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WO2025010504A1 - Lipides diester, nanoparticule lipidique contenant des lipides diester et formulations associées - Google Patents

Lipides diester, nanoparticule lipidique contenant des lipides diester et formulations associées Download PDF

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Publication number
WO2025010504A1
WO2025010504A1 PCT/CA2024/050922 CA2024050922W WO2025010504A1 WO 2025010504 A1 WO2025010504 A1 WO 2025010504A1 CA 2024050922 W CA2024050922 W CA 2024050922W WO 2025010504 A1 WO2025010504 A1 WO 2025010504A1
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lipid
mol
independently
lipid nanoparticle
compound
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Rajesh Krishnan Gopalakrishna Panicker
Yury Karpov
Natalia Martin Orozco
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Providence Therapeutics Holdings Inc
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Providence Therapeutics Holdings Inc
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Priority claimed from CA3205968A external-priority patent/CA3205968A1/en
Priority claimed from CA3216061A external-priority patent/CA3216061A1/en
Application filed by Providence Therapeutics Holdings Inc filed Critical Providence Therapeutics Holdings Inc
Publication of WO2025010504A1 publication Critical patent/WO2025010504A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/08—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to an acyclic carbon atom of an acyclic unsaturated carbon skeleton
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001104—Epidermal growth factor receptors [EGFR]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
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    • C12N2760/00011—Details
    • C12N2760/10011—Arenaviridae
    • C12N2760/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates to diester lipids, and their use for preparing systems for encapsulating cargos such as nucleic acid sequences, polypeptides or peptides. More particularly, the present disclosure relates to ionizable diester compounds useful to prepare lipid nanoparticles (LNPs). The present disclosure also relates to LNPs comprising such di ester lipids.
  • the present disclosure also relates to lipids nanoparticles (LNPs) for the delivery of cargos such as nucleic acid sequences, polypeptides or peptides and methods of use of these LNPs for the treatment of diseases, disorders and/or conditions.
  • LNPs lipids nanoparticles
  • Lipid nanoparticles usually contain four ingredients: an ionizable lipid, a phospholipid, cholesterol and a PEGylated lipid.
  • a major component of LNPs is the ionizable lipid.
  • the phospholipid supports the formation of a lipid bilayer while cholesterol can stabilize the lipid bilayer.
  • the PEGylated lipid being amphiphilic, remains on the surface of LNPs to provide colloidal stability by steric shielding. Designing new ionizable lipids with suitable efficacy, stability and/or biodegradability to allow the preparation of LNPs is needed.
  • nucleic acids e.g., siRNA, mRNA, circular RNA, DNA, etc.
  • nucleic acids can be more effective when compared to protein- based therapies.
  • new delivery systems such as new LNPs, for both nucleic acid and protein therapeutics.
  • the present disclosure provides new lipid compounds, more particularly lipid compounds comprising at least two ester functions, referred to as “diester lipids” in the present disclosure.
  • the present disclosure also provides LNPs, more particularly LNPs formulated with diester lipid compounds.
  • Particles such as nanoparticles, comprising the compounds, constructs comprising the nanoparticles and cargos, wherein the cargo can be a small molecule, an antibody, a polynucleotide, or a polypeptide, methods of using the particles/constructs, and methods of preparing the compounds, particles and constructs are also provided.
  • R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;
  • X is -CH 2 -, -NH- or -NR7-; n is a number from 0 to 10; p is a number from 0 to 2; R5 and R6 are independently H, or an optionally substituted linear C1-C4 alkyl group;
  • a compound selected from the group consisting of Compounds 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217 and 218 of Table 1 of the present disclosure, or a pharmaceutically acceptable salt thereof, preferably Compounds 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 216, 217 and 218 of Table 1, or a pharmaceutically acceptable salt thereof.
  • the compound of the present disclosure i.e., the di ester lipid compound of the present disclosure, or the pharmaceutically acceptable salt thereof can be in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.
  • lipid nanoparticle comprising at least one compound of the present disclosure, i.e., the di ester lipid compound of the present disclosure, or the pharmaceutically acceptable salt thereof.
  • lipid nanoparticle comprising:
  • R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • R.2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • m is a number from 1 to 12;
  • the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid; (c) from 0 to about 50 mol % of the helper lipid; (d) from 0 to about 5 mol % of the polymer- conjugated lipid; and (e) from 0 to about 5 mol % of the hydrophobic component.
  • the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from 0 to about 5 mol % of the polymer-conjugated lipid; and (e) from 0 to about 5 mol % of the hydrophobic component.
  • the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and (e) from 0 to about 5 mol % of the hydrophobic component.
  • the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and (e) from about 0.1 to about 5 mol % of the hydrophobic component.
  • the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 30 to about 50 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and (e) from about 0.1 to about 5 mol % of the hydrophobic component.
  • the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 30 to about 45 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and (e) from about 0 to about 5 mol % of the hydrophobic component.
  • the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 30 to about 45 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and (e) from about 0.1 to about 5 mol % of the hydrophobic component.
  • a pharmaceutical composition comprising a lipid nanoparticle as defined herein, and a pharmaceutical acceptable excipient.
  • the present application relates to a method for delivering a cargo to a cell comprising contacting the cell with a lipid nanoparticle as defined herein, wherein the lipid nanoparticle comprises the cargo.
  • the present application relates to there is provided a use of a lipid nanoparticle as defined herein, for delivering a cargo to a cell, wherein the lipid nanoparticle comprises the cargo.
  • a vaccine comprising a lipid nanoparticle as defined herein, wherein the lipid nanoparticle comprises at least one cargo, preferably the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide, more preferably the cargo comprises at least one nucleic acid such as mRNA.
  • a method of vaccinating a subject against an infectious agent comprising:
  • a method of treating cancer in a subject comprising administering a lipid nanoparticle as defined herein to the subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.
  • lipid nanoparticle as defined herein for use in the treatment of cancer in a subject, wherein the lipid nanoparticle comprises an anti- cancer cargo or a cargo triggering an immune response against cancer cells.
  • lipid nanoparticle as defined herein for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.
  • lipid nanoparticle as defined herein for the preparation of a medicament for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.
  • lipid nanoparticle as defined herein for use in transfection of targeted cells, e.g. in transfecting human cells, including stem cells.
  • lipid nanoparticle as defined herein for use in gene replacing therapy.
  • Figure 1 depicts a cryo-TEM image of LNPs-01 formulated with ionizable diester lipid 201, DSPC, cholesterol and PEG2k-DMG.
  • Figure 2 depicts a cryo-TEM image of LNPs-02 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and squalene.
  • Figure 3 depicts a cryo-TEM image of LNPs-03 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and cardiolipin.
  • Figure 4 shows a graph representing the in vitro comparison of LNPs-07 containing ionizable lipid 201 and LNPs-10 containing Dlin-MC3-DMA in Huh-7 cells.
  • the spike protein produced is quantified using ELISA after 18 hours of DP transfection in Huh-7 cells.
  • Figure 5 shows a graph representing the in vitro comparison of LNPs-07, LNPs-08 and LNPs-09 containing ionizable lipid 201 and LNPs-10, LNPs-11 and LNPs-12 containing Dlin-MC3-DMA in Huh-7 cells.
