NL2026825B1 - Multicomponent delivery system for polyanionic cargo compound delivery - Google Patents

Abstract

The present disclosure relates to multicomponent delivery systems for delivering polyanionic cargo compounds such as nucleic acids. The present disclosure also relates to methods of preparing and using the multicomponent delivery systems.

Description

MULTICOMPONENT DELIVERY SYSTEM FOR POLYANIONIC CARGO COMPOUND DELIVERY FIELD OF THE DISCLOSURE

[0001] The present disclosure generally relates to multicomponent delivery systems for delivering polyanionic cargo compounds such as nucleic acids, and methods of making and using the multicomponent delivery systems.

BACKGROUND

[0002] Therapeutic nucleic acids include, e.g., mRNA, small interfering RNA (siRNA), small activating RNA (saRNA), micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, and immune stimulating nucleic acids. These nucleic acids act via a variety of mechanisms. A safe and effective delivery system is required for the nucleic acids to be therapeutically useful. Viral vectors are relatively efficient gene delivery systems, but suffer from a variety of limitations, such as the potential for reversion to the wild-type as well as immune response concerns. Furthermore, viral systems are rapidly cleared from the circulation, limiting transfection to organs such as the lungs, liver, and spleen. In addition, these systems induce immune responses that compromise delivery with subsequent injections.

[0003] Thus, nonviral gene delivery systems are being developed such as reverse micelles, anionic liposomes, cationic liposomes, and polymer liposomes. There still remains a strong need in the art for methods and compositions that can be optimized for delivering various types of nucleic acid material to a range of different target cells.

BRIEF SUMMARY {0004] The present disclosure provides multicomponent delivery systems comprising at least one cationic lipidated peptoid component for the delivery of polyanionic compounds, such as nucleic acids, with improved efficiency. These polyanionic compounds are also referred to as polyanionic cargo compounds or cargos of the multicomponent delivery system. Other optional components in the multicomponent delivery systems may include anionic or zwitterionic components, lipid components, and shielding components.

[0005] In yet another aspect of the present disclosure, provided herein are delivering a polyanionic cargo compound to a cell comprising contacting the cell with a complex formed by the multicomponent delivery system and the polyanionic cargo compound.

[0006] The disclosure provides a system comprising at least one cationic component, wherein the cationic component is a lipidated peptoid.

[0007] The lipidated peptoid can be a tertiary amino lipidated and/or PEGylated cationic peptoid or a lipitoid.

[0008] The molar percentage of the lipidated peptoid can be 10.0-99.5 mol®%.

[0009] The system can further comprise at least one shielding component.

[0010] The shielding component preferably comprises a poly(ethylene glycol) (PEG) moiety, for example a PEGylated lipid or a PEGylated lipidated peptoid, and can be DMG-PEG or Compound 107 from Table 1.

[0011] The molar percentage of the shielding component can be 1-5 mol%. {0012] The system can further comprise at least one anionic or zwitterionic component, for example wherein the zwitterionic component comprises a phospholipid.

[0013] The zwitterionic component can be selected from a phospholipid, a lipitoid, or a mixture thereof.

[0014] The zwitterionic component can be a DOPE, DSPC or Compound 105. f0015] The molar percentage of the anionic or zwitterionic component can be in the range of about 10 to about 60 mol%.

[0016] The system can further comprises at least one lipid component, for example wherein the lipid component comprises a sterol.

[0017] The lipid component can be a cholesterol, Compound 89 or Compound 90 from Table

1.

[0018] The molar percentage of the lipid component can be about 50 to about 85 mol%. {0019] The system can comprise a mixture of an anionic or zwitterionic component and a lipid component.

[0020] The molar percentage of the mixture of the anionic or zwitterionic component and the lipid component can be about 60 to about 80 mol%.

[0021] The system can comprise at least 99 mol% cationic component and less than 1 mol% shielding component, and preferably comprises Formula F1 as disclosed herein.

[0022] The system can comprise less than 20 mol% cationic component, less than 5 mol% shielding component, and more than 75 mol% a mixture of a zwitterionic component and a lipid component, and preferably comprises Formula F2 or Formula F4 as disclosed herein.

[0023] The system can comprise between 30-45 mol% cationic component, between 50-70 mol% a mixtare of a zwitterionic component and a lipid component, and between 1.5-4.5 mol% shielding component, and preferably comprises Formula F3 or Formula F5 as disclosed herein. {0024] The system can comprise between 15-35 mol% cationic component, between 60-80 mol% a mixture of a zwitterionic component and a lipid component, and between 1.5-3.0 mol% shielding component, and preferably comprises Formula F2 or Formula F3.

[0925] The disclosure also includes a acomplex comprising this system and a polyanionic compound, preferably wherein the polyanionic compound is a nucleic acid, more preferably wherein the nucleic acid is an mRNA encoding a protein.

[0026] The disclosure also includes a method of delivering a polyanionic compound to a cell comprising contacting the cell with this complex.

[0027] An additional aspect of this disclosure provides methods of preparing the system and the complex comprising contacting the system and polyanionic compounds.

[0028] The disclosure also includes the following sub-sections:

1. A system for the delivery of polyanionic compounds such as nucleic acids to target cells, the delivery system comprising at least one cationic component, wherein the at least one cationic component comprises a lipidated peptoid.

2. The system of subsection] further comprising one or more of the following: e an anionic or zwitterionic component, * a non-cationic lipid component; e a shielding component.

3. The system of subsection 2, wherein the anionic or zwitterionic component is selected from a phospholipid, a lipitoid, or a mixture thereof, preferably wherein the anionic or zwitterionic component is a DOPE, or DSPC.

4. The system of subsections 2 or 3 wherein the non-cationic lipid component comprises a sterol, for example cholesterol, and/or a neutral peptoid.

5. The system of any of the preceding subsections 2-4 wherein the shielding component comprises a poly(ethylene glycol) (PEG) moiety, preferably wherein the shielding component is a PEGylated lipid or a PEGylated lipidated peptoid, more preferably wherein the shielding component is DMG-PEG, for example DMGPEG2k.

6. The system of subsection 4 or 5, wherein the non-cationic lipid component comprises Compound 90:

A N, 0

N N \ \ or wherein the non-cationic lipid component comprises Compound 89 AY A, |

N N \ \

7. The system according to any of the preceding subsections 2-6 wherein the cationic component comprises compound 112:

HN N N N and the non-cationic lipid component comprises Compound 90 : A A, 0

N N A \ 5 8 The system according to subsection? wherein the mol % of the cationic component is between about 20 to about 50; the mol % of the anionic or zwitterionic component, is between about 0 to about 30; the mol % of the non-cationic lipid component is between about 30 to about 80; and the mol % of the shielding component is between about 0 to about 10.

9. The system according to subsection 7 or 8 wherein the mol % of the cationic component is between about 30 to about 50; the mol % of the anionic or zwitterionic component,is about 0 to about 10; the mol % of the non-cationic lipid component is about 40 to about 70; and the mol % of the shielding component at about to about 5.

10. The system according to any of the subsections 7-9 wherein the mol % of the cationic component is 38; the mol % of the anionic or zwitterionic component is 0; the mol % of the non- cationic lipid component is 59.7; and the mol % of the shielding component is 2.3.

11. The system according to subsection 10, wherein the anionic or zwitterionic component is DOPE, and wherein the shielding component is DMG-PEG2K.

12. The system according to any of the subsections 1-5, wherein the cationic component comprises Compound 93, as referred to in Table 1, and the non-cationic lipid component comprises cholesterol, Compound 90, as referred to in Table 1, Compound 89, as referred to in Table 1, or any combination thereof.

13. The system according to any of the subsections 1-5 wherein the cationic component comprises Compound 79, as referred to in Table 1, and the non-cationic lipid component comprises Compound 90 as referred to in Table 1.

14. The system according to any of the subsections 1-5 wherein the cationic component comprises Compound 24 and the non-cationic lipid component comprises cholesterol, Compound 89, as referred to in Table 1, Compound 90, as referred to in Table 1, or any combination thereof .

15. The system of any of the preceding subsections 2-5 comprising at least about 99 mol% cationic component and less than about 1 mol% shielding component.

16. The system according to subsection 15 wherein the cationic component comprises compounds, 24, 79, 93 or 112, as referred to in Table 1 , or any combination thereof.

17. The system according to subsection 15 or 16 wherein the mol % of the cationic component is 99.1, and the mol % of the shielding component comprising PEG is 0.9.

18. The system according to any of the preceding subsections 2-5 comprising less than about 20 mol% of the cationic component, less than about 5 mol% of the shielding component, and more than about 75 mol% of a mixture of the zwitterionic component and the non-cationic lipid component.

19. The system of subsection 18, wherein the cationic component comprises one or more of the compounds, 24, 79, 93 or 112, as referred to in Table 1 or any combination thereof.

20. The system of subsection 18 or 19, wherein the mol % of the cationic component is 17.9, the mol % of the shielding component comprising PEG is 2.8, the mol % of the non-cationic lipid component is 62.9 , and the mol % of the anionic or zwitterionic component, is 16.4.

21. The system of subsection 18 or 19, wherein the mol % of the cationic component is 17.1, the mol % of the shielding component comprising PEG is 2.7, the mol % of the non-cationic lipid component is 80.2.

22. The system according to any of the subsections 2-5 comprising between about 30 and about 45 mol% of the cationic component, between about 50 and about 70 mol% of a mixture of the anionic or zwitterionic component and the non-cationic lipid component, and between about

1.5 about 4.5 mol% of the shielding component.

23. The system of subsection 22 wherein the cationic component comprises one or more of the compounds 24, 79, 93 or 112, as referred to in Table 1 , or any combination thereof.

24. The system of subsection 22 or 23 wherein the mol % of the cationic component is 32.9, the mol % of the shielding component comprising PEG is 2.0, the mol % of the non-cationic comprising cholesterol is 51.7, and the mol % of the anionic or zwitterionic component is 13.4,

25. The system of subsection 22 or 23 wherein the mol % of the cationic component is 42.3, the mol % of the shielding component comprising PEG is 4.4,, the mol % of the non-cationic lipid 19 component is 0.0 , and the mol % of the anionic or zwitterionic component is 53.3.

26. The system according to any of the subsections 2-5 comprising between about 15 and about 35 mol% of the cationic component, between about 60 and about 80 mol% of a mixture of the zwitterionic component and the non-cationic lipid component, and between about 1.5 and about

3.0 mol% of shielding component.

27. The system of subsection 26 wherein the cationic component comprises one or more of the compounds 24, 79, 93 or 112 as referred to in Table 1, or any combination thereof.

28. The system of subsection 26 or 27, wherein the mol % of the cationic component is 17.9, the mol % of the shielding component comprising PEG is 2.8, the mol % of the non-cationic lipid component is 62.9 , and the mol % of the anionic or zwitterionic component, is 16.4.

29. The system of subsection 26 or 27 wherein the mol % of the cationic component is 32.9, the mol % of the shielding component comprising PEG is 2.0, the mol % of the non-cationic lipid component is 51.7 , and the mol % of the anionic or zwitterionic component is 13.4.

30. A complex comprising the system of any of the preceding subsections and a polyanionic compound, preferably a nucleic acid.

31. The complex of subsection 30 wherein the polyanionic compound is an mRNA encoding a protein.

32. The complex according to subsections 30 or 31 wherein the mass ratio of the cationic component to the nucleic acid is between 0.5:1 and 20:1, between 0.5:1 and 10:1, between 0.5:1 and 5:1, between 1:1 and 20:1, between 1:1 and 10:1, between 1:1 and 5:1, between 2:1 and 20:1, between 2:1 and 10:1, between 2:1 and 5:1, for example wherein the complex comprises the lipidated cationic peptoids and the nucleic acid at a mass ratio of 3:1.

33. The complex according to subsection 32 wherein the cationic component is compound 112 as referred to in Table 1, and the non-cationic lipid component is Compound 90 as referred to in Table 1.

34. The complex according to subsection 33 wherein the shielding component is DMG- PEG2k.

35. The complex according to any of the subsections 31-34 wherein mass ratio of the cationic compound to the nucleic acid is 10:1.

36. The complex according to subsection 35, wherein the mass ratio of the shielding component to the nucleic acid 1s 1.4:1.

37. The complex according to subsections 35 or 36 wherein the mass ratio component of the non-cationic lipid component to the nucleic acid is 5.4:1.

38. The complex according to any of the subsections 35-37 wherein a charge ratio between the cationic component and the nucleic acid is 6:1. 39, The complex according to any of the subsections 30-38, wherein mass ratio of the cationic compound to the nucleic acid is 10:1, the mass ratio of the shielding component to the nucleic acid is 1.4 :1, or 1.38:1, and the mass ratio component of the non-cationic lipid component to the nucleic acid is 5.4:1 or 5.36: 1. 40 The complex according to any of the subsections 30-39, comprising the system according to any of the subsections 7-11.

41. The system according to any of the subsections 7-11 for use in delivering a therapeutic nucleic acid, preferably an mRNA.

42. The system according to any of the subsections 1-29 or the complex according to any of the subsections 30-40 for use as a medicament.

43. The system according to any of the subsections 1-29 or the complex according to any of the subsections 30-40 for use for use in the treatment of any of the conditions referred to in the description herein.

44. A method of forming the complex of any of the subsections 30-40 comprising the step of contacting a system according to any of the subsections 1-29, with a polyanionic compound, for example a nucleic acid, preferably an mRNA encoding a protein.

