituted pseudouridine (e.g., 1-methyl- pseudouridine or 1-ethyl-pseudouridine ), and/or a 5-substituted cytidine (e.g., 5-methyl- cytidine).
• a 5-substituted uridine e.g., 5 -methoxy uridine
• the shortest length of a polynucleotide can be the length of the polynucleotide sequence that is sufficient to encode for a dipeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a tripeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a tetrapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a pentapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a hexapeptide.
• the length of the polynucleotide sequence is sufficient to encode for a heptapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for an octapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a nonapeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode for a decapeptide.
• a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the polynucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides.
• the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides.
• the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides.
• the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.
• Nucleic acids and polynucleotides may include one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).
• all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the poly A tail, and any combination of (a, b, c, or d above) comprise naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).
• Nucleic acids and polynucleotides may include one or more alternative components, as described herein, which impart useful properties including increased stability and/or the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.
• an altemative polynucleotide or nucleic acid exhibits reduced degradation in a cell into which the polynucleotide or nucleic acid is introduced, relative to a corresponding unaltered polynucleotide or nucleic acid.
• These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotides, and/or viability of contacted cells, as well as possess reduced
• Polynucleotides and nucleic acids may be naturally or non-naturally occurring.
• Polynucleotides and nucleic acids may include one or more modified (e.g., altered or altemative) nucleobases, nucleosides, nucleotides, or combinations thereof.
• the nucleic acids and polynucleotides useful in the nanoparticle compositions described herein can include any useful modification or alteration, such as to the nucleobase, the sugar, or the intemucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone).
• alterations e.g., one or more alterations are present in each of the nucleobase, the sugar, and the intemucleoside linkage.
• Alterations according to the present disclosure may be alterations of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), e.g., the substitution of the 2 ' -OH of the ribofuranosyl ring to 2 ' -H, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or hybrids thereof. Additional alterations are described herein.
• Polynucleotides and nucleic acids may or may not be uniformly altered along the entire length of the molecule.
• one or more or all types of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
• nucleotide may or may not be uniformly altered in a polynucleotide or nucleic acid, or in a given predetermined sequence region thereof.
• nucleotides X in a polynucleotide are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
• nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.
• An alteration may also be a 5 ' - or 3 ' -terminal alteration.
• the polynucleotide includes an alteration at the 3 ' -terminus.
• the polynucleotide may contain from about 1% to about 100% alternative nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from
• Polynucleotides may contain at a minimum zero and at maximum 100% alternative nucleotides, or any intervening percentage, such as at least 5% alternative nucleotides, at least 10% alternative nucleotides, at least 25% alternative nucleotides, at least 50% alternative nucleotides, at least 80% alternative nucleotides, or at least 90% alternative nucleotides.
• polynucleotides may contain an alternative pyrimidine such as an alternative uracil or cytosine.
• At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in a polynucleotide is replaced with an alternative uracil (e.g., a 5-substituted uracil).
• the alternative uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
• cytosine in the polynucleotide is replaced with an alternative cytosine (e.g., a 5-substituted cytosine).
• an alternative cytosine e.g., a 5-substituted cytosine
• the alternative cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
• nucleic acids do not substantially induce an innate immune response of a cell into which the polynucleotide (e.g., mRNA) is introduced.
• a cell into which the polynucleotide e.g., mRNA
• features of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc., and/or 3) termination or reduction in protein translation.
• the nucleic acids can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, and vectors).
• the nucleic acids may include one or more messenger RNAs (mRNAs) having one or more alternative nucleoside or nucleotides (i.e., alternative mRNA molecules).
• a nucleic acid (e.g. mRNA) molecule, formula, composition or method associated therewith comprises one or more polynucleotides comprising features as described in WO2002/098443, WO2003/051401, WO2008/052770,
• the alternative nucleosides and nucleotides can include an alternative nucleobase.
• a nucleobase of a nucleic acid is an organic base such as a purine or pyrimidine or a derivative thereof.
• a nucleobase may be a canonical base (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties, e.g., increased stability such as resistance to nucleases.
• Non-canonical or modified bases may include, for example, one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and/or thio substitutions; one or more fused or open rings; oxidation; and/or reduction.
• Alternative nucleotide base pairing encompasses not only the standard adenine- thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and/or alternative nucleotides including non-standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non- standard base structures.
• non-standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.
• the nucleobase is an alternative uracil.
• Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine ( ⁇ ), pyridin-4- one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s 2 U), 4-thio- uracil (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uracil (ho 5 U), 5-aminoallyl- uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m U), 5-methoxy- uracil (mo 5 U), uracil 5-oxyacetic acid (cmo 5 U), uracil 5-oxyacetic acid methyl ester (mc
• 2- thio-dihydropseudouridine 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uracil (acp U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ⁇ ), 5- (isopentenylaminomethyl)uracil (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm 5 s 2 U), 5,2'-0-dimethyl-uridine (m 5 Um), 2-thio-2'-0_methyl-uridine (s 2 Um), 5- methoxycarbonylmethyl-2'-0-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-0-methyl- ur
• the nucleobase is an alternative cytosine.
• nucleobases and nucleosides having an alternative cytosine include 5-aza-cytosine, 6-aza- cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl- cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5- iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1 -methyl-pseudoisocytidine, pyrrolo- cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio- pseudoisocy tidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl
• the nucleobase is an alternative adenine.
• Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6- chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenine (ml A), 2-methyl-adenine (m2A), N6- methyl-adenine (m6A),
• the nucleobase is an alternative guanine.
• Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQl
• the alternative nucleobase of a nucleotide can be independently a purine, a pyrimidine, a purine or pyrimidine analog.
• the nucleobase can be an alternative to adenine, cytosine, guanine, uracil, or hypoxanthine.
• the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and
• each letter refers to the representative base and/or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine).
• Nucleosides include a sugar molecule (e.g., a 5-carbon or 6-carbon sugar, such as pentose, ribose, arabinose, xylose, glucose, galactose, or a deoxy derivative thereof) in combination with a nucleobase, while nucleotides are nucleosides containing a nucleoside and a phosphate group or alternative group (e.g., boranophosphate, thiophosphate, selenophosphate, phosphonate, alkyl group, amidate, and glycerol).
• a sugar molecule e.g., a 5-carbon or 6-carbon sugar, such as pentose, ribose, arabinose, xylose, glucose, galactose, or a deoxy derivative thereof
• nucleotides are nucleosides containing a nucleoside and a phosphate group or alternative group (e.g., boranophosphate, thio
• a nucleoside or nucleotide may be a canonical species, e.g., a nucleoside or nucleotide including a canonical nucleobase, sugar, and, in the case of nucleotides, a phosphate group, or may be an alternative nucleoside or nucleotide including one or more alternative components.
• alternative nucleosides and nucleotides can be altered on the sugar of the nucleoside or nucleotide.
• the alternative nucleosides or nucleotides include the structure:
• each of m and n is independently, an integer from 0 to 5,
• each of U and U' independently, is O, S, N(R u ) nu , or C(R u ) nu , wherein nu is an integer from 0 to 2 and each R u is, independently, H, halo, or optionally substituted alkyl;
• each of R 1' , R 2 , R 1" , R 2 , R 1 , R 2 , R 3 , R 4 , and R 5 is, independently, if present, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent; wherein the combination of R 3
• R , R , R , R , or R e.g., the combination of R and R , the combination of R and R , the combination of R and R , the combination of R and R , or the combination of R 5 and R 3
• R e.g., the combination of R and R , the combination of R and R , the combination of R and R , the combination of R and R , or the combination of R 5 and R 3
• R e.g., the combination of R and R , the combination of R and R , the combination of R and R , or the combination of R 5 and R 3
• R e.g., the combination of R and R , the combination of R and R , the combination of R and R , or the combination of R 5 and R 3
• optionally substituted heterocyclyl e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl
• R 5 with one or more of R 1 , R 1 , R 2 , or R 2 can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); and wherein the combination of R and one or more of R , R , R , R , R , or R can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); each of m' and
• each of Y 1 , Y 2 , and Y 3 is, independently, O, S, Se,— NR N1 — , optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;
• each Y 4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
• each Y 5 is, independently, O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene; and
• B is a nucleobase, either modified or unmodified.
• the 2'-hydroxy group (OH) can be modified or replaced with a number of different substituents.
• Exemplary substitutions at the 2'-position include, but are not limited to, H, azido, halo (e.g., fluoro), optionally substituted C 1-6 alkyl (e.g., methyl); optionally substituted C 1-6 alkoxy (e.g., methoxy or ethoxy); optionally substituted Ce- ⁇ aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C6-10 aryl-C ⁇ alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -
• n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); "locked" nucleic acids (LNA) in which the 2'-hydroxy is connected by a C 1-6 alkylene or Ci-6 heteroalkylene bridge to the 4 ' - carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein.
• LNA locked nucleic acids
• RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
• exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (that also has a phosphoramidate backbone));
• the sugar group contains one or more carbons that possess the opposite stereochemical configuration of the corresponding carbon in ribose.