  • LNPs-07 and LNPs-10 Four components LNPs (LNPs-07 and LNPs-10) were compared to five components LNPs containing either cardiolopin (LNPs-08 and LNPs-11) or squalene (LNPs-09 and LNPs-12) as fifth component.
  • the spike protein produced is quantified using ELISA after 18 hours of LNPs transfection in Huh-7 cells.
  • Figure 6 shows a graph that represents the quantity of blood glucose over a period of time in mice that were dosed with BMDC+3gp peptides + CpG or formulation buffer on day zero and day 0+4.
  • the blood glucose of mice is measured for 30 days from day 0.
  • the Bone marrow dendritic cells (BMDC) were used as positive control.
  • Figure 7a shows a graph that represents the in vivo testing of LNPs made from ionizable lipid 201 in RIP-gp mice.
  • the mice were dosed with LNPs-02.
  • the blood glucose of mice is measured for 30 days from day 0.
  • T-cell activation induced by LNPs leads to diabetes.
  • Figure 7b shows a graph that represents the in vivo testing of LNPs made from ionizable lipid 201 in RIP-gp mice.
  • the mice were dosed with LNPs-03.
  • the blood glucose of mice is measured for 30 days from day 0. T-cell activation induced by LNPs leads to diabetes.
  • Figures 8a/8b/8d show graphs representing the in vivo testing of LNPs made from ionizable lipids 201 in RIP-gp mice.
  • the mice were dosed with LNPs-02 (Figure 8a), LNPs- 03 ( Figure 8b) and LNPs-13 ( Figure 8d).
  • the blood glucose of mice is measured for more than 30 days from day 0.
  • T-cell activation induced by LNPs leads to diabetes.
  • Figure 8c represents the diabetes incidence.
  • Figures 9-13 summarize the stability data of LNPs made from ionizable lipid 201.
  • the LNPs are either four components or five components with squalene or cardiolipin. All LNPs were stored at -80 °C and samples were withdrawn on third and sixth month of manufacturing and tested for particle size (Figure 9), poly dispersity index (PDI) ( Figure 10), encapsulation efficiency (EE) ( Figure 11), mRNA integrity (purity) ( Figure 12) and mRNA concentration ( Figure 13) and compared to the same parameters at the time of manufacturing (time zero).
  • PDI poly dispersity index
  • EE encapsulation efficiency
  • mRNA integrity Purity
  • Figure 13 mRNA concentration
  • Figure 14 shows an in vivo comparison of LNPs made from ionizable lipid 201 and Dlin-MC3-DMA in mice.
  • LNPs-07 and LNPs-10) were compared to five component LNPs containing either squalene (LNPs-09 and LNPs-12) or cardiolipin (LNPs-08 and LNPs-11) as fifth component.
  • the spike antibody produced is quantified using ELISA after two weeks of second dose of LNPs.
  • Figure 15 depicts a cryo-TEM image of LNPs-13 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and squalene.
  • Figure 16 depicts a cryo-TEM image of LNPs-14 formulated with ionizable di ester lipid 201, DSPC, cholesterol, PEG2k-DMG and Withaferin A.
  • Figure 17 depicts a cryo-TEM image of LNPs-15 formulated with ionizable di ester lipid 201, DSPC, cholesterol, PEG2k-DMG and a-tocopherol.
  • Figure 18 depicts a cryo-TEM image of LNPs-16 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and
  • Figure 19 depicts a cryo-TEM image of LNPs-17 formulated with ionizable di ester lipid 201, DSPC, cholesterol, PEG2k-DMG and retinol.
  • Figure 20A shows Spike-specific IgG antibody levels detected by using an ELISA assay 14 days after a second immunization in mice with LNPs-07, LNPs-08 and LNPs-18.
  • Figure 20B is a graph showing the Neutralizing Antibody (NAb) response against Wuhan-Hu-1/D614G measured 14 days after a second immunization in mice with doses of 2.5 pg LNPs-07, LNPs-08 and LNPs-18.
  • NAb Neutralizing Antibody
  • Figure 21 is a graph showing the Neutralizing Antibody (NAb) response against Wuhan-Hu-1/D614G with LNPs-18 at different mRNA doses.
  • Figure 22 shows level of the antigen-specific IFNy secreting cells measured in splenocytes 14 days after the second immunization in mice with LNPs-07, LNPs-08 and LNPs- 18
  • Figure 23 shows the level of the antigen-specific IL-4 secreting cells measured in splenocytes 14 days after the second immunization in mice with LNPs-07, LNPs-08 and LNPs- 18
  • Figure 24 shows the MC38gp tumor growth control after immunization in mice with LNPs-02 and LNPs-03 made from ionizable lipid 201.
  • Figure 25 shows the results of Day 8 CD8+ T cell induction in wild-type mice using LNPs-02, LNPs-03 and LNPs-13 made from ionizable lipid 201.
  • Figure 26 shows the results of Day 12 CD45+ T cell induction in wild-type mice using LNPs-02 and LNPs-03 made from ionizable lipid 201 (gp-33 tetramer strain and gp-34 tetramer strains).
  • Figures 27A and 27B illustrate A: representative MRI images of tumors implanted in C3H-CL1-F2 (female) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control, at days 30 and 43 post-tumor implantation; and B: overall survival rate of C3H-CL1-F2 (female) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control.
  • Figure 28 illustrates the overall survival rate of C3H-CL1-M1 (male) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control.
  • Figure 29 illustrates representative MRI images of tumors implanted in C3H-CL1- M1 (male) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control, at days 30 and 43 post-tumor implantation.
  • Figure 30 illustrates the mice survival rate of untreated mice (buffer), of mice treated with BMDC, of mice treated with LNPs-02 and of mice treated with LNPs-03 in MC38gp Model.
  • Figure 31 represents a comparison of the Wt EGFR and the EGFRvIII sequences.
  • Figures 32A-E shows the in vivo EGFRvIII-induced GBM mouse model and vaccination with LNPs containing EGFRvIII mRNA.
  • Figure 32A shows the survival rate of vaccinated mice.
  • Figure 32B shows the flow cytometry analysis of EGFRvIII-specific CTLs in the spleens from vaccinated mice.
  • Figures 32C-32E show the quantification of EGFRvIII- specific CTLs in the spleens from vaccinated mice in frequencies and absolute numbers.
  • Figure 33 shows anti- EGFRvIII IgG antibody levels by ELISA in EGFRvIII- induced GBM following vaccination with LNPs.
  • Figure 34 shows the flow cytometry analysis of the transfection efficiency of LNPs containing tdTomato mRNA in human PBMCs.
  • Figure 35 shows the flow cytometry analysis of the transfection efficiency of LNPs containing tdTomato mRNA in human PBMCs preincubated with either ApoE or autologous human plasma.
  • Figure 36 shows the transfection efficiency of LNPs containing tdTomato mRNA in monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs) differentiated from human PBMCs with, before and after maturation/polarization.
  • MDDCs monocyte-derived dendritic cells
  • MDMs monocyte-derived macrophages
  • nucleic acid therapy includes the use of small interfering (siRNA) to reduce the translation of messenger RNA (mRNA), mRNA as a way to produce a target of interest, circular RNA (oRNA) which can provide continuous production of a polypeptide or peptide or can be a sponge to compete with other RNA molecules, and viral vectors to provide a continuous production of a target of interest.