DETAILED DESCRIPTION

[0029] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary examples.

[0030] The cellular and in vivo delivery of oligonucleotides remains a significant hurdle in the field of gene therapy. Therefore, chemical strategies to enhance the bioavailability of this class of drugs remain in significant demand. Notably, prior systems in the area of chemical agents for nucleic acid delivery have focused on cationic lipid nanoparticles (LNPs), and polycationic polymers. LNPs have traditionally included 4 lipid-like components which utilize hydrophobic interactions to self-assemble into lamellar or non-lameliar particles and encapsulate anionic oligonucleotides. Polymeric delivery agents typically utilize molecules with numerous cationic charges to electrostatically bind the nucleic acid into a nano-sized coacervate. These cationic materials sometimes include additional elements such as hydrophilic shielding (such as poly(ethylene glycol) (PEG), etc.) or lipid elements through block co-polymer structures. These domains are most commonly incorporate onto the same polymer in what can be described as a “jack of all trades” strategy in an attempt to balance the many necessary functions for nucleic acid delivery. However, while lipid nanoparticle formulations have often melded multiple functions (such as cationic nucleic acid binding, PEG shielding, and lipid fluidity, etc.) by lipid anchoring into the LNP, relatively little innovation has taken place to systematically incorporate multiple polycationic elements with specific functions into a single particle. Components of the Multicomponent Delivery System

[0031] The present disclosure provides a multicomponent polyanionic compound delivery vehicle system utilizing a plurality of components to contribute specific features to the resulting complex. “Multicomponent” as used herein refers to a composition or complex with more than one structurally different compound (also referred to as component). The multicomponent delivery system comprises complexes of at least one peptoid compound. “Peptoid™ as used herein refers to peptidomimetic compounds wherein the nitrogen atom of the peptide backbone are substituted with side chains. Peptoids where the side chains on the nitrogen atom comprise lipids are referred to as “lipidated peptoids”. “Nucleic acids” as used herein refers to any oligonucleotide compounds, such as but not limited to naturally occurring nucleic acids such as DNA, RNA, and/or hybrids thereof, as well as unnaturally occurring variations with unnatural backbone and modified backbone linkages such as phosphorothioate, unnatural and modified bases, and unnatural and modified termini. Non-limiting examples of nucleic acids include mRNA, small interfering RNA (siRNA), small activating RNA (saRNA), micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, and immune stimulating nucleic acids.

[0032] In some examples, the multicomponent delivery system comprises lipidated cationic peptoid compounds. The lipidated cationic peptoid compounds have cationic anchoring groups that associate with the nucleic acids through electrostatic interactions between the cationic anchoring groups and the negative phosphodiester backbone of the nucleic acids.

[0033] In some examples, the multicomponent delivery system comprises cationic peptoid compounds in combination with further components. These further components each serve a specific function or functions in the delivery vehicle such that all functionality for cellular delivery does not need to be accomplished by a single entity. These functions can be isolated, or can be served in concert through cooperation between two or more components. The further components may comprise additional peptoid compounds including cationic peptoids, neutral lipidated peptoids, anionic peptoids, or zwitterionic peptoids. They could further comprise lipids, such as structural lipids, phospholipids, and/or shielding lipids to prevent aggregation. The further components may comprises other peptoids that function as structural lipids, phospholipids, and/or shielding lipids. Cationic Component Cationic Anchor

[0034] The multicomponent delivery system in the present disclosure comprises one or more compounds that have a cationic anchoring portion which contributes to their association with nucleic acid cargos and other components. The compounds that have a cationic anchoring portion may be lipidated cationic peptoids that can form complexes with counterbalance the negative charge on the polyanionic cargoes, such as nucleic acids, thus promoting uptake of the cargoes into the target cell. The lipidated cationic peptoids as described herein have a net zero charge or a net positive charge. In some examples wherein the multicomponent delivery system has one or more cationic compounds, the one or more lipidated cationic peptoids independently have a net zero charge or a net positive charge. In certain examples, the one or more lipidated cationic peptoids independent have a net positive charge of at least +1. It should be recognized that the net charge present on the one or more lipidated cationic peptoids may vary depending upon environmental conditions. For example, in some examples, the one or more lipidated cationic peptoids independently have a stable, net positive charge at physiologically relevant pH ranges. For example, physiological pH is at least about 5.5 and typically at least about 6.0. More typically, physiological pH is at least about 6.5. Usually, physiological pH is less than about 8.5 and typically less than about 8.0. More typically, physiological pH is less than about 7.5.

[0035] In some variations of the foregoing, the multicomponent delivery system comprises

10.0-99.5 mol% (molar percentage) of lipidated cationic peptoids of the total number of moles of the multicomponent delivery system. In the present disclosure, when the mol% of a component is discussed, the polyanionic cargo compound is not calculated in the total number of moles. The mol% of a component is the number of moles of this component divided by the total number of moles of all the components of the multicomponent delivery system.

[0036] In certain variations, the multicomponent delivery system comprises between 10.1-20.0 mol%, 20.1-30.0 mol%, 30.1-40.0 mol%, 40.1-50.0 mol%, 50.1-60.0 mol%, 60.1-70.0 mol%, 70.1-

80.0 mol%, 80.1-90.0 mol%, or 90.1-99.5 mol% of lipidated cationic peptoids of the total number of moles of the multicomponent delivery system.

[0037] In some examples, the lipidated cationic peptoids utilized in the compositions as provided herein may include cationic peptoid-phospholipid conjugate constructs, also known as “lipitoids.” ln some examples, the lipidated cationic peptoids utilized in the multicomponent delivery system may include N-substituted cationic peptide compounds possessing lipid moieties and/or (oligo- and/or poly)ethylene glycol moieties throughout the peptide backbone, herein referred to as “tertiary amino lipidated and/or PEGylated cationic peptoids.” In some examples, the multicomponent delivery system of the present disclosure comprises complexes comprising one or more tertiary amino lipidated and/or PEGylated cationic peptoids. In other examples, the multicomponent delivery system comprises complexes comprising one or more lipitoids. In still other examples, the multicomponent delivery system comprises complexes comprising one or more tertiary amino lipidated and/or PEGylated cationic peptoids, one or more lipitoids, or any combinations thereof.

[0038] In some examples, the tertiary amino lipidated and/or PEGylated cationic peptoids comprise an oligopeptide backbone, wherein the oligopeptide backbone comprises repeating subunits of N-substituted cationic amino acid residues optionally interleaved with N-substituted neutral (“spacer”) and/or lipid amino acid residues. The oligopeptide backbone is further capped at the N- and/or C-terminus by amino acid residues that are N-substituted with lipid moieties (“N- lipidated™) and/or N-substituted with oligoethylene glycol and/or polyethylene glycol (“N- PEGylated™).

[0039] In some examples, the cationic components are tertiary amino lipidated and/or PEGylated cationic peptoids selected from any tertiary amino lipidated and/or PEGylated cationic peptide compounds disclosed in WO2020/069445, such as tertiary amino lipidated and/or PEGylated cationic peptide compounds of formula (I) or salts thereof: R2 R2 O R3 R2 O R+ R3 O RRO RRO RRO RRO gab af end dL Edd RP RP RP RP RP r RP RP q) wherein: m is an integer from 0 to 10; n is an integer from 0 to 5; s is an integer from (0 5; t is an integer from ( to 10; wherein at least one of m, n, s, and t is nonzero; r is an integer from 1 to 20; each o is independently an integer 0, 1, or 2;

each q is independently an integer 0, 1, or 2; each p is independently an integer 1 or 2; R' is -H, alkyl, alkylaryl, -COR™ or a lipid moiety, wherein R" is ~H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl; each R7 is independently an ethylene glycol moiety of the formula ~CH,CH,O{CH,CH,0),CH;, and wherein each u is independently an integer from 2 to 200; each R? is independently a lipid moiety; each R* is independently a neutral spacer moiety or a lipid moiety; each R'is independently a cationic moiety; each R° is independently an ethylene glycol (EG) moiety of the formula -CH-CH:O(CH;CH;0),CH;, and wherein each v is mndependently an integer from 2 to 200; R’ is —H, alkyl, acyl, -OH, -OR™, -NH,, -NHR", or a lipid moiety, wherein R™ is alkyl, acyl, or a lipid moiety; and each R° and R" are independently ~H, C,-C,-alkyl, or a side chain moiety found on a naturally- or non-naturally-occurring amino acid; Neutral Spacer Moieties

[0040] Within the oligopeptide backbone of tertiary amino lipidated and/or PEGylated cationic peptoids, the cationic amino acid residues may be optionally interleaved with neutral spacer amino acid residues, possessing a neutral spacer moiety at the N-position. The neutral amino acid residues may be useful to modulate the spatial distribution of the positive charge in the tertiary amino lipidated and/or PEGylated cationic peptide compounds for improved electrostatic interactions with the polyanionic cargo compounds, including polynucleotides, to be complexed with the cationic peptide compounds.

[0041] In each repeating subunit of the tertiary amino lipidated and/or PEGylated cationic peptoids, a neutral amino acid residue may present on either N- or C-terminal end of the cationic amino acid residue as one or more R* groups. In some examples wherein a subunit comprises a neutral spacer moiety R’, the corresponding o and/or ¢ for each neutral spacer moiety present represent the respective numbers of neutral spacer residues bonded to the N- and C-terminal ends of the cationic amino acid residue(s) within the subunit r. In some examples, each ¢ is independently an integer 0, 1, or 2. In other examples, each g is independently an integer 0, 1, or 2.

[0042] Each neutral spacer amino acid residue comprises a neutral spacer moiety R* at the N- position. As with the cationic moieties described herein, it should be recognized that each neutral spacer moiety R* is independently selected within the repeating subunit of the cationic peptide compounds as well as amongst the repeating subunits r of the oligopeptide backbone.

[0043] It should also be recognized that neutral spacer moieties may include any substituents that are neutral, or have zero net charge, at physiologically relevant pH ranges. In some examples, each neutral moiety R* is independently a C‚-C;-alkyl substituted by cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, wherein each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl is optionally substituted with one or more substituents ~OH, halo, or alkoxy. In still some examples, each neutral spacer moiety R* is independently selected from the AIO Oe Pu group consisting of: , , N, \=N and 0 ~O N NH in certain examples, each neutral spacer moiety R* is . Lipid Moieties

[0044] In addition to the optional interleaving of neutral amino acid residues with cationic amino acid residues within the oligopeptide backbone of tertiary amino lipidated and/or PEGylated cationic peptoids, N-lipidated amino acid residues, possessing a lipid moiety at the N-position, may also optionally be interleaved with the cationic {and optional neutral spacer) amino acid residues. In some examples wherein the tertiary amino lipidated and/or PEGylated cationic peptoid comprises N-lipidated amino acid residues, the tertiary amino lipidated and/or PEGylated cationic peptoid is N-lipidated. Similar to the neutral amino acid residues, the N-lipidated amino acid residues within the oligopeptide backbone may be useful to modulate the spatial distribution of the positive charge in the tertiary amino lipidated and/or PEGylated cationic peptoids as well as augment their lipophilicity for improved encapsulation of polyanionic materials and endocellular delivery. The spacing of lipids along the peptoid backbone may also influence the lipid fluidity/crystallinity which is known to influence cellular uptake and endosomal release.

[0045] As with the neutral spacer residues, in each repeating subunit of the tertiary amino lipidated and/or PEGylated cationic peptoids, the N-lipidated amino acid residue may present on either N- or C-terminal end or both ends of the cationic amino acid residue as one or more R* groups. In some examples wherein a subunit r comprises a lipid moiety RY, the corresponding o and/or g for each lipid moiety present may also represent the respective numbers of lipidated residues bonded to the N- and C-terminal ends of the cationic amino acid residue(s) within the subunit +. In some examples, each o is independently an integer 0, 1, or 2. In other examples, each q is independently an integer 0, 1, or 2.

[0046] Each N-lipidated amino acid residue comprises a lipid moiety R* at the N-position. As with the cationic and neutral moieties described herein, it should be recognized that each lipid moiety R* is independently selected within the repeating subunit of the cationic peptide compounds as well as amongst the repeating subunits r of the oligopeptide backbone.

[0047] Suitable lipid moieties may include, for example, optionally substituted branched or straight chain aliphatic moieties, or optionally substituted moieties derived from natural lipid compounds, including fatty acids, sterols, and isoprenoids.

[0048] In some examples, the lipid moieties may include branched or straight chain aliphatic moieties having from about 6 to about 50 carbon atoms or from about 10 to about 50 carbon atoms, i0 optionally comprising one or more heteroatoms, and optionally comprising one or more double or triple bonds {i.e., saturated or mono- or poly-unsaturated). In certain examples, the lipid moieties may include optionally substituted aliphatic, straight chain or branched moieties, each hydrophobic tail independently having from about 8 to about 30 carbon atoms or from about 6 to about 30 carbon atoms. In certain examples, the lipid moieties may include, for example, aliphatic carbon chains derived from fatty acids and fatty alcohols. In some examples, each lipid moiety R* is independently Cs-C:4-alkyl or Cs-C4-alkenyl, wherein the Cs-Cyy-alkenyl is optionally mono- or poly-unsaturated. In some examples, each lipid moiety R* is a Ce-Cg alkyl or C4-Cg alkenyl. In certain examples, each lipid moiety R* is C4-C2 alkyl. In still other examples, each lipid moiety R* is a Cyg-alkyl, such as n-decyl. In other examples, each lipid moiety R” is independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl.