• a polynucleotide molecule can include nucleotides containing, e.g., arabinose or L-ribose, as the sugar.
• the polynucleotide includes at least one nucleoside wherein the sugar is L-ribose, 2 ' -O-methy l-ribose, 2 ' -fluoro-ribose, arabinose, hexitol, an LNA, or a PNA.
• nucleotides can be altered on the internucleoside linkage (e.g., phosphate backbone).
• phosphate backbone in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably.
• Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.
• the alternative nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein.
• alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
• Phosphorodithioates have both non-linking oxygens replaced by sulfur.
• the phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged
• the alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH 3 ), sulfur (thio), methyl, ethyl, and/or methoxy.
• a borane moiety BH 3
• sulfur (thio) thio
• methyl ethyl
• methoxy e.g., methoxy of two non-bridging oxygens at the same position
• two non-bridging oxygens at the same position e.g., the alpha (a), beta ( ⁇ ) or gamma ( ⁇ ) position
• the replacement of one or more of the oxygen atoms at the a position of the phosphate moiety is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages.
• Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
• internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.
• Polynucleotides may contain an internal ribosome entry site (IRES).
• IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
• a polynucleotide containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multicistronic mRNA).
• a second translatable region is optionally provided.
• IRES sequences that can be used according to the present disclosure include without limitation, those from picomaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
• picomaviruses e.g., FMDV
• CFFV pest viruses
• PV polio viruses
• ECMV encephalomyocarditis viruses
• FMDV foot-and-mouth disease viruses
• HCV hepatitis C viruses
• CSFV classical swine fever viruses
• MLV murine leukemia virus
• SIV simian immune deficiency viruses
• CrPV cricket paralysis viruses
• a polynucleotide (e.g., an mRNA) may include a 5 ' -cap structure.
• the 5 ' -cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for
• the cap further assists the removal of 5 ' -proximal introns removal during mRNA splicing.
• Endogenous polynucleotide molecules may be 5 ' -end capped generating a 5 ' -ppp-5 ' -triphosphate linkage between a terminal guanosine cap residue and the 5 ' -terminal transcribed sense nucleotide of the polynucleotide.
• This 5 ' -guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
• the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5 ' end of the polynucleotide may optionally also be 2'-0-methylated.
• 5 ' -decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation.
• Alterations to polynucleotides may generate a non-hydrolyzable cap structure preventing decapping and thus increasing polynucleotide half-life. Because cap structure hydrolysis requires cleavage of 5 ' -ppp-5 ' phosphorodiester linkages, alternative nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 ' -ppp-5 ' cap.
• a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 ' -ppp-5 ' cap.
• guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.
• Additional alterations include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 ' -terminal and/or 5 ' -anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxy group of the sugar.
• Multiple distinct 5 ' -cap structures can be used to generate the 5 ' -cap of a polynucleotide, such as an mRNA molecule.
• 5 ' -Cap structures include those described in International Patent Publication Nos.
• Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type, or physiological) 5 ' -caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and/linked to a polynucleotide.
• the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5 ' -5 ' -triphosphate group, wherein one guanosine contains an N7-methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5 ' -triphosphate-5 ' -guanosine, m 7 G-3'mppp-G, which may equivalently be designated 3' 0-Me-m7G(5 ' )ppp(5 ' )G).
• the 3'-0 atom of the other, unaltered, guanosine becomes linked to the 5 ' -terminal nucleotide of the capped polynucleotide (e.g., an mRNA).
• the N7- and 3'-0-methlyated guanosine provides the terminal moiety of the capped polynucleotide (e.g., mRNA).
• mCAP which is similar to ARCA but has a 2'-0- methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5 ' -triphosphate-5 ' -guanosine, m 7 Gm-ppp-G).
• a cap may be a dinucleotide cap analog.
• the dinucleotide cap analog may be modified at different phosphate positions with a
• boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. 8,519,110, the cap structures of which are herein incorporated by reference.
• a cap analog may be a N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and/or described herein.
• Non-limiting examples of N7- (4-chlorophenoxy ethyl) substituted dinucleotide cap analogs include a N7-(4- chlorophenoxyethyl)-G(5 )ppp(5 ')G and a N7-(4-chlorophenoxyethyl)-m3 '-OG(5 ')ppp(5 ')G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al.
• a cap analog useful in the polynucleotides of the present disclosure is a 4-chloro/bromophenoxy ethyl analog.
• cap analogs allow for the concomitant capping of a polynucleotide in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5 ' -cap structures of polynucleotides produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
• Alternative polynucleotides may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5 ' -cap structures.