  • small interfering siRNA
  • mRNA messenger RNA
  • oRNA circular RNA
  • viral vectors to provide a continuous production of a target of interest.
  • nucleic acids are unstable and easily degraded so they need to be formulated to prevent the degradation and to aid in the intracellular delivery of the nucleic acids.
  • the present disclosure relates to novel diester lipid compounds and compositions comprising the same, more particularly nanoparticles based on these diester compounds, capable of encapsulating a cargo such as a biologically active and therapeutic agent.
  • the present disclosure also relates to novel lipid nanoparticle compositions that may have improved stability, and/or increased efficacy such as increased immunogenicity when it is used in vaccines, and/or facilitate the intracellular delivery of biologically active and therapeutic agents.
  • the lipid nanoparticle compositions may have low or reduced toxicity.
  • the present disclosure relates also to pharmaceutical compositions that comprise such lipid compositions, and that are useful to deliver therapeutically effective amounts of biologically active agents into the cells of patients.
  • biologically active agents include but are not limited to: (1) proteins including immunoglobin proteins, (2) polynucleotides such as genomic DNA, cDNA, or mRNA, (3) antisense polynucleotides, and (4) low molecular weight compounds, whether synthetic or naturally occurring, such as the peptide hormones and antibiotics.
  • Lipid means an organic compound that comprises an ester of fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • Lipid particle or “lipid nanoparticle (i.e., “LNP”) means a lipid formulation that can be used to deliver a cargo, such as a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a therapeutic nucleic acid e.g., mRNA
  • target site of interest e.g., cell, tissue, organ, and the like.
  • the lipid particle can be used to encapsulate a nucleic acid.
  • the lipid nanoparticle can be formed from an ionizable lipid, a neutral lipid (e.g., a phospholipid), a polymer- conjugated lipid that can prevent aggregation of the nanoparticle (e.g., a PEG-lipid), and optionally a helper lipid (e.g., cholesterol).
  • a therapeutic nucleic acid e.g., mRNA
  • the lipid nanoparticle can comprise another component such as a hydrophobic component to improve LNP internalization, immune activation and/or antibody production.
  • Lipid nanoparticles generally comprise cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from non-specific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP -forming composition.
  • cholesterol saids in stability and promotes membrane fusion
  • a phospholipid which provides structure to the LNP bilayer and also may aid in endosomal escape
  • PEG polyethylene glycol
  • ionizable lipid complexes negatively charged RNA and enhances endosomal escape
  • Lipid nanoparticles typically can have a particle size, e.g., expressed as a mean diameter, ranging from 30 nm to 200 nm, from 40 nm to 180 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from 60 nm to 120 nm, from 60 nm to 110 nm, from 60 nm to 100 nm, from 60 nm to 90 nm, from 70 nm to 110 nm, from 70 nm to 100 nm, from 80 nm to 100 nm, from 90 nm to 100 nm, from 70 to 90 nm, from 80 nm to 90 nm, or from 70 nm to 80 nm.
  • a particle size e.g., expressed as a mean diameter, ranging from 30 nm to 200 nm, from 40 nm to 180 nm, from 50 nm to 150 nm, from 60 nm to 130
  • the particle size can be about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm and are substantially non-toxic.
  • nucleic acids when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.
  • the present disclosure relates to compounds that are ionizable lipids, more particularly ionizable diester lipids.
  • the ionizable lipids may be cationic lipids.
  • ionizable lipid compounds of the present disclosure comprise at least two ester bonds (-CO-O- or -O-CO-). Ester bonds present the particularity of being biodegradable.
  • compounds of the present disclosure can further comprise one secondary amino group.
  • compounds of the present disclosure further comprise at least one terminal amino group, wherein the amino group may be substituted with at least one lower alkyl group (e.g., C1-C3 alky groups), which may be further substituted.
  • the terminal amino group can be NH2, a primary amino group, a secondary amino group, or a tertiary amino group.
  • the terminal amino group can be NfCHsh.
  • the ionizable lipids of the present disclosure can be characterized in that the two ester bonds are separated by two tertiary carbon atoms, a first one of the two tertiary carbon atoms being substituted with an alkyl chain and the second one of the two tertiary carbon atoms being substituted with a hydrocarbon chain bearing the terminal amino group.
  • the hydrocarbon chain bearing the terminal amino group optionally comprises a nitrogen atom within the chain, this nitrogen atom being itself optionally substituted.
  • the ionizable lipid compound of the present disclosure can have a structure of Formula (I): or a pharmaceutically acceptable salt thereof, wherein
  • R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;
  • m in the structure of Formula (I), can be an integer from 1 to 12, or m can be an integer from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or m can be 1 or 2. In some embodiments, in the structure of Formula (I), m can be 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12.
  • n in the structure of Formula (I), can be an integer from 0 to 10, or n can be an integer from 0 to 9, or from 0 to 8, or from 0 to 7, or from 0 to 6, or from 0 to 5, or from 0 to 4, or from 0 to 3, or from 0 to 2, or n can be 0, 1 or 2. In some embodiments, in the structure of Formula (I), n can be 0, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10.
  • p can be an integer from 0 to 2, or p can be 0 or 1, or p can be 1 or 2, or p can be 0, or 1, or 2.
  • X is -CH2-.
  • X is -NH-.
  • X is -NR7- and R7 is a linear or branched C1-C6 alkyl.
  • X is -NR7- and R7 is a linear or branched C1-C4 alkyl.
  • X is -NR7- and R7 is C1-C2 alkyl.
  • X is -NMe-.
  • alkyl, alkenyl and/or alkynyl groups in the substituents of the Formula (I) when any of the alkyl, alkenyl and/or alkynyl groups in the substituents of the Formula (I) is substituted, these groups can independently be substituted with one or more halogen, hydroxyl, acetoxy, alkoxycarbonyl, formyl, acyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety or an heteroaromatic moiety.
  • any alkyl, alkenyl and/or alkynyl group groups in the substituents of the Formula (I) are substituted, these groups can be independently substituted with one or more halogen, hydroxyl, acetoxy, amino, cyano, nitro, azido, or sulfhydryl.
  • any alkyl, alkenyl and/or alkynyl group groups in the substituents of the Formula (I) are substituted, these groups can be independently substituted with one or more hydroxyl or acetoxy.
  • the ionizable diester lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein
  • R1 and R.4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • R.2 and Rs are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;
  • X is -CH 2 -, -NH- or -NR 7 -; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently an optionally substituted C1-C4 alkyl group;
  • R 7 is a linear or branched C1-C4 alkyl.