[0049] In yet other examples which may be combined with any of the preceding examples, each lipid moiety R* is independently TTT “UT

TTT NNN 5 Leia i i ee |

EO ~~

ANN NNN NNN NJ

XX eS NTE U en , ‚or esse ee 5 . In still yet other examples which may be combined with any of the preceding examples, each lipid moiety R* is independently a lipid of the Oo. 9 o._R® Oo. formula 0 ‚ 0, O or © ‚ wherein R* is a branched or straight chain aliphatic moieties having from about 6 to about 50 carbon atoms or from about 10 to about 50 carbon atoms, optionally comprising one or more heteroatoms, and optionally comprising one or more double or triple bonds. In certain examples,

CI each lipid moiety R*is independently Oo ‚

IT Oo *

NOS SSN YO oO oO YU pot 0 ‚or Oo .

[0050] Natural lipid moieties employed in the practice of the present invention can be derived from, for example, phospholipids, glycerides (such as di- or tri-glycerides), glycosylglycerides, sphingolipids, ceramides, and saturated and unsaturated sterols, isoprenoids, and other like natural lipids.

10051] Other suitable lipid moieties may include lipophilic carbocyclic or aromatic groups such as optionally substituted aryl, cycloalkyl, cycloalkylalkyl, or arylalkyl moieties, including for example naphthalenyl or ethylbenzyl, or lipids comprising ester functional groups including, for example, sterol esters and wax esters. In still other examples, the lipid moiety R° is JL or — )

[0052] In some examples, the cationic components are lipitoids selected from any lipitoids disclosed in WO2020/069445, such as lipitoids of formula (11):

L RY °O R™ © ROA Oop OD NAY 7, \ 2 0 009 Mg "

an, wherein: xX is a integer from 1 to 100; each R° is independently a lipid moiety; and each R" is independently a cationic or neutral spacer moiety. Electrostatic binding

[0053] In some examples, the cationic component, such as lipidated cationic peptoids, has electrostatic binding portions. The electrostatic binding portions can contain much of the same elements are the cationic anchor, but typically a larger number of those groups to facilitate oligonucleotide condensation. In some examples, the lipidated cationic peptoids contains spacer groups or other functionality specifically to modulate interaction with oligonucleotide. This can be through incorporation of aromatic groups for pi-pi stacking interactions, hydrophobic or hydrophilic groups, or elements to buffer cationic charge. In some cases, the lipidated cationic peptoids can have the ability to degrade through either a hydrolytic or self-immolative mechanism to facilitate cargo release. Anionic and/or zwitterionic components

[0054] In some examples, the multicomponent delivery system comprises anionic components, zwitterionic components, or a mixture thereof. The functions of the anionic components include but not limited to buffering the particle zeta potential without affecting cargo ratios and/or contributing to particle endosomal escape through protonation at low pH in the endosome. The zwitterionic components have similar functions. Further, the zwitterionic components could hold particles together by interacting with both the cationic peptoid as well as the polyanionic cargo compounds. Including anionic components also allows for the creation of core-shell structures where first a net positive zeta potential particle is made (e.g., by mixing cationic lipidated peptoid and the cargo at a positive +/- charge ratio) and then coated with the anionic components. These negatively charged multicomponent system particles would avoid reticuloendothelial system (RES) clearance better than positively charged ones.

[0955] In some examples, the anionic and/or zwitterionic components are peptoids. In some examples, the anionic and/or zwitterionic components are lipidated peptoids. In some examples, the anionic and zwitterionic components are Compound 105. Phospholipids

[0056] Phospholipids are zwitterionic compounds and may be incorporated into the multicomponent delivery system of the present disclosure. Phospholipids provide further stabilization to complexes in solution as well as facilitate cell endocytosis, by virtue of their amphipathic character and ability to disrupt the cell membrane. In some examples, the compositions provided herein comprises complexes comprising one or more phospholipids as zwitterionic components.

[0057] In some examples, the multicomponent delivery system as described herein comprises one or more phospholipids. In some examples, the multicomponent delivery system comprises

10.0-60.0 mol% of one or more phospholipids of the total moles of the multicomponent delivery system. In certain variations, the multicomponent delivery system comprises 0-10.0 mol%, 10.1-

20.0 mol%, 20.1-30.0 mol%, 30.1-40.0 mol%, 40.1-50.0 mol®%, or 50.1-60.0 mol% of one or more phospholipids of the total moles of the multicomponent delivery system. In some examples, the one or more phospholipids are selected from the group consisting of 1.2-ditinoleoyl-sn-glyeero-3- phosphocholine {DLPC), {,2-dbmyristovi-sa-glycero-phosphochoting (DMPC), 1, 2-dickeoyi-sn- glyoero-3-phosphocholine (DOP), 1,2-dipalmitoyl-sn-giycero-3-phospbocholise (DPPC), 1.2- distearoyi-sn-glycero-3-phasphocholine (DSPC), 1 2-divndecanoyl-sn-giyeero-phosphocholine {(BUPCY, palmitoyl-2-oleovl-sn-glveero-3-phosphocheline (POPC), 1,2-di-O-ociadecenyi-sn- givegro-3-phosphocholine (18:0 Diether PC), L-oleoyl-2-cholesterylhemisacomoyl-sn-glycero-3- phosphocholine (QChemsPC,, I-hexadecyl-sn-glycers-3-phosphorholine {C16 Lyso PLD, 1,2- diinalensyl-sn-glycero-3-nhospbochoine, 1,2 Hargchidonoyl-sn-giycero-3-phosphocholine, 1,7 didocosahexsenoylsn-giycerc-2-phosphocholine, 1 2-dicteoylsn-glycero-3-phosphoetbanclamnme (DOPE), 2-dipalmitoyl-sn-glveere-3-phosphoethanolamine (DPPE), 1,2-diphytanoyvi-sn-glycero- 3-phosphosthanclamine (ME 16,0 PE}, 1, 2-dietearoylen-giycers-3-phosphocthanolsntne, 1,2- iljnoleoyi-sn-glycern-3-phosphoethanolamine, 1.2-diiinoienovi-so-giveere-3- phiosphoethanolamine, 1,2-diarschidonovlsn-giycero-3-phosphoethanolanune, 1,2- didocasahexaenovian-glycere-3-phosphoethancdamine, 1.2-doleoybsn-glycero-3-phosphorac-(i- glveerol) sodium ssn (DOPG), sphingomyelin, and mixtures theroof. In certain examples, the phospholipid is DOPE.

[0058] Alternatively, the multicomponent delivery system as described herein comprises lipidated peptoids, wherein the lipid moieties of the lipidated peptoids are phospholipids. In some examples, the lipid components are lipitoids. In some examples, the multicomponent delivery system comprises 10.0-60.0 mol% of one or more lipitoids of the total moles of the multicomponent delivery system. In certain variations, the multicomponent delivery system comprises 0-10.0 mol%, 10.1-20.0 mol%, 20.1-30.0 mol%, 30.1-40.0 mol%, 40.1-50.0 mol%, or

50.1-60.0 mol% of one or more lipitoids of the total moles of the multicomponent delivery system. In some examples, the phospholipid moiety of the lipitoids is selected from the group consisting of i 2-dilincleovisn-glyoera-3-phosphocholine (DLPU), LZ dimyristoybsn-glycero-phosphochshne (DMPC, 1 2-dioleoyi-an-glyeero-3-phosphocholine (DOPO), 1.2-dipaboitoyl-sn-glyeero-3- phosphocholing (DPPC), 1 2-distearoyl-sn-glycere-3-phosphocholine (DSP, 1 2-diundecanoyi-

sn-givcero-phosphoecholine (DUPT), Lpahmtoyl2-clezoyl-sr-glycero-3-phosphocholine (POPC), 12 di octadecenyb-sn-glycero-3-mhosphocholine (18:4 Diether PC), 1-oleoyi-2- chalesteryibemisnccinoyb-sn-giycero-3-phospbocholine (OClbersPC), L-hezadecybsn-glycero-3- phosphacholine {U 16 Lyso PC), 1,2-diiinclenoybsn-giycero-3-pbosprocbholine, 1,2- diarachidonoyl-srglycero-S-pbosphocholine, 2-didocosabexaenoyi-sn-glycero-3- vhosphocholine, 12-dioleoyl-sn-glycers-3-phosphosthanclamine (DOPE), 1, 2-dipalmitoyh-sn- givcero-3-phospboerhanolamine (DPPH), 1,2-diphytanoylsn-glycerc-3-phosphoethancianins: (ME

10.0 PE), 1 Z-distearoyksn-glycerc-3-phosphoethanolamine, 1,2 dimsalcoyl-sn-glycero-3- phosphoethanolamine, 1.2-Hinclenoybsn-glycero-3-phosphoethanolanune, 12 draracindonoyl-sn- glveero-3-phusphoethanolamine, 1.2-didocosahexaenoyvi-sn-glyeero-3-phosphoethanciamine, 1,2- disleoyl-sn-glycero-2mospho-rac-(l-giyceroD sodivm salt (DOPE), sphingomyelin, and mixtures thereof. In certain examples, the phospholipid is DOPE.

Lipid component for membrane association

[0059] In some examples, the multicomponent delivery system includes one or more lipid component comprising lipid moieties. The lipid component can be designed to degrade or hydrolyze to facilitate in vivo clearance of the multicomponent delivery system. The lipid moieties of the lipid components can be either strait-chain or branched, saturated, unsaturated, or aromatic. The lipid moieties of the structural lipid components may be naturally-occurring lipids, In some examples, the lipid moieties of the lipid components are non-cationic. Alternatively, the lipid component may be a lipidated peptoids comprising lipid moieties at the N-position.

[0060] In some examples, the multicomponent delivery system includes one or more structural lipid components. In some examples which may be combined with any of the foregoing examples, the multicomponent delivery system comprises 0-85.0 mol% one or more structural lipid components of the total moles of the multicomponent delivery system. In certain variations, the multicomponent delivery system comprises between 0-10.0 mol%, 10.1-20.0 mol%, 20.1-30.0 mol%, 30.1-40.0 mol%, 40.1-50.0 mol%, 50.1-60.0 mol%, 60.1-70.0 mol%, 70.1-80.0 mol%, or

80.1-85.0 mol% of structural lipid component of the total number of moles of the multicomponent delivery system.

[0061] In some examples, the lipid component is neutral.

[0062] In some examples, the lipid component is Compound 89 or 90. Sterols {0063] In some examples, the multicomponent delivery system as described herein comprises one or more sterols. In some examples, the one more sterols are selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigrnasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain examples, the sterol is cholesterol. In some examples, the multicomponent delivery system comprises 0-85.0 mol% cholesterol of the total moles of the multicomponent delivery system. In some examples, the multicomponent delivery system comprises 50.0-85.0 mol% cholesterol of the total moles of the multicomponent delivery system. In certain variations, the multicomponent delivery system comprises 0-10.0 mol%, 10.1-20.0 mol%, 20.1-30.0 mol%, 30.1-40.0 mol%, 40.1-50.0 mol%,

50.1-60.0 mol%, 60.1-70.0 mol%, 70.1-80.0 mol%, or 80.1-85.0 mol% of cholesterol of the total moles of the multicomponent delivery system. In certain variations, the multicomponent delivery system comprises at least 50 mol% of cholesterol of the total moles of the multicomponent delivery system.

[0064] In some examples, the multicomponent delivery system as described herein comprises one or more lipidated peptoids, wherein the lipid moieties of the lipidated peptoids are sterols. In some examples, the sterols are selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof. In certain examples, the lipid moieties of the lipidated peptoids are cholesterol. Such peptoids are referred to as “cholesteroids.” In some examples, the multicomponent delivery system comprises (-85.0 mol% cholesteroids of the total moles of the multicomponent delivery system. In certain variations, the multicomponent delivery system comprises 0-10.0 mol%, 10.1-20.0 mol%, 20.1-30.0 mol%, 30.1-40.0 mol%, 40.1-50.0 mol%,

50.1-60.0 mol%, 60.1-70.0 mol%, 70.1-80.0 mol%, or 80.1-85.0 mol% of cholesteroids of the total moles of the multicomponent delivery system. In certain variations, the multicomponent delivery system comprises at least 50 mol% of cholesteroids of the total moles of the multicomponent delivery system.

[0065] In some examples, the multicomponent delivery system comprises one or more phospholipids, cholesterol, lipitoids, cholesteroids, or a mixture thereof as structural lipid component.

[0066] In some examples, the multicomponent delivery system comprises molecules similar to cholesterol or phospholipid, such as sphingosine or phosphoinositides.

Particle stabilization and/or shielding component

[0067] Stabilization of the delivery vehicles can be accomplished through anchoring of hydrophilic polymers or other molecules to the surface of the delivery vehicles, including but not limited to poly(ethylene glycol), poly(propylene glycol), polysaccharides, and/or poly(phosphate)s. These groups can vary in molecular weight and/or length to modulate shielding properties. The shielding component can also be anchored into the particle via a lipid, or an anionic component to attach to a positive zeta potential multicomponent delivery system.

[0068] In some examples, the shielding component is cationic. In some examples, the shielding component is neutral. In some examples, the shielding component is zwitterionic. In some examples, the shielding component is anionic. In certain examples, the shielding component is a peptoid.