• the phrase "more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function, and/or structure as compared to synthetic features or analogs of the prior art, or which outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects.
• Non-limiting examples of more authentic 5 ' -cap structures useful in the polynucleotides of the present disclosure are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5 ' -endonucleases, and/or reduced 5 ' - decapping, as compared to synthetic 5 ' -cap structures known in the art (or to a wild-type, natural or physiological 5 ' -cap structure).
• recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 ' -5 ' -triphosphate linkage between the 5 ' -terminal nucleotide of a polynucleotide and a guanosine cap nucleotide wherein the cap guanosine contains an N7-methylation and the 5 ' -terminal nucleotide of the polynucleotide contains a 2'-0-methyl.
• Capl structure Such a structure is termed the Capl structure.
• cap results in a higher translational-competency, cellular stability, and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 ' cap analog structures known in the art.
• Other exemplary cap structures include 7mG(5 ')ppp(5 ')N,pN2p (Cap 0),
• the alternative polynucleotides may be capped post-transcriptionally, and because this process is more efficient, nearly 100% of the alternative polynucleotides may be capped. This is in contrast to -80% when a cap analog is linked to an polynucleotide in the course of an in vitro transcription reaction.
• 5 ' -terminal caps may include endogenous caps or cap analogs.
• a 5 ' -terminal cap may include a guanosine analog.
• Useful guanosine analogs include inosine, Nl-methyl- guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine.
• a polynucleotide contains a modified 5 ' -cap.
• a modification on the 5 ' -cap may increase the stability of polynucleotide, increase the half-life of the
• the modified 5 ' - cap may include, but is not limited to, one or more of the following modifications: modification at the 2 ' - and/or 3 ' -position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH 2 ), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety.
• GTP capped guanosine triphosphate
• CH 2 methylene moiety
• G nucleobase
• a 5 -UTR may be provided as a flanking region to polynucleotides (e.g., mRNAs).
• a 5 ' -UTR may be homologous or heterologous to the coding region found in a polynucleotide.
• Multiple 5 ' -UTRs may be included in the flanking region and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and/or after codon optimization.
• each 5'-UTR (5 '-UTR-005 to 5 '-UTR 68511) is identified by its start and stop site relative to its native or wild type (homologous) transcript (ENST; the identifier used in the ENSEMBL database).
• ENST wild type (homologous) transcript
• 5 ' -UTRs which are heterologous to the coding region of an alternative polynucleotide (e.g., mRNA) may be engineered.
• the polynucleotides e.g., mRNA
• the polynucleotides may then be administered to cells, tissue or organisms and outcomes such as protein level, localization, and/or half-life may be measured to evaluate the beneficial effects the heterologous 5 ' -UTR may have on the alternative polynucleotides (mRNA).
• Variants of the 5 ' -UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G. 5 ' -UTRs may also be codon-optimized, or altered in any manner described herein.
• the 5 ' -UTR of a polynucleotides may include at least one translation enhancer element.
• translation enhancer element refers to sequences that increase the amount of polypeptide or protein produced from a polynucleotide.
• the TEE may be located between the transcription promoter and the start codon.
• the polynucleotides (e.g., mRNA) with at least one TEE in the 5 ' -UTR may include a cap at the 5 ' -UTR. Further, at least one TEE may be located in the 5 ' -UTR of polynucleotides (e.g., mRNA) undergoing cap-dependent or cap-independent translation.
• TEEs are conserved elements in the UTR which can promote translational activity of a polynucleotide such as, but not limited to, cap-dependent or cap- independent translation.
• the conservation of these sequences has been previously shown by Panek et al. (Nucleic Acids Research, 2013, 1-10) across 14 species including humans.
• the TEEs known may be in the 5 -leader of the Gtx homeodomain protein (Chappell et al, Proc. Natl. Acad. Sci. USA 101 :9590-9594, 2004, the TEEs of which are incorporated herein by reference).
• TEEs are disclosed in US Patent Publication Nos. 2009/0226470 and 2013/0177581, International Patent Publication Nos. WO2009/075886, WO2012/009644, and WO 1999/024595, US Patent Nos. 6,310,197 and 6,849,405, the TEE sequences disclosed in each of which are incorporated herein by reference.
• the TEE may be an internal ribosome entry site (IRES), HCV-IRES or an IRES element such as, but not limited to, those described in US Patent No. 7,468,275, US Patent Publication Nos. 2007/0048776 and 2011/0124100 and International Patent Publication Nos. WO2007/025008 and WO2001/055369, the IRES sequences of each of which are incorporated herein by reference.
• the IRES elements may include, but are not limited to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci.

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