  • the ionizable diester lipid can have a structure of Formula (I) wherein
  • R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • R22 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • m is a number from 1 to 12;
  • X is -CH 2 -, -NH- or -NR 7 -; n is a number from 0 to 8; p is 0 or 1 ;
  • R5 and R6 are independently an optionally substituted C1-C4 alkyl group
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein:
  • R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • R2 and R3 are independently H or an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;
  • X is -CH2-, -NH- or -NR 7 -; n is a number from 0 to 8; p is 0 or 1 ;
  • R5 and R6 are independently an optionally substituted C1-C4 alkyl group
  • R 7 is a C1-C2 alkyl.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein: R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;
  • R2 and R3 are independently H or an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;
  • X is -CH 2 -, -NH- or -NR7-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently an optionally substituted C1-C4 alkyl group;
  • R7 is a C1-C2 alkyl; wherein when any alkyl, alkenyl and/or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, amino, cyano, nitro, azido, or sulfhydryl.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein:
  • R1 and R4 are independently linear or branched C8-C20 alkyl, linear or branched C8-C20 alkenyl, or linear or branched C8-C20 alkynyl group;
  • R2 and R3 are independently H, linear or branched C8-C20 alkyl, linear or branched C8- C20 alkenyl, or linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;
  • X is -CH2-, -NH- or -NMe-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently a C1-C4 alkyl group optionally substituted with hydroxyl or acetoxy.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein:
  • R1 and R4 are independently linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8- C20 alkynyl group;
  • R2 and R3 are independently H, linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8-
  • C20 alkynyl group m is a number from 1 to 8; X is -CH 2 -, -NH- or -NMe-; n is a number from 0 to 8; p is a number from 0 to 1 ; and R5 and R6 are independently a C1-C4 alkyl group.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein:
  • R1 and R4 are independently linear C8-C20 alkyl or linear C8-C20 alkenyl
  • R2 and R3 are independently H or linear C8-C20 alkyl or linear C8-C20 alkenyl; m is a number from 1 to 8;
  • X is -CH 2 -, -NH- or -NMe-; n is a number from 0 to 8; p is a number from 0 to 1 ; and R5 and R6 are independently C1-C4 alkyl.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein R1 and R4 are identical.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein R2 and R3 are H or linear C8-C18 alkyl, preferably H or C10-C16 alkyl, more preferably H or C14 alkyl.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein R2 and R3 are H.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein anRd5 R6 are independently a C1-C2 alkyl group.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formula (I) wherein aRn5d R6 are identical.
  • the ionizable diester lipid compound can have a structure of Formula (II), (12), (13), (14), (15), (16), (17), or (18):
  • q can be a number from 0 to 11, from 0 to 10, from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or q can be 0 or 1, or q can be 1 or 2, or q can be 0, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11.
  • s in the structure of Formulas (17), can be a number from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or s can be 0 or 1, or s can be 1 or 2, or s can be 0, or 1, or 2, or 3, or 4, or 5.
  • t in the structure of Formulas (18), t can be a number from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or t can be 0 or 1, or t can be 1 or 2, or t can be 2 or 3, or t can be 3 or 4, or 1, or 2, or 3, or 4, or 5, or 6.
  • X is independently -CH2-.
  • X is independently -NH-.
  • X is independently -NMe-.
  • the ionizable lipid compound, or the pharmaceutically acceptable salt thereof can have a structure of Formulas (13), (14), (15), (16), (17), or (18) wherein X is -CH2-.
  • the ionizable diester lipid compound can have a structure of Formula (Ila): number from 1 to 8; and q is a number from 0 to 11.
  • the ionizable diester lipid compound can have a structure of Formula (lib): or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.
  • the ionizable diester lipid compound can have a structure of Formula (lie): or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.
  • the ionizable diester lipid compound can have a structure of Formula (I2a):
  • the ionizable diester lipid compound can have a structure of
  • the ionizable diester lipid compound can have a structure of Formula (I3a): or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.
  • the ionizable diester lipid compound can have a structure of Formula (I4a):
  • the ionizable diester lipid compound can have a structure of Formula (I5a):
  • the ionizable diester lipid compound can have a structure of Formula (I6a): number from 1 to 8; and q is a number from 0 to 8.
  • the ionizable diester lipid compound can have a structure of Formula (I7a):
  • the ionizable diester lipid compound can have a structure of number from 1 to 8; and t is a number from 0 to 6.
  • (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a), or (I8a) and/or in the compounds represented in Table 1, can independently have the E or Z configuration.
  • the ionizable diester lipid compounds of the present disclosure can be selected from the group consisting of Compounds 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217 and 218 of Table 1, or a pharmaceutically acceptable salt thereof.
  • the ionizable di ester lipid compounds can be in the form of any enantiomer and/or any diastereoisomer thereof, or any mixture thereof. [0138] Table 1: Non-Limiting Examples of Ionizable Lipid Compounds
  • the compounds of Table 1 can be in the form of mixtures of cis or trans geometric isomers.
  • the ionizable diester lipid compounds of Table 1 can be in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.
  • compound is meant to embrace all stereoisomers, geometric isomers, tautomers, and isotopes of a depicted or described structure associated with the compound.
  • the terms “optional” or “optionally” refer to a feature or substituent that may or may not occur.
  • “optionally substituted alkyl” encompasses both “alkyl” and “substituted alkyl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and/or inherently unstable.
  • the compounds herein described may have asymmetric centers, geometric centers (e.g., double bond), or both. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
  • Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms, by synthesis from optically active starting materials, or through use of chiral auxiliaries.
  • cis and trans geometric isomers of the compounds of the present disclosure may also exist and may be isolated as a mixture of isomers or as separated isomeric forms.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H- imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • each individual hydrogen atom present in formula (200) may be present as a 1 H, 2 H (deuterium) or 3 H (tritium) atom, preferably J H or 2 H.
  • each individual carbon atom present in formula (200) may be present as a 12 C, 13 C or 14 C atom, preferably 12 C.
  • the compounds or structures and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • Neutral lipids can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • the lipid nanoparticle can also include at least one neutral lipid.
  • the neutral lipids may be phospholipids, or derivatives thereof.
  • Examples of phospholipids suitable for use in the present disclosure include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2 -palmitoyl phosphatidylcholine (MPPC), 1-palmi toy 1-2 -myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PS
  • the preferred phospholipids are distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE).
  • DSPC distearoylphosphatidylcholine
  • DOPE dioleoylphosphatidylethanolamine
  • the preferred phospholipids are DSPC, DOPC, DMPC and PE.
  • Helper lipids are DSPC, DOPC, DMPC and PE.
  • the lipid nanoparticle can also include at least one helper lipid.
  • helper lipids are lipids that enhance transfection, such as transfection of the lipid nanoparticle including the payloads and cargos. The mechanism by which the helper lipid enhances transfection may include enhancing particle stability and/or enhancing membrane fusogenicity. Helper lipids include steroids and alkyl resorcinols.
  • Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl- 2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.
  • the preferred helper lipid is cholesterol.
  • the lipid nanoparticle can further include at least one polymer- conjugated lipid.
  • the polymer-conjugated lipid comprises a polymer conjugated to at least one lipid.
  • the polymer-conjugated lipid can comprise at least one component that reduces aggregation of particles, at least one component that decreases clearing of the lipid nanoparticle from circulation in a subject, at least component that increases the lipid nanoparticle’s ability to traverse mucus layers, at least one component that decreases a subjects immune response to administration of the lipid nanoparticle, at least one component that modifies membrane fluidity of the lipid nanoparticle, at least one component that contributes to the stability of the lipid nanoparticle, or any combination thereof.