PEGylated lipids and PEGylated lipidated peptoids

[0069] In some examples, the multicomponent delivery system described herein comprises one or more PEGylated compounds. The one or more PEGylated compounds may be PEGylated lipids or PEGylated lipidated peptoids. It should be recognized that the term “PEGylated lipid” may also be interchangeably referred to as a “PEG lipid” or “PEG-modified lipid”. As described herein, PEGylated lipid may be understood to include any lipid or Hpid-like compounds covalently bound to a polyethylene glycol moiety. “PEGylated lipidated peptoids” may also be interchangeably referred to as a “PEG lipidated peptoids” or “PEG-modified lipidated peptoids”.

[0070] In some examples, the multicomponent delivery system comprise 0.5-5 mol% (such as

0.5-1.0 mol%, 1.1-2.0 mol®%, 2.1-3.0 mol%, 3.1-4.0 mol%, 4.1-5.0 mol%) one or more shielding component of the total weight of the multicomponent delivery system. In certain examples, the multicomponent delivery system comprises (.5-5 mol% {such as 0.5-1.0 mol%, 1.1-2.0 mol%, 2.1-

3.0 mol%, 3.1-4.0 mol%, 4.1-5.0 mol%) one or more PEGylated compounds of the total moles of the multicomponent delivery system. In still other examples, the multicomponent delivery system therein comprises 0.5-5 mol% (such as 0.5-1.0 mol%, 1.1-2.0 mol%, 2.1-3.0 mol%, 3.1-4.0 mol%,

4.1-5.0 mol%) 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) of the total moles of the multicomponent delivery system.

[0071] It should be recognized that particular molecular weights of the PEG chain in the foregoing PEGylated lipids and/or PEGylated lipidated peptoids may be especially advantageous for incorporation into the complexes of the present disclosure. For example, in some examples, the PEG chain has a molecular weight between 350 and 6,000 g/mol, between 1,000 and 5,000 g/mol, or between 2,000 and 5,000 g/mol. In certain examples, the PEG chain of the PEG lipid has a molecular weight of about 350 g/mol, 500 g/mol, 600 g/mol, 750 g/mol, 1,000 g/mol, 2,000 g/mol, 3,000 g/mol, 5,000 g/mol, or 10,000 g/mol. In certain other examples, the PEG chain of the PEGylated lipid has a molecular weight of about 500 g/mol, 750 g/mol, 1,000 g/mol, 2,000 g/mol or 5,000 g/mol. The PEG chain can be branched or linear. For example, in certain examples, the PEGylated lipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000).

{0072] Suitable lipid moieties for the PEGylated lipid or the PEGylated lipidated peptoids may include, for example, optionally substituted branched or straight chain aliphatic moieties, or optionally substituted moieties derived from natural lipid compounds, including fatty acids, sterols, and isoprenoids.

[0973] In some examples, the lipid moieties may include branched or straight chain aliphatic moieties having from about 6 to about 50 carbon atoms or from about 10 to about 50 carbon atoms, optionally comprising one or more heteroatoms, and optionally comprising one or more double or triple bonds (i.e., saturated or mono- or poly-unsaturated). In certain examples, the lipid moieties may include optionally substituted aliphatic, straight chain or branched moieties, each hydrophobic tail independently having from about 8 to about 30 carbon atoms or from about 6 to about 30 carbon atoms. In certain examples, the lipid moieties may include, for example, aliphatic carbon chains derived from fatty acids and fatty alcohols. In some examples, each lipid moiety is independently Cs-C4-alkyl or Cs-C:4-alkenyl, wherein the Cg-C4-alkenyl is optionally mono- or poly-unsaturated.

{0074] Natural lipid moieties employed in the practice of the present invention can be derived from, for example, phospholipids, glycerides (such as di- or tri-glycerides), glycosylglycerides, sphingolipids, ceramides, and saturated and unsaturated sterols, isoprenoids, and other like natural lipids.

[0075] Other suitable lipid moieties may include lipophilic aromatic groups such as optionally substituted aryl or arylalkyl moieties, including for example naphthalenyl or ethylbenzyl, or lipids comprising ester functional groups including, for example, sterol esters and wax esters.

[0076] In some examples, the one or more PEGylated lipids are 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, a PEG- modified dialkylglycerol, and any combinations thereof.

[0077] In some examples, the PEGylated lipids comprise a PEG-modified sterol. In certain examples, the PEGylated lipids comprises PEG-modified cholesterol.

[0078] In some examples, the PEGylated lipid is a PEG-modified ceramide. In certain examples, the PEG-modified ceramine is selected from the group consisting of N-octanoyl- sphingosine-1-{succinyl[methoxy(polyethylene glycol)] }and N-palmitoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)}}, and any combination thereof.

[0079] In some examples, the PEGylated lipids are PEG-modified phospholipid, wherein the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPQ), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterythemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-

dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholime, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero- 3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1- glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In certain examples, the phospholipid is DOPE.

[0080] In some examples, the one or more PEGylated lipids comprise a PEG-modified phosphatidylethanol. In some examples, the PEGylated lipid is a PEG-modified phosphatidylethanol selected from the group consisting of PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), and PEG-modified DOPE (DOPE-PEG).

[0081] In certain examples, the PEGylated lipid is selected from the group consisting of dimyristoylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerol-polyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), and dioleoylglycerol- polyethylene glycol (DOG-PEG). In certain examples, the PEG lipid is DMG-PEG.

[0082] In still further examples, the one or more PEGylated lipidated peptoids comprise a tertiary amino PEGylated cationic peptoids encompassed by formula (1), comprising at least one oligo- or polyethylene glycol moiety. For example, the PEGylated lipidated peptoids may be Compound 48, 56, 64, 72, 106, 107, 108 and 109 in Table 1. By virtue of their flexibility in accommodating both lipid and PEG moieties along the backbone of the peptide chain and depending upon the nature of their specific substituents, the tertiary amino lipidated and/or PEGylated cationic peptoids of formula (I) may serve not only as the cationic components but also as suitable PEGylated compounds to stabilize the multicomponent delivery system. It should be recognized that tertiary amino PEGylated cationic peptoids of formula (I) may be combined with other classes of cationic compounds such as lipitoids or lipid-like compounds as well as other tertiary amino lipidated and/or PEGylated cationic peptide compounds of formula (1).

Other peptoid components

[0083] The multicomponent delivery system comprises various peptoids components that have different functions. Thus, fine tuning of the multicomponent delivery system can be achieved to deliver different nucleic acid cargos to cells. In some examples, one or more of these additional peptoids are cationic. In some examples, one or more of these additional peptoids are neutral. In some examples, one or more of these additional peptoids are zwitterionic. In some examples, one or more of these additional peptoids are anionic. In some examples, at least one of these additional peptoids is not cationic.

[0084] Lipid fluidity/crystallinity of the components is known to influence cellular uptake and endosomal release. In some examples, the multicomponent delivery system comprises end-capped cholesteroids. In some examples, the lipidated peptoids (cationic or non-cationic) comprise aliphatic side chains such as cyclohexane, decalin, adamantane, (+)-dehydroabietylamine, or (-)- cis-myrtanylamine.

[0085] In some examples, peptoid components with sheets and helices in their structures can be included to provide more/less structure to domains. The helices may have hydrophobic and/or 19 hydrophilic faces. As a non-limiting example, low density lipoprotein (LDL) peptoid mimics may be included as a component in the multicomponent delivery system.

[0086] In some examples, the multicomponent delivery system comprises sugar-functionalized peptoids such as mannose-lipid, galactose, or phosphoinositide.

[0087] In some examples, the multicomponent delivery system comprises phosphoinositol compounds. The phosphoinositol compounds may be located on the outside of the multicomponent delivery system. The phosphoinositol compounds may be Phosphatidylinositol monophosphates, such as Phosphatidylinositol 3-phosphate, also known as PtdIns3P or PI(3)P, Phosphatidylinositol 4-phosphate, also known as PtdIns4P or PI(4)P, or Phosphatidylinositol S-phosphate, also known as PtdIns5P or PI(5)P; phosphatidylinositol bisphosphates, such as Phosphatidylinositol 3,4- bisphosphate, also known as PtdIns(3,4)P; or P1(3,4)P», Phosphatidylinositol 3,5-bisphosphate, also known as PtdIns(3,9)P; or PI(3,5)P,, or Phosphatidylinositol 4,5-bisphosphate, also known as PtdIns(4.5)P,, P1(4,5)P, or PIP,. Phosphatidylinositol trisphosphate, such as Phosphatidylinositol 3,4,5-trisphosphate, also known as PtdIns(3,4,5)P; or PI(3,4,5)P..

Endosomal escape/disruption component

[0088] The multicomponent delivery system may optionally comprise components that facilitate endosomal escape, including but not limited to buffering amines or polyamines; nitrogen- containing heterocycle groups and/or nitrogen-containing heteroaryl groups such as imidazoles, pyrroles, pyridines, pyrimidines; maleic acid derivatives; or membrane-lytic peptides.

Targeting components

[0089] The multicomponent delivery system may comprise targeting moeities on the surface of the system. Targeting moieties can be peptides, antibody mimetics, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies), glycoproteins, small molecules, carbohydrates, or lipids. Non- limiting examples of the targeting moiety include a peptide such as somatostatin, octreotide, LHRH, an EGFR-binding peptide, RGD-containing peptides, a protein scaffold such as a fibronectin domain, an aptide or bipodal peptide, a single domain antibody, a stable scFv, or a bispecific T-cell engagers, nucleic acid (e.g., aptamer), polypeptide (e.g., antibody or its fragment), glycoprotein, small molecule, carbohydrate, or lipid. The targeting moiety may be an aptamer being either RNA or DNA or an artificial nucleic acid; small molecules; carbohydrates such as mannose, galactose and arabinose; vitamins such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxal, Hpoic acid, folic acid (folate), riboflavin, biotin, vitamin B12, vitamin A, E, and K; a protein or peptide that binds to a cell-surface receptor such as a receptor for thrombospondin, tumor necrosis factors (TNF), annexin V, interferons, cytokines, transferrin, GM- CSF (granulocyte-macrophage colony-stimulating factor), or growth factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), (platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Therapeutic components

[0090] Small molecule drugs or other biologics can also be incorporated into the multicomponent delivery system. Non-limiting examples include incorporating drugs that disrupt the blood-brain-barrier or enhance cellular uptake; drugs that affect intracellular trafficking or endosomal escape; or drugs that are immunomodulators to affect antigen presentation when the multicomponent delivery system is used in as a vaccine. Non-limiting Examples of Multicomponent Delivery System Formulations

[0091] Table 1 shows non-limiting examples of lipidated peptoids. In one example, the multicomplnent delivery system may comprise at least one of the lipidated peptoids provided in Table 1. Some of the peptoids provided in Table 1 are lipidated cationic peptoids, such as Compound 24, Compound 79, Compound 93 and Compound 112, that may be used as cationic component, or cationic anchors, in the multicomponent delivery system. Some of them are neutral peptoids, such as Compound 89 and Compound 90, and can be used as the non-cationc lipid component in the system. Some of them are zwitterionic, such as Compound 105, and can be used as anionic or zwitterionic component of the system. In one example, a PEG-containing molecule of those shown in Table 1, such as of the lipidated peptoid Compounds 48, 56, 64, and 72, each of which has a PEG moiety (average molecular weight 2000 g/mol) attached to the peptoid, can also be used as a shielding component. In one example, lipidated peptoid Compounds 106, 107, 108 and 109 can also be used as a shielding component.

Table 1. Non-limiting lipidated peptoids

€ ie 0 TT a ey 3 Hee 5

AIEN NH, i on Eese te. 5,0 a ey 3 | Hee 5

TT iz ?, ?. i oh | > 3 > de U, N

EEE

LG Ga Eva 7 NY Tr WT WY WT WT NH,

A T T A ob 8 5 CY 5 5 NN

9 RAS <, YA eh "CY 5 5 NN NH LTE, ay EER,

/ Li > Fo teh en aw

AN A A oan NA NA NJL NJ NJL ‚A 16 NY NY NY NY NY NH, To de Ms \

18 A TEN 2,90 > u > 5 a ® A Ah, .

: LY 5 5 \ iN \

°, Su %, °, Su ob

ETE NL , °, NL AAA, ) 3 7 5 5 NN \ NN ) Ni °, , Yi CAA, . : TEETER 24 pe 9 T°, SU ob 3 5 5 NN \ ) © Sd © SA oN 2% CY 0 or A

EIEN

2g “WY Ay Ay Ay Ay Ay ANY Ay A, Led wos ON

34 49,06 0 gE eier

SN

A ANY 38 BN 5 NN \ NH,

T T° po Tb

ES

|

| | AS

A A

46 AN pp

NL Ee! | A AY | - FT iy

ER

A, NL Ni Ay ONT N 8 AN A, Au “1 4 Hp Hy Ho SNY 5 A

Ù 0 ¢ A, A, O

: TE ie Ni AA,

53 CY NTA NA NY NH,

54 EA YU ob 55 IONA J A A NH,

TER HP <, NR 2h 57 | HINT CY 5 a NN NN NH, oJ °, NL A,

ETT

| A NU Any “ HO AN Cr NM wy N NH, 60 NANA CY 3 3 \ \ NH Oi © Jd ny pre

LR ?, Ni A, 6 ON NY NY NN NY NY NH; LO DN 5 \ \ ae ai ANY A %, JA AA, 64 A LY 5 5 NN \ NH;

A G J Lud HNN NN NY NY NN NY NH, * 5 5 AY AY A 9 NA Tb INN NN NOT NN NY NY NH,

EES HON NY NY NY NY NY NH, HN HL ?, Ni TN NaN Cy NV 3 NN 68

C~ Poe NN AAI Sey Ae ye TY iN N Is °, po ey SN NN NY NN NN NY NH, L$ N N Ek °, NL U dh | - ™ 3 5 NN ) 1 °, A TN te CY 5 3 5 i wr, 72

| ie

AL

AAN | 7 eN

A

8 A

B

Bs \ A 81 T A NH,

Nad, 83 H 5 De

84 AAT py

NN, | AA, 37 Nee OY a aN NY pe

ANS

EE 92 ier

| ps 2, NA

DA ‚ aaa

\ nT ad : djs

ANY 7 \ Ag 0 A | \ | î TTR \ (i a aA 5 ve) 3 0 2 | EN SONY \ No 3 0 2 ty \

HOO Cd, A~N N N N . HN OV UT TY NH; 0“ "OH

UN NN NNN § 9 { Q { Q § 0 L 0 L 0 { Q 3, Q 3, 0 He MAR A A AA AN I AAI AN I AA IA NH An h h 5 5 5 Noun ou 1 1 1, 1 1,

CU UL HN Nn AL Nn JA NL NL NL NA NL H NY NY NY NN NY NY NY NH, 107 3 ö N° N o N° N° 6 5 LoL Lu Low Ww Lu

AAA ANN 108 SUS SD SDE DD, o N° N° NC N° N° N° 1 1 L L 4 1 4 ie ook 0% oH

LE ee UL “en 0 AP ep dep ep dd N° N° N° N° N° 3° 1 1, 1, 1, 1, 1, Oo” “OH 0” “OH

RN A A NHBoc Wo ADDI I,

TO TT

! ro Susu

| A 5 a i nS NAAN AA 118 J J 0

[0092] In one non-limiting example, the multicomponent delivery system comprises at least about 99 mol% cationic component and less than about 1 mol% shielding component (e.g., Formula F1 in Tables 2 and 3). The cationic component may be a lipidated cationic peptoid such as any stuiable compounds in Table 1 (e.g., Compound 24, 79, 93, or 112). The shielding component may be a peptoid or lipid comprising PEG moieties.

[0093] In another non-limiting example, the multicomponent delivery system comprises less than about 20 mol% cationic component, less than about 5 mol% shielding component, and more than 75 mol% a mixture of anionic/zwitterionic component and lipid component (e.g., Formula F2 and Formula F4 in Tables 2 and 3). The cationic component may be a lipidated cationic peptoid such as any cationic compounds in Table 1 (e.g., Compound 24, 79, 93, or 112). The shielding component may be a peptoid or lipid comprising PEG moieties. The lipid component may be a peptoid or lipid comprising phospholipid, cholesterol, or a mixture thereof.

[0094] In yet another non-limiting example, the multicomponent delivery system comprises about 30-45 mol% cationic component, about 50-70 mol% a mixture of anionic/zwitterionic component and lipid component, and about 1.5-4.5 mol% shielding component (e.g., Formula F3 and Formula F5 in Tables 2 and 3). The cationic component may be a lipidated cationic peptoid such as any suitable compounds in Table 1 (e.g., Compound 24, 79, 93, or 112). The shielding component may be a peptoid or lipid comprising PEG moieties. The anionic/zwitterionic component may be a peptoid or lipid comprising phospholipid, or a mixture thereof. The lipid component may be a peptoid or lipid comprising cholesterol, or a mixture thereof.

[0095] In yet another non-limiting example, the multicomponent delivery system comprises about 15-35 mol% cationic component, about 60-80 mol% a mixture of anionic/zwitterionic component and lipid component, and about 1.5-3.0 mol% shielding component (e.g., Formula F2 and Formula F3 in Tables 2 and 3). The cationic component may be a lipidated cationic peptoid such as any suitable compounds in Table 1 (e.g., Compound 24, 79, 93, or 112). The shielding component may be a peptoid or lipid comprising PEG moieties. The anionic/zwitterionic component may be a peptoid or lipid comprising phospholipid, or a mixture thereof. The lipid component may be a peptoid or lipid comprising cholesterol, or a mixture thereof.

[0096] In yet another non-limiting example, the multicomponent delivery system comprises 19 about 15-35 mol% cationic component, about 10-20 mol% an anionic/zwitterionic component,about 50-65 mol% lipid component, and about 1.5-3.0 mol®% shielding component (e.g., Formula F2 and Formula F3 in Tables 2 and 3). The cationic component may be a lipidated cationic peptoid such as any suitable compounds in Table 1 (e.g., Compound 24, 79, 93, or 112). The shielding component may be a peptoid or lipid comprising PEG moieties. The anionic/zwitterionic component may be a peptoid or lipid comprising phospholipid, or a mixture thereof. The lipid component may be a peptoid or lipid comprising cholesterol, or a mixture thereof.

[0097] In yet another non-limiting example, the multicomponent delivery system comprises about 10-20 mol% cationic component, about 75-89 mol% lipid component, and about 1-5 mol% shielding component (e.g., Formula F4 in Tables 2 and 3). The cationic component may be a lipidated cationic peptoid such as any suitable compounds in Table 1 (e.g., Compound 24, 79, 93, or 112). The shielding component may be a peptoid or lipid comprising PEG moieties. The lipid component may be a peptoid or lipid comprising cholesterol, or a mixture thereof.

[0098] In yet another non-limiting example, the multicomponent delivery system comprises about 40-50 mol% cationic component, about 50-59 mol% an anionic/zwitterionic component, and about 1-5 mol% shielding component (e.g., Formula F5 in Tables 2 and 3). The cationic component may be a lipidated cationic peptoid such as any suitable compounds in Table 1 (e.g., Compound 24, 79, 93, or 112). The shielding component may be a peptoid or lipid comprising PEG moieties. The anionic/zwitterionic component may be a peptoid or lipid comprising phospholipid, or a mixture thereof.

[0099] Non-limiting examples of the multicomponent delivery system formulations are included in Table 2 (molecular percentages) and Table 3 (mass ratios and charge ratios). In Formula F2, F3, or F5, the anionic/zwitterionic component (e.g., a lipid component comprising phospholipid) can be but not limited to DOPE, DSPC or Compound 105. In Formula F2, F3 or F4, the non-cationic lipid component comprising cholesterol can be but not limited to cholesterol, or a neutral peptoid such as Compound 89 or Compound 90. The shielding component in Formula Fi, F2, F3, F4 F5, or F6 can be but not limited to DMG-PEG (such as DMD-PEG2k) or Compound

107.

Table 2. Molecular Percentages of the components of the multicomponent delivery system Molecular Cationic Anionic or Non-cationic Shielding Percentages component Zwitterionic lipid component | component {mol%) component Wee ow Table 3. Mass ratios and charge ratios of the components of the multicomponent delivery system Cationic Anionic or Non-cationic Shielding Charge | component Zwitterionic lipid component ratios mass ratio component component mass ratio mass ratio mass ratio Fw fo per fw Woe pp pe wow Methods of Making the Multicomponent Delivery System

[0100] Components of the multicomponent delivery system can be prepared through a variety of physical and/or chemical methods to modulate their physical, chemical, and biological properties. These may involve rapid combination of the lipidated cationic peptide compound (e.g., a tertiary amino lipidated and/or PEGylated cationic peptide or a lipitoid) in water or a water- miscible organic solvent with the desired polyanionic cargo compound (e.g., oligonucleotides or nucleic acids) in water or an aqueous buffer solution. These methods can include simple mixing of the components by pipetting, or microfluidic mixing processes such as those involving T-mixers, vortex mixers, or other chaotic mixing structures. In some examples, the multicomponent delivery system is prepared on a microfluidic platform.

[0101] Itis to be understood that the particular process conditions for preparing the multicomponent delivery system described herein may be adjusted or selected accordingly to provide the desired physical properties of the compositions. For example, parameters for mixing the components of the multicomponent delivery system which may influence the final compositions may include but are not limited to order of mixing, temperature of mixing, mixing speed/rate, flow rate, physical dimensions of the mixing structure, concentrations of starting i0 solutions, molar ratio of components, and solvents used. {0102] Formulation of the multicomponent delivery system can be accomplished in many ways. In some cases, all components will be pre-mixed prior to addition of the nucleic acid cargo, resulting in a (presumably) uniform distribution of components throughout the delivery particle.

[0103] In other cases, the components will be added sequentially to produce a core-shell type structure. For example, a cationic component could be added first to begin particle condensation, followed by a lipid component to allow the particle's surface to associate with target cells, followed by a shielding component to prevent particle aggregation. For example, the cationic lipidated peptoid can be premixed with the nucleic acid cargo to form a core structure. Then the lipid components (such as lipid components comprising phospholipids and cholesterol) are added to influence cell/endosomal membrane association. Since the shielding component is primarily useful on the outside of the multicomponent delivery system, this component could be introduced last, so that it doesn’t disrupt the internal structure of the system, but rather provides a coating of the system after it is formed.

[0104] Additional components in the complexes and composition, such as the additional components of polymers, surface-active agents, targeting moieties, or excipients, may be admixed and combined with the rest of the components before, during or after the principal components of the nucleic acid cargo, the cationic component, the lipid component and the shielding component have been combined. Methods of Using the Multicomponent Delivery System

[0105] The multicomponent delivery system of the present disclosure may be used for delivering one or more polyanionic cargo compounds. The multicomponent delivery system forms a complex with the one or more polyanionic cargo compounds.

[0106] In some examples, the one or more polyanionic cargo compounds comprises a nucleic acid. Nucleic acids, as used herein, include naturally occurring nucleic acids such as DNA, RNA, and/or hybrids thereof as well as unnaturally occurring variations with unnatural backbone and modified backbone linkages such as phosphorothioate, unnatural and modified bases, and unnatural and modified termini. Exemplary nucleic acids include genomic DNA, cDNA, mRNA, miRNA, and siRNA. In certain examples, the nucleic acid cargo is RNA including but not limited to modified mRNAs, self-amplifying RNAs, and circular RNAs.

10107] The nucleic acids may be recombinantly produced or chemically synthesized molecules. A nucleic acid may be single-stranded. double-stranded, triple stranded, and quadraple stranded as well as in more complicated three-dimensional forms including single and double stranded regions.

[0108] Depending upon the type of nucleic acid, the length of the nucleic acid (defined in 19 nucleotide units or base pairs (bp) as appropriate) may vary. In some examples wherein the nucleic acid is mRNA, the mRNA may have from 100 to 10,000 nucleotide units, or from 1,000 to 3,000 nucleotide units. In other examples wherein the nucleic acid is DNA, the DNA may have from 5,000 bp to 20,000 bp, or about 10,000 bp.

[0109] With regard to the polyanionic cargo compounds present in the complexes of the present disclosure, the quantity of polyanionic cargo compounds within the complexes, may be characterized in a number of ways. In some examples, the complexes described herein may be characterized by the ratio of the number cationic groups on the cationic component of the multicomponent delivery system to the number of anionic phosphate groups on the nucleic acid. In some examples, the complex comprises the lipidated cationic peptoids and the nucleic acid at a cation: anion charge ratio of between 0.5:1 and 20:1, between 0.5:1 and 10:1, between 0.5:1 and 5:1, between 1:1 and 20:1, between 1:1 and 10:1, between 1:1 and 5:1, between 2:1 and 20:1, between 2:1 and 10:1, or between 2:1 and 5:1. In certain examples, the complex comprises the lipidated cationic peptoids and the nucleic acid at a cation: anion charge ratio of between 2:1 and 5:1. In still yet other examples, the complex comprises the lipidated cationic peptide compound and the nucleic acid at a cation: anion charge ratio of 3:1.

[0110] Alternatively, the complexes described herein may be characterized by the relative mass ratio of the lipidated cationic peptoids to the polyanionic compound(s) and/or other cargoes in the complex. Mass ratios of the components in the complex can be readily calculated based upon the known concentrations and volumes of stock solutions of each component used in preparing the complex. Moreover, if non-anionic cargoes are present in the complex, mass ratios may provide a more accurate representation of the relative amounts of lipidated cationic peptoids to the overall cargo than cation:anion charge ratios, which do not account for non-anionic material. Specifically, the mass ratio of a component refers to the ratio of the mass of this particular component in the system to the mass of the “cargo” in the system. “Cargo” may refer to the total polyanoic compound(s) present in the system. In one example, the polyanoic compound(s) may refer to nucleic acid(s). In one example, the polyanoic compound(s) refer to mRNA(s) encoding at least one protein.

[0111] In some examples, the complex comprises the one or more lipidated cationic peptoids and the cargos comprising one or more polyanionic cargo compounds and/or non-anionic cargo compounds at a mass ratio of between 0.5:1 and 20:1, between 0.5:1 and 10:1, between 0.5:1 and 5:1, between 1:1 and 20:1, between 1:1 and 10:1, between 1:1 and 5:1, between 2:1 and 20:1, between 2:1 and 10:1, or between 2:1 and 5:1. In certain examples, the complex comprises the one or more lipidated cationic peptoids and the cargos comprising one or more polyanionic cargo compounds and/or non-anionic cargo compounds at a mass ratio of between 2:1 and 5:1. In still yet i0 other examples, the complex comprises the one or more lipidated cationic peptoids and the cargos comprising one or more polyanionic cargo compounds and/or non-anionic cargo compounds at a mass ratio of 3:1.