  • the lipid nanoparticle may be essentially devoid of polymer-conjugated lipid. In some embodiments, the lipid nanoparticle may contain no amount of polymer-conjugated lipid.
  • the polymer present in the polymer-conjugated lipid may comprise at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.
  • the lipid conjugated to the polymer may be selected from, but is not limited to, at least one of the ionizable, neutral, or helper lipids listed previously.
  • the polymer conjugated to at least one lipid is PEG and the polymer-conjugated lipid can be referred to as “PEG-lipid”.
  • the at least one PEG-lipid may be selected from, but is not limited to at least one of Siglec-IL-PEG-DSPE, R)-2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE Cl 8, PEG-DSPE, PEG- DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG Cl 4, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG-
  • the preferred polymer-conjugated lipids are polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-oxazoline) (POZ), polyamide (ATTA), cationic polymer, poly sarcosine (Psar), poly glutamic acid (PGA) and 1 ,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol (PEG-DMG).
  • the preferred polymer-conjugated lipids are PEG-lipids selected from PEG-DMG or PEG-DSG.
  • the preferred PEG-lipid is PEG2k-DMG.
  • the average molecular weight of the polymer moiety (e.g., PEG) of the polymer-conjugated lipid may be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer may be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, ,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to0,000, 7,000 to 20,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000, 9,000 to 20,000,.500 to 20,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000,2,000 to 20,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000,4.500 to 20,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to
  • the lipid nanoparticles can also include another type of lipids, referred to as “hydrophobic components” in the present disclosure.
  • the hydrophobic component is defined as a component, which, during the lipid nanoparticle formation, is incorporated in the lipid bilayer due to its hydrophobic nature.
  • the hydrophobic component may be selected from the group consisting of cardiolipin, squalene, vitamin A and derivatives thereof, 0-carotene, withaferin A and a-tocopherol.
  • the hydrophobic component may be selected from the group consisting of cardiolipin, squalene, vitamin A, retinol, 0-carotene, withaferin A and a-tocopherol.
  • lipid nanoparticles can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-lipid nanoparticle environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space.
  • Lipid nanoparticle membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.
  • lipid nanoparticles may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.
  • the present disclosure also provides lipid nanoparticle comprising a cargo or payload.
  • the term “cargo” or “payload” can refer to one or more molecules or structures encompassed in a lipid nanoparticle for delivery to or into a cell or tissue.
  • cargo can include a nucleic acid, a polypeptide, peptide, protein, a liposome, a label, a tag, a small chemical molecule, a large biological molecule, and any combinations or fragments thereof.
  • the region of the construct which comprises or encodes the cargo or payload is referred to as the “cargo region” or the “payload region”.
  • the lipid nanoparticle comprises at least one ionizable lipid of the present disclosure discussed in Section II, such as the ionizable lipid of general Formula (I), or more particularly, the compounds in Table 1.
  • the diester bonds of the lipids provide biodegradability and biocompatibility, such ester bonds are stable at physiological pH, but can be enzymatically hydrolyzed within tissues and cells.
  • the length of the Rl, R2, R3 and R4 groups in Formula (I) can also be adjusted to reach the desired zeta potential, particle size or membrane rigidity.
  • the lipid nanoparticle may comprise any lipid described in the disclosure.
  • the lipid may be any cationic lipid described in the disclosure.
  • the lipid nanoparticle may comprise neutral lipids.
  • the neutral lipid may be a phospholipid, or a derivative thereof.
  • the lipid may be any phospholipid described in the disclosure.
  • the lipid may be any cholesterol derivative described in the disclosure.
  • a polymer e.g., PEG
  • a PEG-lipid may be used in the lipid nanoparticle and can be any PEG-lipid conjugate described in the disclosure.
  • the lipid nanoparticles can be characterized by their shape.
  • the lipid nanoparticles are essentially spherical.
  • the lipid nanoparticles are essentially rod-shaped (i.e., cylindrical).
  • the lipid nanoparticles are essentially disk shaped.
  • the term “nanoparticle” as used herein refers to any particle ranging in size from 10-1000 nm.
  • the nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,
  • a population of lipid nanoparticles such as those resulting from the same formulation, may be characterized by measuring the uniformity of size, shape, or mass of the particles in the population, uniformity may be expressed in some embodiments as the polydispersity index (PI) of the population. In some embodiments uniformity may be expressed in some embodiments as the disparity (D) of the population.
  • PI polydispersity index
  • D disparity
  • PI polydispersity index
  • D disparity
  • a population of lipid nanoparticles resulting from a given formulation can have a PI of between about 0.1 and 1.
  • a population of lipid nanoparticles resulting from a giving formulation can have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1. In some embodiments, a population of lipid nanoparticles resulting from a given formulation can have a PI ofbetween about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1.
  • the lipid nanoparticle may have PI ranging between about 0.01 to 0.3, 0.02-0.3, 0.03-0.3, 0.04-0.3, 0.05-0.3, 0.06-0.3, 0.07-0.3, 0.08-0.3, 0.09-0.3, 0.
  • the total mole percentage of the lipid(s) in the LNP is between about 10% to about 95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31% to about 40%, between about 41% to about 50%, between about 51% to about 60%, between about 61% to about 70%, between about 71% to about 80%, between about 81% to about 90%, or between about 91% to about 95%.
  • the lipids of the present disclosure may be incorporated into lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid, or any combination thereof, wherein the neutral lipid, helper lipid, polymer-conjugated lipid are as defined herein.
  • the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one helper lipid.
  • the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid, at least one helper lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one neutral lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one polymer-conjugated lipid.
  • the LNP may be comprised of at least one neutral lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one helper lipid and at least one polymer- conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid. In some embodiments, a LNP may be comprised of a helper lipid. In some embodiments, the LNP may be comprised of a polymer-conjugated lipid.
  • a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid; and at least one hydrophobic component, wherein the ionizable lipid, neutral lipid, helper lipid, polymer-conjugated lipid and hydrophobic component are as defined herein.
  • the lipid nanoparticle can further comprise an adjuvant, a cell targeting component, a fat-soluble vitamin, an immunomodulating substance or a component that promotes absorption of drugs.
  • the adjuvant can be squalene
  • the cell targeting component can be cardiolipin
  • the fat-soluble vitamin can be vitamin A or E
  • the immunomodulating substance can be withaferin
  • the component that promote absorption of drugs can be caffeine.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, can be from 0.1 to 100 mol%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is between 10%-95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31% to about 40%, between about 41% to about 50%, between about 51% to about 60%, between about 61% to about 70%, between about 71% to about 80%, between about 81% to about 90%, or between about 91% to about 95%.
  • the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 40 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 20 to 60 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 50 to 85 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of less than about 20 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than about 60 mol% or about 85 mol%.
  • the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises an ionizable lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is from about 30% to 100%, from about 40% to 100%, from about 30% to about 90%, from about 30% to about 85%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 65%, or from about 30% to about 60%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II),
  • the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, or from about 40% to about 60%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is from about 40 to about 47 mol % of the ionizable lipid, preferably from about 40 to about 45 mol % of the ionizable lipid.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12),
  • the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 65%, or from about 50% to about 60%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) or the compounds in Table 1, in the lipid nanoparticle, is about 55% to about 90%, from about 55% to about 85%, from about 55% to about 80%, from about 55% to about 75%, from about 55% to about 70%, from about 55% to about 65%, or from about 55% to about 60%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 60% to about 90%, from about 60% to about 85%, from about 60% to about 80%, from about 60% to about 75%, from about 60% to about 70%, or from about 60% to about 65%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II),
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12),
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 75% to about 90%, from about 75% to about 85%, or from about 75% to about 80%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 80% to about 90%, or from about 80% to about 85%.
  • the total mole percentage of the ionizable lipid such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 85% to about 90%.
  • the ionizable lipid mol % of the lipid nanoparticle can be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol %.
  • transfer vehicle variability between lots can be less than 15%, less than 10% or less than 5%.
  • the neutral lipid when the lipid nanoparticle comprises at least one neutral lipid, can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 5 to 35 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 5 to 25 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of more than about 25 mol% or about 35 mol%.
  • the lipid nanoparticle comprises at least one neutral lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiment, the lipid nanoparticle comprises at least one neutral lipid in an amount of at most bout 10 mol%.
  • the total mole percentage of the neutral lipid in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 18%, from about 5% to about 16%, from about 5% to about 14%, from about 5% to about 12%, from about 5% to about 10%, or from about 5% to about 8%.
  • the total mole percentage of the neutral lipid in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 18%, from about 7% to about 16%, from about 7% to about 14%, from about 7% to about 12%, from about 7% to about 10%, or from about 7% to about 8%.
  • the total mole percentage of the neutral lipid in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 18%, from about 9% to about 16%, from about 9% to about 14%, from about 9% to about 12%, or from about 9% to about 10%.
  • the total mole percentage of the neutral lipid in the lipid nanoparticle is about 11% to about 20%, from about 11% to about 18%, from about 11% to about 16%, from about 11% to about 14%, or from about 11% to about 12%.
  • the total mole percentage of the neutral lipid in the lipid nanoparticle is about 13% to about 20%, from about 13% to about 18%, from about 13% to about 16%, or from about 13% to about 14%.
  • the total mole percentage of the neutral lipid in the lipid nanoparticle is about 15% to about 20%, or from about 17% to about 18%.
  • the neutral lipid mol % of the lipid nanoparticle can be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol %.
  • the helper lipid when the lipid nanoparticle comprises at least one helper lipid, can be present in the lipid nanoparticle in an amount of about 0. 1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of at most 50 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 20 to 45 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 25 to 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than about 20 mol%.
  • the lipid nanoparticle comprises at least one helper lipid in an amount of more than about 45 mol% or about 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%.
  • the lipid nanoparticle comprises at least one helper lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
  • the helper lipid mol % of the lipid nanoparticle can be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol %.
  • the total mole percentage of the helper lipid in the lipid nanoparticle is about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, or from about 20% to about 25%.
  • the total mole percentage of the helper lipid in the lipid nanoparticle is about 25% to about 50%, from about 25% to about 45%, from about 25% to about 40%, from about 25% to about 35%, or from about 25% to about 30%.
  • the total mole percentage of the helper lipid in the lipid nanoparticle is about 30% to about 50%, from about 30% to about 45%, from about 30% to about 40%, or from about 30% to about 35%.
  • the total mole percentage of the helper lipid in the lipid nanoparticle is about 35% to about 50%, from about 35% to about 45%, or from about 35% to about 40%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 40% to about 50%, or from about 40% to about 45%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 45% to about 50%.
  • the polymer-conjugated lipid when the lipid nanoparticle comprises at least one polymer- conjugated lipid, can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 0.5 to 15 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 15 to 40 mol%.
  • the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than about 0.1 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of at most about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%.
  • the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
  • the polymer-conjugated lipid mol % of the lipid nanoparticle can be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol %.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 0.1% to about 10%, from about 0.1% to about 9%, from about 0.1% to about 8%, from about 0.1% to about 7%, from about 0.1% to about 6%, from about 0. 1% to about 5%, or from about 0. 1% to about 4%, from about 0. 1% to about 3%, from about 0. 1% to about 2%, or from about 0. 1% to about 1%.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 0.5% to about 10%, from about 0.5% to about 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%, from about 0.5% to about 5%, or from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, or from about 0.5% to about 1%.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 1% to about 10%, from about 1% to about 9%, from about 1% to about 8%, from about 1% to about 7%, from about 1% to about 6%, from about 1% to about 5%, from about 1% to about 4%, from about 1% to about 3%, or from about 1% to about 2%.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 2% to about 10%, from about 2% to about 9%, from about 2% to about 8%, from about 2% to about 7%, from about 2% to about 6%, from about 2% to about 5%, from about 2% to about 4%, or from about 2% to about 3%.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 3% to about 10%, from about 3% to about 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, or from about 3% to about 4%.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, or from about 4% to about 5%.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 5% to about 10%, from about 5% to about 9%, from about 5% to about 8%, from about 5% to about 7%, or from about 5% to about 6%.
  • the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 6% to about 10%, from about 6% to about 9%, from about 6% to about 8%, or from about 6% to about 7%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 7% to about 10%, from about 7% to about 9%, or from about 7% to about 8%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%.
  • the total mole percentage of the polymer- conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%. In some embodiments, the total mole percentage of the PEG-lipid in the lipid nanoparticle is about 9% to about 10%.
  • the lipid nanoparticle can comprise a hydrophobic component.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle at most about 20%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, or from about 1% to about 5%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 3% to about 20%, from about 3% to about 15%, from about 3% to about 10%, or from about 3% to about 5%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is at most about 5 %.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 15%, or from about 7% to about 10%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 15%, or from about 9% to about 10%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 11% to about 20%, or from about 11% to about 15%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 13% to about 20%, or from about 13% to about 15%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 15% to about 20%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 17% to about 20%.
  • the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 19% to about 20%.
  • the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • the at least one ionizable lipid is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie). (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • the lipid nanoparticle is comprised of about 35-45 mol% of at least one ionizable lipid, about 25-35 mol% of at least one neutral lipid (e.g., a phospholipid), about 20-30 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • the lipid nanoparticle is comprised of about 45-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid, about 5-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 35-45 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-3 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 15-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.01-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • at least one neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • the lipid nanoparticle is comprised of about 10-75 mol% of at least one ionizable lipid, about 0.5-50 mol% of at least one neutral lipid (e.g., a phospholipid), about 5-60 mol% of at least one helper lipid (e.g., cholesterol), and about 0.1-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • at least one neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • the lipid nanoparticle is comprised of about 25-75 mol% of at least one ionizable lipid, about 0.1-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 5-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-35 mol% of at least one helper lipid (e.g., cholesterol), and about 5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • the lipid nanoparticle is comprised of about 20-60 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-55 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-15 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).
  • the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol).
  • a hydrophobic component e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol.
  • the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol).
  • a hydrophobic component e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol.
  • the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol).
  • a hydrophobic component e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol.
  • the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol).
  • a hydrophobic component e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol.
  • the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol).
  • at least one neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • hydrophobic component e.g., squalene, cardiolipin, vitamin A,
  • the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol).
  • at least one neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • hydrophobic component e.g., squalene, cardiolipin, vitamin A,
  • the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol).
  • a hydrophobic component e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a-tocopherol.