[0112] In certain examples wherein the complex comprises a nucleic acid as cargo, the complex comprises the lipidated cationic peptoids and the nucleic acid at a mass ratio of between

0.5:1 and 20:1, between 0.5:1 and 10:1, between 0.5:1 and 5:1, between 1:1 and 20:1, between 1:1 and 10:1, between 1:1 and 5:1, between 2:1 and 20:1, between 2:1 and 10:1, or between 2:1 and 5:1. In certain examples, the complex comprises the lipidated cationic peptoids and the nucleic acid at a mass ratio of between 2:1 and 5:1. In still yet other examples, the complex comprises the lipidated cationic peptoids and the nucleic acid at a mass ratio of 3:1.

[0113] In still other examples, the amount of polyanionic cargo compounds present in the complexes may be characterized by a mass ratio of the multicomponent delivery system (e.g., lipidated cationic peptoids, phospholipid, cholesterol, and/or the shielding component in total) to the one or more polyanionic cargo compounds. In some examples, the mass ratio of the multicomponent delivery system to the one or more polyanionic cargo compounds is between 0.5:1 and 20:1, between 0.5:1 and 10:1, between 0.5:1 and 5:1, between 1:1 and 20:1, between 1:1 and 10:1, between 1:1 and 5:1, between 2:1 and 20:1, between 2:1 and 10:1, or between 2:1 and 5:1. In certain examples, the mass ratio of the multicomponent delivery system to the one or more polyanionic cargo compounds is between 5:1 and 8:1 or between 6:1 and 7:1.

[0114] In some examples wherein the nucleic acid is an mRNA encoding a protein or a peptide. The peptide may be an oligopeptide or a polypeptide. In certain examples, the mRNA is an mRNA encoding a polypeptide. In yet further examples, the mRNA is an mRNA encoding a protein. As described above, the mRNA may be naturally-occurring (e.g., isolated tumor RNA) or may be synthetic (e.g., produced by in vitro transcription). For synthetic or unnaturally occurring variations of mRNA, the mRNA may comprise an unnatural backbone with modified backbone linkages such as phosphorothioate, unnatural and modified bases, and/or unnatural and modified termini. In certain examples wherein the nucleic acid is an mRNA, the mRNA may comprise special sequences such as self-amplifying sequences or internal ribosome entry sites.

[0115] In some examples, the one or more polyanionic cargo compounds may include anionic or polyanionic cargo compounds that are not nucleic acids. Suitable anionic compounds may include but are not limited to proteins, polyphosphates, or heparins. In some examples, the one or more polyanionic cargo compounds comprises one or more proteins. In one examples, the one or more polyanionic cargo compounds comprises Cas9 protein. In other examples, the one or more polyanionic cargo compounds comprises polyphosphates. In yet other examples, the one or more polyanionic cargo compounds comprises heparins or other glycosaminoglycan derivatives.

[0116] In some examples, the combined delivery of two or more particular nucleic acids together may be especially useful for therapeutic applications. For example, in some examples, the one or more polyanionic cargo compounds includes a combination of sgRNA (single guide RNA) as a CRISPR sequence and mRNA encoding Cas9. In still further examples, the nucleic acids may also be complexed with proteins such as with the CRISPR/Cas9 ribonucleoprotein complex.

[0117] In other examples, the methods of delivering a polyanionic cargo compound to a cell comprise contacting the cell with the multicomponent delivery system and one or more polyanionic cargo compounds, wherein the cell is contacted in vitro, ex vivo, or in vivo.

[0118] In some examples wherein the cell is contacted in vitro, the cell is a Hel.a cell. In other examples wherein the cell is contacted in vive, the multicomponent delivery system of the present disclosure is administered to a mammalian subject. A mammalian subject may include but is not limited to a human or a mouse subject. In yet other examples wherein the cell is contacted ex vivo, the cell is obtained from a human or mouse subject.

[0119] In some examples, the one or more polyanionic cargo compounds may be delivered for therapeutic uses. Non-limiting therapeutic uses include cancer, infectious diseases, autoimmune disorders, and neurological disorders. In certain examples, the complex comprising the multicomponent delivery system and the polyanionic cargo compound is used as a vaccine.

[0120] In some examples of the foregoing methods wherein the cell is contacted in vivo, the complex comprising the multicomponent delivery system and the nucleic acid cargos as described. herein may be administered by injection (intravenous (IV), subcutaneous (SC), intramuscular (IM), or intrathecal injection. In other examples, the complex comprising multicomponent delivery system and the nucleic acid cargos as described herein are administered by bolus injection or intravenous infusion. In other examples wherein the cell is contacted in vivo, the complexes are administered by nasal or oral inhalation. In some examples wherein the cell is contacted in vivo, the complexes are administered orally. In still other examples wherein the cell is contacted in vivo,

the complexes are administered via absorption into the mucous membrane (including topical, intra- anal, buccal, intravaginal, etc.).

[0121] It should be understood that clinical applications, such as the diagnostic, prophylactic and therapeutic examples disclosed above, may involve dosing regimens (e.g., dosage levels and time courses for administration) which may be varied as appropriate to the specific multicomponent delivery system and the nucleic acid cargos being used, the route of administration, the subject to which the complexes and compositions are being administered, and/or the desired physiological effect. For example, in some examples, the methods of the present disclosure comprise administering the multicomponent delivery system and the nucleic acid cargos ata dose of about 0.001 mg/kg to about 2 mg/kg of bodyweight. {0122] For clinical applications, the complex comprising the multicomponent delivery system and the polyanionic cargo compound is further mixed with at least one pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For the complex administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or tale. If desired, the oral composition, such as tablets, may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

Cancer

[0123] Various cancers may be treated with the polyanionic cargo compounds delivered by the multicomponent system of the present disclosure. As used herein, the term “cancer” refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.

[0124] As a non-limiting example, the carcinoma which may be treated may be Acute granufocytic leukemia, Acute lymphocytic leukemia, Acute myelogenous leukemia, Adenocarcinoma, Adenosarcoma, Adrenal cancer, Adrenocortical carcinoma, Anal cancer, Anaplastic astrocytoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal cell carcinoma, B- Cell lymphoma), Bile dact cancer, Bladder cancer, Bone cancer, Bowel cancer, Brain cancer, Brain stem glioma, Brain tumor, Breast cancer, Carcinoid tumors, Cervical cancer, Cholangiocarcinoma, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Colon cancer, Colorectal cancer, Craniopharyngioma, Cutaneous lymphoma, Cutaneous melanoma, Diffuse astrocytoma, Ductal carcinoma in situ, Endometrial cancer, Ependymoma, Epithelioid sarcoma,

{0 Esophageal cancer, Ewing sarcoma, Extrahepatic bile duct cancer, Eye cancer, Fallopian tube cancer, Fibrosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumors, General, Germ cell tumor, Glioblastoma multiforme, Glioma, Hairy cell leukemia, Head and neck cancer, Hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, Hodgkin's lymphoma, Hypopharyngeal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Intestinal Cancer, Intrahepatic bile duct cancer, Invasive / infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma, Leptomeningeal metastases, Leukemia, Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ, Low-grade astrocytoma, Lung cancer, Lymph node cancer, Lymphoma, Male breast cancer, Medullary carcinoma, Medulloblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesenchymal chondrosarcoma, Mesenchymous, Mesothelioma, Metastatic breast cancer, Metastatic melanoma, Metastatic squamous neck cancer, Mixed gliomas, Mouth cancer, Mucinous carcinoma, Mucosal melanoma, Multiple myeloma, Nasal cavity cancer, Nasopharyngeal cancer, Neck cancer, Neuroblastoma, Neuroendocrine tumors, Non-Hodgkin lymphoma, Non-Hodgkin's lymphoma,

Non-small cell lung cancer, Oat cell cancer, Ocular cancer, Ocular melanoma, Oligodendroglioma, Oral cancer, Oral cavity cancer, Oropharyngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian primary peritoneal carcinoma, Ovarian sex cord stromal tumor, Paget's disease, Pancreatic cancer, Papillary carcinoma, Paranasal sinus cancer, Parathyroid cancer, Pelvic cancer, Penile cancer, Peripheral nerve cancer, Peritoneal cancer, Pharyngeal cancer, Pheochromocytoma, Pilocytic astrocytoma, Pineal region tumor, Pineoblastoma, Pituitary gland cancer, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell cancer, Renal pelvis cancer, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sarcoma, bone, Sarcoma, soft tissue, Sarcoma, uterine, Sinus cancer, Skin cancer, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Spinal cancer, Spinal column cancer, Spinal cord cancer, Spinal tumor, Squamous cell carcinoma, Stomach cancer, Synovial sarcoma, T-cell lymphoma), Testicular cancer, Throat cancer, Thymoma/ thymic carcinoma, Thyroid cancer, Tongue cancer, Tonsil cancer, Transitional cell cancer, Transitional cell cancer, Transitional cell cancer, Triple-negative breast cancer, Tubal cancer, Tubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer, Uterine sarcoma, Vaginal cancer, and Vulvar cancer. Infectious Diseases {0125] In some examples, the complex comprising the polyanionic cargo compounds and the multicomponent system of the present disclosure is used to treat infectious diseases, such as microbial infection, e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection. Non-limiting examples of infectious diseases include hepatitis (such as HBV infection or HCV infection), RSV, influenza, adenovirus, rhinovirus, or other viral infections. Vaccines

[0126] In some examples, the complex comprising the polyanionic cargo compounds and the multicomponent system of the present disclosure is used as vaccines. The polyanionic cargo may be or may encode an immunogen, antigen or neoantigen. f0127] The pharmaceutical composition comprising the complex may further comprise one or more immunologic adjuvants. As used herein, the term “immunologic adjuvant” refers to a compound or a mixture of compounds that acts to accelerate, prolong, enhance or modify immune responses when used in conjugation with an immunogen (e.g., neoantigens). Adjuvant may be non- immunogenic when administered to a host alone, but that augments the host's immune response to another antigen when administered conjointly with that antigen. Specifically, the terms “adjuvant” and “immunologic adjuvant” are used interchangeably in the present invention. Adjuvant-mediated enhancement and/or extension of the duration of the immune response can be assessed by any method known in the art including without limitation one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant/antigen combination versus those produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells recognizing the antigen or the adjuvant; and (iti) an increase in the level of one or more cytokines. f0128] Adjuvants may be aluminum based adjuvants including but not limiting to aluminum hydroxide and aluminum phosphate; saponins such as steroid saponins and triterpenold saponins; bacterial flagellin and some cytokines such as GM-CSF. Adjuvants selection may depend on antigens, vaccines and routes of administrations. f0129] In some examples, adjuvants improve the adaptive immune response to a vaccine antigen by modulating innate immunity or facilitating transport and presentation. Adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs). Adjuvants may be ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity. In other instances, adjuvants may signal via proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation. This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes and comprises colloids and molecular assemblies exhibiting complex, heterogeneous structures (Powell et al., Clin Exp. Vaccine Res., Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes. 2015, 4(1):23-45). f0130] In one example, the composition farther comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant.

Autoimmune diseases

[0131] Various autoimmune diseases and autoimmune-related diseases may be treated with the polyanionc compounds delivered by the multicomponent system of the present disclosure. As used herein, the term “autoimmune disease” refers to a disease in which the body produces antibodies that attack its own tissues. As a non-limiting example, the autoimmune disease may be Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal & neuronal neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic fatigue syndrome’**, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’s disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed ryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressler’s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia**, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's Granulomatosis), Graves’ disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, 1gG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosis, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic’s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, 11, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren’s syndrome, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac’s syndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo, and Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis (GPA).

Neurological diseases

[0132] Various neurological diseases may be treated with the polyanionc compounds delivered by the multicomponent delivery system of the present disclosure. As a non-limiting example, the neurological disease may be Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi- Goutieres Syndrome Disorder, AIDS - Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation,

Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit- Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Behcet's Disease, Bell's Palsy, Benign Essential

Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy

(CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavernous Malformation, Cerebral

Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital

Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia -Multi-

Infarct, Dementia - Semantic, Dementia Subcortical, Dementia With Lewy Bodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome,

Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalopathy, Encephalopathy (familial infantile), Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Erb's Palsy, Erb- Duchenne and Dejerine-Klumpke Palsies, Essential Tremor, Extrapontine Myelinolysis, Fabry Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis,

Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant

Syndrome, Foot Drop, Friedreich's Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-Barré

Syndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington's Disease, Hydranencephaly, Hydrocephalus, Hydrocephalus - Normal

Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaacs’ Syndrome, Joubert Syndrome,

Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Klüver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut

Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus - Neurological Sequelae, Lyme Disease - Neurological Complications, Machado-Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes Disease, Meralgia Paresthetica, Metachromatic

Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke, Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidosis, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia - Congenital, Myasthenia Gravis,

Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy, Myopathy- Congenital, Myopathy -Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, Neurological Consequences of Cytomegalovirus Infection,

Neurological Manifestations of Pompe Disease, Neurological Sequelae Of Lupus, Neuromyelitis

Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy- Hereditary, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse

Syndrome, Pain - Chronic, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir 11 Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's

Disease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Post infectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive

Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen's Encephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease, Refsum Disease - Infantile, Repetitive Motion Disorders, Repetitive Stress Injuries,

Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seitelberger Disease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren's

Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Shortlasting, Unilateral,

Neuralgiform (SUNCT) Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible

Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal

Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of the Central and Peripheral Nervous Systems, Von Economo’s Disease, Von Hippel-Lindau Disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple's Disease, Williams Syndrome, Wilson Disease, Wolman’s Disease, X-Linked Spinal and Bulbar Muscular Atrophy. Definitions

[0133] “Alkenyl” as used herein refers to an unsaturated linear or branched univalent hydrocarbon chain or combination thereof, having at least one site of olefinic unsaturation (i.e.

having at least one moiety of the formula C=C) and having the number of carbon atoms designated (i.e. C:-C19 means two to ten carbon atoms). The alkenyl group may be in “cis” or “trans” configurations, or alternatively in “E” or “Z” configurations. Particular alkenyl groups are those having 2 to 20 carbon atoms (a “C»-Cy alkenyl”), having 2 to 8 carbon atoms (a “C,-Cy alkenyl™), having 2 to 6 carbon atoms (a “C,-Cg alkenyl”), or having 2 to 4 carbon atoms (a “C,-C, alkenyl”).