  • the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and/or a- tocopherol).
  • at least one neutral lipid e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • about 0-10 mol% of at least one polymer-conjugated e.g., a PEG-lipid
  • about 4-10 mol% of at least one hydrophobic component e.g
  • the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolip
  • the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, [3-carotene, and/or a-tocophe
  • the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1- 4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, f>- carotene, withaferin A and/or a-tocopherol.
  • ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (
  • the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, f>- carotene, withaferin A and/or a-tocopherol).
  • a neutral e.g., a phospholipid
  • helper lipid e.g., cholesterol
  • polymer-conjugated lipid e.g., a PEG-lipid
  • hydrophobic component e.g., squalene, cardiolipin, vitamin A
  • the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG- lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, [3-carotene and/or a-tocopherol.
  • the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, [3-carotene, withaferin A and/or a-tocopherol.
  • the lipid nanoparticle can comprise from about 40 to about 100 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (Il), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b) (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from 0 to about 10 mol % of the neutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the nano
  • the lipid nanoparticle can comprise from about 40 to about 99 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the nanoparticle
  • the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the nanoparticle
  • the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the nanoparticle
  • the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the nano
  • the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer-conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the
  • the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 30 to about 50 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer-conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the
  • the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 30 to about 45 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer-conjugated lipid; and from 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the mol % are based on the total lipids present in the nano
  • the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of a phospholipid as the neutral lipid; from about 30 to about 50 mol % of a sterol as the helper lipid; from about 1 to about 4 mol % of a PEG-lipid as the polymer-conjugated lipid; and from 0.
  • the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15),
  • mol % 1 to about 5 mol % of squalene, cardiolipin, a-tocopherol, withaferin A, vitamin A, or a combination thereof as the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.
  • the lipid nanoparticle may fully or partially encapsulate a cargo or payload. In some embodiments, essentially 0% of the cargo present in the final formulation is exposed to the environment outside of the lipid nanoparticle (i.e., the cargo is fully encapsulated. In some embodiments, the cargo is associated with the lipid nanoparticle but is at least partially exposed to the environment outside of the lipid nanoparticle. In some embodiments, the lipid nanoparticle may be characterized by the % of the cargo not exposed to the environment outside of the lipid nanoparticle, e.g., the encapsulation efficiency.
  • an encapsulation efficiency of about 100% refers to a lipid nanoparticle formulation where essentially all the cargo is fully encapsulated by the lipid nanoparticle, while an encapsulation rate of about 0% refers to a lipid nanoparticle where essential none of the cargo is encapsulated in the lipid nanoparticle, such as with a lipid nanoparticle where the cargo is bound to the external surface of the lipid nanoparticle.
  • a lipid nanoparticle may have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15% less than about 10%, or less than 5%.
  • an lipid nanoparticle may have an encapsulation efficiency of between about 90 to 100%, 80 to 100%, 70 to 100%, 60 to 100%, 50 to 100%, 40 to 100%, 30 to 100%, 20 to 100%, 10 to 100%, 80 to 90%, 70 to 90%, 60 to 90%, 50 to 90%, 40 to 90%, 30 to 90%, 20 to 90%, 10 to 90%, 70 to 80%, 60 to 80%, 50 to 80%, 40 to 80%, 30 to 80%, 20 to 80%, 10 to 80%, 60 to 70%, 50 to 70%, 40 to 70%, 30 to 70%, 20 to 70%, 50 to 70%, 40 to 70%, 30 to 70%, 20 to 70%, 10 to 70%, 40 to 70%, 30 to 70%, 20 to 70%, 10 to 70%, 40 to 50%, 30 to 50%, 20 to 50%, 10 to 50%, 30 to 40%, 20 to 40%, 10 to 40%, 20 to 30%, 10 to 30%, and 10 to 20%.
  • the weight ratio of the lipid nanoparticle (including all the lipids) and the cargo or payload is between about 100: 1 to about 1: 1, such as between about 100: 1 to about 90: 1, between about 89: 1 to about 80: 1, between about 79: 1 to about 70: 1, between about 69: 1 to about 60: 1, between about 59: 1 to about 50: 1, between about 49: 1 to about 40: 1, between about 39: 1 to about 30: 1, between about 29: 1 to about 20: 1, between about 19:1 to about 10:1, and between about 9:1 to about 1:1.
  • the lipid nanoparticle further comprises an originator construct or a benchmark construct with at least one cargo or payload.
  • the cargo or payload can be a small molecule, an antibody, a polynucleotide or a polypeptide.
  • the cargo or payload can comprise at least one nucleic acid, such as mRNA.
  • the cargo or payload may be any DNA, plasmid, RNA or polypeptide described herein.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a coding RNA.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a non-coding RNA.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is an oRNA.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is an mRNA.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a personalized vaccine mRNA.
  • a personalized vaccine mRNA Non-limiting example includes 3-GP.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a Covid- 19 vaccine mRNA.
  • a Covid- 19 vaccine mRNA Non-limiting example includes PTX-CB and coronavirus spike protein.
  • the at least one RNA compound is comprised of a functional RNA where the RNA results in at least one change in a cell, tissue, organ and/or organism.
  • Said changes in state may include, but are not limited to, altering the expression level of a polypeptide, altering the translation level of a nucleic acid, altering the expression level of a nucleic acid, altering the amount of a polypeptide present in a cell, tissue, organ and/or organism, changing a genetic sequence of a cell, tissue, organ and/or organism, adding nucleic acids to a target genome, subtracting nucleic acids from a target genome, altering physiological activity in a cell, tissue, organ and/or organism or any combination thereof.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is DNA.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are DNA.
  • the DNA may be the same DNA or different DNA.
  • the DNA are the same.
  • the DNA are different.
  • the DNA are different but encode the same payload or cargo.
  • the DNA are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are DNA.
  • the DNA may be the same DNA or different DNA.
  • the DNA are the same.
  • the DNA are different.
  • two DNA are the same and one is different.
  • the first DNA is different from the second and third DNA.
  • the first DNA, second DNA and third DNA are all different.
  • the first DNA is different from the second and third DNA, but they all encode the same payload or cargo.
  • the first DNA is different from the second and third DNA but the second and third DNA encode the same payload or cargo.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a polypeptide.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are polypeptides.
  • the polypeptides may be the same polypeptide or different polypeptides.
  • the polypeptides are the same.
  • the polypeptides are different.
  • the polypeptides are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are polypeptides.
  • the polypeptides may be the same polypeptide or different polypeptides.
  • the polypeptides are the same.
  • the polypeptides are different.
  • two polypeptides are the same and one is different.
  • the first polypeptide is different from the second and third polypeptides.
  • the first polypeptide, second polypeptide and third polypeptide are all different.
  • the first polypeptide is different from the second and third polypeptides, but they all encode the same payload or cargo.
  • the first polypeptide is different from the second and third polypeptides but the second and third polypeptides encode the same payload or cargo.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a peptide.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are peptides.
  • the peptides may be the same peptide or different peptides.
  • the peptides are the same.
  • the peptides are different.
  • the peptides are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are peptides.
  • the peptides may be the same peptide or different peptides.
  • the peptides are the same.
  • the peptides are different.
  • two peptides are the same and one is different.