Examples of alkenyl include, but are not limited to, groups such as ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (or allyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2- methylbuta-1,3-dienyl, homologs and isomers thereof, and the like.

[0134] The term "alkyl" refers to and includes saturated linear and branched univalent hydrocarbon structures and combination thereof, having the number of carbon atoms designated (ie. C-C)y means one to ten carbons). Particular alkyl groups are those having 1 to 20 carbon atoms (a “Cy-Cy alkyl”). More particular alkyl groups are those having 1 to 8 carbon atoms (a “C;- Cg alkyl”), 3 to 8 carbon atoms (a “Cs-Cy alkyl”), 1 to 6 carbon atoms (a “C-Cg alkyl”), 1 to 5 carbon atoms (a “C-Cs alkyl”), or 1 to 4 carbon atoms (a “CC; alkyl”). Examples of alkyl include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0135] “Alkylene” as used herein refers to the same residues as alkyl, but having bivalency. Particular alkylene groups are those having 1 to 6 carbon atoms (a “C-C alkylene”), 1 to 5 carbon atoms (a “C,-Cs alkylene”), 1 to 4 carbon atoms (a “C;-C, alkylene”) or 1 to 3 carbon atoms (a “Cy-Cs alkylene”). Examples of alkylene include, but are not limited to, groups such as methylene (-CH;-), ethylene {-CH;CH;-), propylene (-CH,CH,CH,-), butylene (-CH.CH,CH,CH,-), and the like.

[0136] “Alkyny!l” as used herein refers to an unsaturated linear or branched univalent hydrocarbon chain or combination thereof, having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C=C) and having the number of carbon atoms designated

{ie C;-C1 means two to ten carbon atoms). Particular alkynyl groups are those having 2 to 20 carbon atoms {a “C,-Cay alkynyl”), having 2 to 8 carbon atoms (a “C.-C; alkynyl”), having 2 to 6 carbon atoms {a “C--Cg alkynyl”), or having 2 to 4 carbon atoms (a “C,-C; alkynyl”). Examples of alkynyl include, but are not limited to, groups such as ethynyl (or acetylenyl), prop-1-ynyl, prop-2- ynyl (or propargyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs and isomers thereof, and the like.

[0137] The term "aryl" refers to and includes polyunsaturated aromatic hydrocarbon groups. Aryl may contain additional fused rings (e.g., from 1 to 3 rings), including additionally fused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings. In one variation, the aryl group contains from 6 to 14 anmular carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, and the like. {0138] “Carbonyl!” refers to the group C=0.

[0139] “Complex” as used herein includes any chemical association between two or more molecules, which may be mediated by ionic interactions, hydrogen bonding, van der Waals interactions, metal-ligand coordination, other chemical forces, and combinations of one or more of the foregoing. The complexes may form higher order structures including, for example, polyplexes, coacervate complexes, nanocomplexes, nanoparticles, and microparticles.

[0140] The term "cycloalkyl" refers to and includes cyclic univalent hydrocarbon structures, which may be fully saturated, mono- or polyunsaturated, but which are non-aromatic, having the number of carbon atoms designated (e.g., C;-Cio means one to ten carbons). Cycloalkyl can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantly, but excludes aryl groups. A cycloalkyl comprising more than one ring may be fused, spiro or bridged, or combinations thereof. A preferred cycloalkyl is a cyclic hydrocarbon having from 3 to 13 annular carbon atoms. A more preferred cycloalkyl is a cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a "C;-Cy cycloalkyl"). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, and the like.

[0141] “Halo” or “halogen” refers to elements of the Group 17 series having atomic number 9 to 85. Preferred halo groups include fluoro, chloro, bromo and iodo. Where a residue is substituted with more than one halogen, it may be referred to by using a prefix corresponding to the number of halogen moieties attached, e.g., dihaloaryl, dihaloalkyl, trihaloaryl etc. refer to aryl and alkyl substituted with two (“di”) or three (“tri”) halo groups, which may be but are not necessarily the same halo; thus 4-chloro-3-fluorophenyl is within the scope of dihaloaryl. An alkyl group in which each hydrogen is replaced with a halo group is referred to as a “perhaloalkyl.” A preferred perhaloalkyl group is trifluoroalkyl. Similarly, “perhaloalkoxy” refers to an alkoxy group in which a halogen takes the place of each H in the hydrocarbon making up the alkyl moiety of the alkoxy group. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF;).

[0142] The term "heteroaryl" refers to and includes unsaturated aromatic cyclic groups having from 1 to 10 annular carbon atoms and at least one annular heteroatom, including but not limited to heteroatoms such as nitrogen, oxygen and sulfur, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule at an annular carbon or at an annular heteroatom. Heteroaryl may contain additional fused rings (e.g., from 1 to 3 rings), including additionally fused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings. Examples of heteroaryl groups include, but i0 are not limited to, pyridyl, pyrimidyl, thiophenyl, furanyl, thiazolyl, and the like. {0143] The term “heterocycle” or “heterocyclyl” refers to a saturated or an unsaturated non- aromatic group having from 1 to 10 annular carbon atoms and from 1 to 4 annular heteroatoms, such as nitrogen, sulfur or oxygen, and the like, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heterocyclyl group may have a single ring or multiple condensed rings, but excludes heteroaryl groups. A heterocycle comprising more than one ring may be fused, spiro or bridged, or any combination thereof. In fused ring systems, one or more of the fused rings can be aryl or heteroaryl. Examples of heterocyclyl groups include, but are not limited to, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, thiazolinyl, thiazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, 2,3- dihydrobenzo[b]thiophen-2-yl, 4-amino-2-oxopyrimidin-1(2H)-yl, and the like.

[0144] “Oxo” refers to the moiety =O.

[0145] “Thiocarbonyl” refers to the group C=S.

[0146] “Optionally substituted” unless otherwise specified means that a group may be unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4 or 5) of the substituents listed for that group in which the substituents may be the same of different. In one example, an optionally substituted group has one substituent. In another example, an optionally substituted group has two substituents. In another example, an optionally substituted group has three substituents. In another example, an optionally substituted group has four substituents. In some examples, an optionally substituted group has 1 t0 2,2 10 5,3 105,2 t0 3,2 t0 4,3 104, 1 t0 3, 1 to 4 or 1 to 5 substituents.

[0147] The term "substituted" refers to the replacement of one or more hydrogen atoms of a moiety with a monovalent or divalent radical. “Optionally substituted” indicates that the moiety may be substituted or unsubstituted. Suitable substituent groups include, for example, hydroxyl, nitro, amino (e.g., -NH, or dialkyl amino), imino, cyano, halo (such as F, Cl, Br, I), haloalkyl (such as -CCl; or -CF3), thio, sulfonyl, thicamido, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, alkyl, alkoxy, alkoxy-alkyl, alkylcarbonyl,

alkylcarbonyloxy (-OCOR), aminocarbonyl, arylcarbonyl, aralkylcarbonyl, carbonylamino, heteroarylcarbonyl, heteroaralkyl-carbonyl, alkylthio, aminoalkyl, cyanoalkyt, carbamoyl (-NHCOOR- or -OCONHR-), urea (-NHCONHR-), aryl and the like, where R is any suitable group, €.g., alkyl or alkylene. In some examples, the optionally substituted moiety is optionally substituted only with select radicals, as described. In some examples, the above groups (e.g., alkyl groups) are optionally substituted with, for example, alkyl {e.g., methyl or ethyl), haloalkyl (e.g., -CCls, -CH,CHCL or -CFy), cycloalkyl (e.g., -CsHs, -C4Hs, -CsHs), amino (e.g., -NH, or dialkyl amino), alkoxy (e.g., methoxy), heterocycloalkyl (e.g., as morpholine, piperazine, piperidine, azetidine), hydroxyl, and/or heteroaryl (e.g., oxazolyl). In some examples, a substituent group is itself optionally substituted. In some examples, a substituent group is not itself substituted. The group substituted onto the substitution group can be, for example, carboxyl, halo, nitro, amino, cyano, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, aminocarbonyl, -SR, thioamido, - SO;H, -SO:R or cycloalkyl, where R is any suitable group, e.g., a hydrogen or alkyl.

[0148] The terms “substantially” and “about” used throughout this Specification are used to describe and account for small fluctuations. For example, they can refer to less than or equal to +5%, such as less than or equal to £2%, such as less than or equal to £1%, such as less than or equal to 20.5%, such as less than or equal to 20.2%, such as less than or equal to £0.1%, such as less than or equal to £0.05%.

EXAMPLES

[0149] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation. Example 1 — Synthesis of exemplary tertiary amino lipidated cationic peptoids {0150] The following example describes the general protocol for synthesis of the tertiary amino lipidated and/or PEGylated cationic peptoids of formula (1) as described herein. R2 R2 O R3 R2 O R* R2 O RRO RRO RRO RRO RNG {Ön 6-GN- Or —CHN—G—CrN—0 CR RP RP RP RP RP r RP RP @

[0151] In the description provided below, all R* and R” are -H. All polymers are synthesized using bromoacetic acid and primary amines. FIGS. 2B-2E provides some of the exemplary substituents of the primary amines at R*, R’, R*, R®, and R° to prepare the tertiary amino lipidated and/or PEGylated cationic peptide compounds of the present disclosure.

[0152] An Fmoc-Rink amide resin is used as the solid support. The Fmoc group on the resin is deprotected with 20% (v/v) piperidine-dimethylformamide (DMF). The amino resin is then amidated with bromoacetic acid. This is followed by amination of the a-carbon by nucleophilic displacement of the bromide with a primary amine. The two steps are successively repeated to produce the desired cationic peptide sequence.

[0153] All reactions and washings are performed at room temperature unless otherwise noted. Washing of the resin refers to the addition of a wash solvent (usually DMF or dimethylsulfoxide (DMSO)) to the resin, agitating the resin so that a uniform slurry is obtained, followed by thorough draining of the solvent from the resin. Solvents are removed by vacuum filtration through the [ritted bottom of the reaction vessel until the resin appeared dry. In all the syntheses, resin shuries are agitated via bubbling argon up through the bottom of the fritted vessel.

[0154] Initial Resin Deprotection, A fritted reaction vessel is charged with Fmoc-Rink amide resin. DMF is added to the resin and this solution is agitated to swell the resin. The DMF is then drained. The Fmoc group is removed by adding 20% piperidine in DMF to the resin, agitating the resin, and draining the resin. 20% piperidine in DMF is added to the resin and agitated for 15 minutes and then drained. The resin is then washed with DMF, six times.

[0155] Acylation/Amidation. The deblocked amine is then acylated by adding bromoacetic acid in DME to the resin followed by N,N-diisoprooplycarbodiimide (DIC) in DMF (FIG. 3A). This solution is agitated for 30 minutes at room temperature and then drained. This step is repeated a second time. The resin is then washed with DMF twice and DMSO once. This is one completed reaction cycle.

[0156] Nucleophilic Displacement/Amination. The acylated resin is treated with the desired primary or secondary amine to undergo nucleophilic displacement at the bromine leaving group on the a-carbon (FIG. 3B). This acylation/displacement cycle is repeated (FIG. 3C) until the desired peptide sequence is obtained (FIG. 3D).

[0157] Peptide Cleavage from Resin. The dried resin is placed in a glass scintillation vial containing a teflon-coated micro stir bar, and 95% trifluoroacetic acid (TFA) in water is added. The solution is stirred for 20 minutes and then filtered through solid-phase extraction (SPE) column fitted with a polyethylene frit into a polypropylene conical centrifuge tube.

[0158] The resin is washed with 1 mL 95% TFA. The combined filtrates are then lyophilized three times from 1:1 acetonitrile:water. The lyophilized peptide (FIG. 3E) is redissolved to a concentration of 5 mM in 5% acetonitrile in water.

[0159] Purification and Characterization. The redissolved crude peptide is purified by preparative HPLC. The purified peptide is characterized by LC-MS analysis.

Example 2 - Synthesis and characterization of representative amino lipidated peptoids

[0160] Amino lipidated peptoids were synthesized by the submonomer method described above in Example 1 with bromoacetic acid and N,N’ -diisopropylcarbodiimide (DIC). Polystyrene- supported MBHA Fmoc-protected Rink amide (200 mg representative scale, 0.64 mmol/g loading, Protein Technologies) resin was used as a solid support. For bromoacetylation, resin was combined with a 1:1 mixture of 2 M bromoacetic acid and 2M N,N’ -diisopropylcarbodiimide (DIC) for 5 minutes. Amine displacement was carried out using a 1M solution of amine in DME for 1 hour. Following synthesis, crude peptoids were cleaved from resin using 5 mL of a mixture of 95:2.5:2.5 triflaoroacetic acid (TFA): water:triisopropylsilane for 40 minutes at room temperature. Resin was removed by filtration and the filtrate concentrated using a Biotage V10 evaporator. The crude peptoids were further concentrated by lyophilization from a 25% solution of MeCN in water. Purity and identity were assayed with a Waters Acquity UPLC system with Acquity Diode Array UV detector and Waters SQD2 mass spectrometer on a Waters Acquity UPLC Peptide BEH C4 Column over a 5-95% gradient. Select crude peptoids were purified by preparative Waters Prepl50LC system with Waters 2489 UV/Visible Detector on a Waters Xbridge BEH300 Prep C4 column using a 5-40% acetonitrile in water with 0.1% TFA gradient over 30 minutes.

[0161] Table 1 shows representative amino lipidated peptoids prepared by the method described in Example 2. Example 3 - Synthesis and characterization of representative multicomponent delivery systems

[0162] Non-limiting exemplary multicomponent delivery system formulations are described in Table 4. Multicomponent delivery systems FORM-A (control), B, C, D,E, F, G,H, 1, J, and K were combined with firefly luciferase (Fluc) mRNA at ratios detailed in Table 4 to form nanoparticle compositions to be evaluated for therapeutic and/or prophylactic purposes in vitro or in vivo. The w/w in Table 4 is the ratio of that component to the mRNA by mass, with the mass ratio defined above.

[0163] The resulting nanoparticles were evaluated by dynamic light scattering (DLS) in order to determine the volume average particle size/diameter (nm) and the size polydispersity index (PDI) within the formulation. Table 5 shows the DLS-based size measurements. Table 4. Compositions of non-limiting examples of multicomponent delivery system formulations Anionic/ Non- Catio | Anionic/ Zwitteri catio Shield nic Zwitterioni | onic Neutral nic ing Name | Cationic W/W c w/w (lipid) Lipid | Shielding | w/w

I Lm

FORM a I = I FORM | Compound Cholester DMG- FORM | Compound Compoun DMG- FORM | Compound Compound Cholester DMG- FORM | Compound Cholester Compound Jom |e FORM | Compound Compoun DMG- Jom a we FORM | Compound Compoun DMG- FORM | Compound Cholester Compound Jon | FORM | Compound Compoun DMG- FORM | Compound Compoun DMG- FORM | Compound DMG- Table 5. Physical properties of the formulations wma FORM-B 122.8 0.14

[0164] The efficacy of mRNA delivered in the multicomponent delivery systems was evaluated in vitro based on their ability to deliver the Fluc reporter gene to cultured cells. The multicomponent delivery systems were combined with Fluc mRNA at the given w/w ratios, and the resulting particles were added to cultured cells at a dose of 50 ng/well (in 150 pL total volume). The resulting luciferase expression (RLU) was measured by a luminescence plate reader after 8 hours of treatment. Table 6 shows the observed in vitro Fluc expression.

[0165] In vivo local expression of Fluc following IM administration of the resulting particles to mice at a dose of 0.05 mg/kg via a tail-vein injection. The resulting bioluminescence was quantified after 6 hours. OV A-specific T-cell responses were quantified by analyzing single-cell suspensions of the spleens of mice vaccinated with OVA mRNA and CpG as an adjuvant. Suspensions were stained with OV A-specific tetramer and gated on CD8+ T-cells. Antibody responses were quantified by ELISA against the OV A protein. Dosing for vaccination studies was administered in the hind thigh muscle in 40 ul. total volume on Day 0 and Day 7. Immune responses were (cellular and humoral) were measured on Day 14. Table 7 shows the observed in vivo Fluc expression and immune response generation. Table 6. In vitro expression from formulations RL Expression hPBMC Expression FORM-D 1287.5 16

Table 7. In vivo expression from formulations OVA Binding Total Fluc Photon Antibodies {endpoint FORM-F 9313000 6.69

Claims (44)

Conclusions A system for the delivery of polyanionic compounds, such as nucleic acids, to target cells, wherein the delivery system comprises at least one cationic component, wherein the at least one cationic component comprises a lipidated peptoid. The system of claim 1, further comprising one or more of the following: an anionic or zwitterionic component, + a non-cationic Hpide component; a shielding component. The system of claim 2, wherein the anionic or zwitterionic component comprises a phospholipid, a lipidoid or a mixture thereof, preferably wherein the anionic or zwitterionic component is a DOPE or DSPC. The system of claim 2 or 3, wherein the non-cationic lipid component comprises a sterol, e.g. cholesterol, and/or a neutral peptoid. A system according to any one of claims 2-4, wherein the shielding component comprises a poly(ethylene glycol) (PEG) moiety, preferably wherein the shielding component is a PEGylated lipid or a PEGylated lipidated peptoid, more preferably wherein the shielding component is DMG-PEG, for example DMGPEG2K. The system of claim 4 or 5, wherein the non-cationic lipid component comprises: Compound 90 A nN, 0 N N H NN AA Ae, or wherein the non-cationic lipid component comprises: Compound 89 A A, | N N The system of any one of claims 2-6, wherein the cationic component comprises: Compound 112 a AHA AA AA, J J 0 and the non-cationic lipid component comprises: Compound 90 A N, 0 N N \ \ The system of claim 7, wherein the mol% of the cationic component is between about and about 50; the mole % of the anionic or zwitterionic component is between about 0 and about 30; the mole % of the non-cationic lipid component is between about 30 and about 80; and the mol% of the shielding component is between about 0 and about 10. The system of claim 7 or 8, wherein the mol% of the cationic component is between about 30 and about 50; the mole % of the anionic or zwitterionic component is between about 0 and about 10; the mole % of the non-cationic lipid component is between about 40 to about 70; and the mol% of the shielding component is between about 0 and about 5. The system of any one of claims 7-9, wherein the mol% of the cationic component is 38; the mole % of the anionic or zwitterionic component is 0; the mole % of the non-cationic lipid component is 59.7; and the mol% of the shielding component is 2.3. The system of claim 10, wherein the anionic or zwitterionic component comprises DOPE, and wherein the shielding component comprises DMG-PEG2K. The system of any one of claims 1-5, wherein the cationic component comprises: Compound 93 NL NL NL HN NY NY NH, 0 0 NS NS and the non-cationic lipid component includes: cholesterol, Compound 90 N \ \ 2 Connection 89 A A, | N \ \ or any combination thereof. The system of any one of claims 1-5, wherein the cationic component comprises: Compound 79 N N N AIA AT A and N and the non-cationic lipid component includes: Compound 90 A N, 0 N N \ \ The system of any one of claims 1-5, wherein the cationic component comprises: Compound 24 NH, NH, N 0 0 O N 0 0 0 0 TAS SS Sy 0O N oO 0 0 Oo oO NH, and the non-cationic lipid component includes: cholesterol, Compound 89 A A, | N N \ \ 10 . Connection 90 A, nN, 0 N N \ or any combination thereof. A system according to any one of claims 2-5, comprising at least about 99 mol% cationic component and less than about 1 mol% shielding component. The system of claim 15, wherein the cationic component comprises: Compound 24 NH 2 NH, 0 0 0 N 0 0 0 0 HA AIA AHA AIHA HA AH Oo \ © Oo Oo Oo Oo NH, Connection 79 a and Connection 93 NN Oo Oo oy Connection 112 HoN NJ NA NA A a NY NN NH, JS | or any combination thereof. The system of claim 15 or 16, wherein the mole % of the cationic component is 99.1 and the mole % of the shielding component comprising PEG is 0.9. The system of any one of claims 2-5, comprising less than about 20 mole percent of the cationic component, less than about 5 mole percent of the shielding component, and greater than about 75 mole percent of a mixture of the zwitterionic component and the non-cationic lipid component. The system of claim 18, wherein the cationic component comprises: Compound 24 NH, NH, N 0 0 O N 0 0 0 0 Mi Hs Ae Ae Aes Aes Ses A, 0O N oO 0 0 Oo oO Connection 79 9 J, 2 J, 2 eT eN Connection 93 N N 0 0 N 2 5 . Compound 112 a ANIA AHA A, or any combination thereof. The system of claim 18 or 19, wherein the mol% of the cationic component is about 17.9, the mole % of the shielding component comprising PEG is about 2.8, the mole % of the non-cationic lipid component is about 62.9 and the mole % of the anionic or zwitterionic component is about 16.4. The system of claim 18 or 19, wherein the mole % of the cationic component is about 17.1, the mole % of the shielding component comprising PEG is about 2.7 and the mole % of the non-cationic lipid component is about 80 .2. The system of any one of claims 2-5, comprising between about 30 and about 45 mol% of the cationic component, between about 50 and about 70 mol% of a mixture of the anionic or zwitterionic component and the non-cationic lipid component, and between about 1.5 and about 4.5 mole percent of the shielding component. The system of claim 22, wherein the cationic component comprises: Compound 24 NH, NH, \ 0 0 0 N 0 0 0 0 HN N N N N N N Ar Hy Hy Hy Hey Ar A, 0O N oO 0O oO 0O oO NH, Connection 79 a and Connection 93 He 0 0 oy Connection 112 HoN NJ NA NA JS | or any combination thereof. The system of claim 22 or 23, wherein the mole % of the cationic component is about 32.9, the mole % of the shielding component comprising PEG is about 2.0, the mole % of the non-cationic lipid component is about 51 .7 and the mole % of the anionic or zwitterionic component is about 13.4. The system of claim 22 or 23, wherein the mole % of the cationic component is about 42.3, the mole % of the shielding component comprising PEG is about 4.4, the mole % of the non-cationic lipid component is 0 and the mole % of the anionic or zwitterionic component is about 53.3. The system of any one of claims 2-5, comprising between about 15 and about 35 mole percent of the cationic component, between about 60 and about 80 mole percent of a mixture of the zwitterionic component and the non-cationic lipid component, and between about 1.5 and about 3.0 mole percent of the shielding component. The system of claim 26, wherein the cationic component comprises: Compound 24 NH, NH, N 0 0 0 N 0 0 0 0 MN Hs A Ae A Ae Aes, 0O N oO 0O oO 0O oO Compound 79 Ho 0 3 0 3 0 SAA AI AH eN Connection 93 N N N 0 0 NS Nn Connection 112 HoN N N N MAN Ay Ay Ae, or any combination thereof. The system of claim 26 or 27, wherein the mole % of the cationic component is about 17.9, the mole % of the shielding component comprising PEG is about 2.8, the mole % of the non-cationic lipid component is about 62.9 and the mole % of the anionic or zwitterionic component is about 16.4. The system of claim 26 or 27, wherein the mole % of the cationic component is about 32.9, the mole % of the shielding component comprising PEG is about 2.0, the mole % of the non-cationic lipid component is about is 51.7 and the mole % of the anionic or zwitterionic component is about 13.4. A complex comprising the system according to any one of the preceding claims and a polyanionic compound, preferably a nucleic acid. The complex of claim 30, wherein the polyanionic compound is an mRNA encoding a protein. The complex of claim 30 or 31, wherein the mass ratio of the cationic component to the nucleic acid is between about 0.5:1 and about 20:1, between about 0.5:1 and about 10:1, between about 0.5 :1 and about 5:1, between about 1:1 and about 20:1, between about 1:1 and about 10:1, between about 1:1 and about 5:1, between about 2:1 and about 20: 1, is between about 2:1 and about 10:1, between about 2:1 and about 5:1, for example wherein the complex comprises the cationic components and the nucleic acid in a mass ratio of about 3:1. The complex of claim 32, wherein the cationic component is: Compound 112 HaN NA NA NJL NN NY NY NH, yi 0 and the non-cationic lipid component is: Compound 90 A, A, | nA NL H NY NOT NH, \ \ The complex of claim 33, wherein the shielding component comprises DMG-PEG2k. The complex of any one of claims 31-34, wherein the mass ratio of the cationic component to the nucleic acid is about 10:1. The complex of claim 35, wherein the mass ratio of the shielding component to the nucleic acid is 1.4:1. The complex of claim 35 or 36, wherein the mass ratio of the non-cationic lipid component to the nucleic acid is about 5.4:1. The complex of any one of claims 35-37, wherein a charge ratio between the cationic component and the nucleic acid is about 6:1. The complex of any one of claims 30-38, wherein the mass ratio of the cationic component to the nucleic acid is about 10:1, the mass ratio of the shielding component to the nucleic acid is about 1.4:1, the mass ratio of the non -cationic lipid component to the nucleic acid is about 5.4:1 and the mass ratio of the anionic or zwitterionic component to the nucleic acid is 0. A complex according to any one of claims 30-39 comprising the system according to any one of claims 7-11. A system according to any one of claims 7-11 for use in delivering a therapeutic nucleic acid, preferably an mRNA. 42. A system according to any one of claims 1 to 29 or the complex according to any one of claims 30 to 40 for use as a medicament. The system of any one of claims 1 to 29 or the complex of any one of claims 30 to 40 for use in the treatment of any of the conditions referred to herein. A method of forming the complex according to any one of claims 30-40, comprising contacting a system according to any one of claims 1-29 with a polyanionic compound, e.g. the nucleic acid, preferably an mRNA encoding a protein.