  • the first peptide is different from the second and third peptides.
  • the first peptide, second peptide and third peptide are all different.
  • the first peptide is different from the second and third peptides, but they all encode the same payload or cargo.
  • the first peptide is different from the second and third peptides but the second and third peptide encode the same payload or cargo.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is plasmid.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is RNA.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are RNAs.
  • the RNAs may be the same RNA or different RNAs.
  • the RNAs are the same.
  • the RNAs are different.
  • the RNAs are different but encode the same payload or cargo.
  • the RNAs are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are RNAs.
  • the RNAs may be the same RNA or different RNAs.
  • the RNAs are the same.
  • the RNAs are different.
  • two RNAs are the same and one is different.
  • the first RNA is different from the second and third RNAs.
  • the first RNA, second RNA and third RNA are all different.
  • the first RNA is different from the second and third RNAs, but they all encode the same payload or cargo.
  • the first RNA is different from the second and third RNA but the second and third RNAs encode the same payload or cargo.
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is DNA.
  • the RNA and DNA may encode the same peptide or polypeptide or may encode different peptides or polypeptides.
  • the RNA and DNA may encode the same peptide or polypeptide.
  • the RNA and DNA may encode different peptides or polypeptides.
  • RNA and DNA are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • a payload or cargo e.g., heavy chain or light chain of an antibody
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is a peptide.
  • the RNA may encode the same peptide as the peptide cargo/payload the RNA may encode a different peptide.
  • the RNA encodes the same peptide.
  • the RNA encodes a different peptide.
  • the RNA and peptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is a polypeptide.
  • the RNA may encode the same polypeptide as the polypeptide cargo/payload the RNA may encode a different polypeptide.
  • the RNA encodes the same polypeptide.
  • the RNA encodes a different polypeptide.
  • the RNA and polypeptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is DNA, and one is a peptide.
  • the DNA may encode the same peptide as the peptide cargo/payload the DNA may encode a different peptide.
  • the DNA encodes the same peptide.
  • the DNA encodes a different peptide.
  • the DNA and peptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is DNA, and one is a polypeptide.
  • the DNA may encode the same polypeptide as the polypeptide cargo/payload the DNA may encode a different polypeptide.
  • the DNA encodes the same polypeptide.
  • the DNA encodes a different polypeptide.
  • the DNA and polypeptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).
  • the lipid nanoparticles described herein may be formed using techniques known in the art.
  • an organic solution containing the lipids is mixed together with an acidic aqueous solution containing the originator construct or benchmark construct in a microfluidic channel resulting in the formation of targeting system (lipid nanoparticle and the benchmark construct).
  • a lipid nanoparticle formulation may be prepared by the methods described in International Publication No W02008103276, the content of which is herein incorporated by reference in its entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. W02019131770, the content of which is herein incorporated by reference in its entirety.
  • a lipid nanoparticle formulation may be prepared by the methods described in International Publication No. WO2020237227, the content of which is herein incorporated by reference in its entirety.
  • a lipid nanoparticle formulation may be prepared using the methodologies and devices described in US20240181406A1, the content of which is herein incorporated by reference in its entirety.
  • a lipid nanoparticle formulation may be prepared using the FDmiX mixing systems developed by FDX Fluid Dynamix GmbH (See https://www.fdx.de/en/fdmix/. the content of which is herein incorporated by reference in its entirety).
  • a lipid nanoparticle formulation may be prepared by a so-called “Point-of-Care” mixing method, which consists in preparing separately a solution of a cargo, such as mRNA for instance, that can be frozen or lyophilized for storage, and a solution of lipid. Mixing the lipid solution and the mRNA solution via transferring the mRNA to a sealed vial containing the lipid followed by vigorous shaking of the vial provides mRNA encapsulated in LNPs ready for using/dosing. Point-of-Care mixing can produce mRNA-LNPs with comparable quality and efficacy to mRNA-LNPs obtained with conventional methods (conventional mRNA-LNPs).
  • Point-of-Care mixing method can produce mRNA-LNPs with comparable quality and efficacy to mRNA-LNPs obtained with conventional methods (conventional mRNA-LNPs).
  • mRNA-LNPs Manufacturing of conventional mRNA-LNPs is a sensitive process and usually involves specialized equipment and analytical techniques.
  • the mRNA- LNPs are sensitive to minor changes in the manufacturing process. Long-term storage of mRNA-LNPs requires cryo-storage facilities. Still long-term stability of mRNA-LNPs may not be ensured.
  • Point-of-Care mixing the LNPs are not manufactured ahead of time of administration.
  • the mRNA and lipid solution can be manufactured, filled and released using conventional small-molecule pharmaceutical manufacturing units. Both the mRNA and lipid solution are stable under frozen conditions.
  • compositions or constructs comprising the lipid nanoparticles of the present disclosure, wherein the lipid nanoparticles may comprise, encode or be conjugated to a cargo or payload to produce the constructs.
  • the cargo or payload is or encodes a biologically active molecule such as, but not limited to a therapeutic protein.
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • the cargo or payload is or encodes one or more prophylactically- or therapeutically-active proteins, polypeptides, or other factors.
  • the cargo or payload may be or encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer.
  • the cargo or payload may be or encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., cargo or payload is or encodes Dystrophin), cardiovascular disease (e.g., cargo or payload is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., cargo or payload is or encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., cargo or payload is or encodes GlyRal), an enkephalin, or a glutamate decarboxylase (e.g., cargo or payload is or encodes GAD65, GAD67, or another isoform), lung disease (e.g., cargo or payload is or encodes CFTR), hemophilia (e.g., cargo or pay load is or encodes Factor VIII or Factor IX), neoplasia (e.g.,
  • muscular dystrophy
  • Neuregulin (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplxl), Tphl Tryptophan hydroxylase, Tph2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAO A, DTNBPI, Dao (Daol)), trinucleotide repeat disorders (e.g., HTT (Huntington's Dx), SBMA/SMAXI/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Machado-Joseph's Dx), ATXNI and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atnl (DRPLA Dx), CBP (Creb-
  • the cargo or payload is or encodes a factor that can affect the differentiation of a cell.
  • a factor that can affect the differentiation of a cell.
  • the expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, mir-302/367, or other miRNAs can cause the cell to become an induced pluripotent stem (iPS) cell.
  • iPS induced pluripotent stem
  • the cargo or payload is or encodes a factor for transdifferentiating cells.
  • factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurrl, LmxlA, Bm2, Myth, NeuroDl, FoxA2 for neural cells; and Hnf4a, Foxal, Foxa2 or Foxa3 for hepatic cells.
  • the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a polypeptide, protein or peptide.
  • a cargo or payload which is a polypeptide, protein or peptide.
  • polypeptide generally refers to polymers of amino acids linked by peptide bonds and embraces “protein and “peptides.”
  • Polypeptides for the present disclosure include all polypeptides, proteins and/or peptides known in the art. Non-limiting categories of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic polypeptides, and chimeric polypeptides.
  • peptide generally refers to shorter polypeptides of about 50 amino acids or less. Peptides with only two amino acids may be referred to as “dipeptides.” Peptides with only three amino acids may be referred to as “tripeptides.” Polypeptides generally refer to polypeptides with from about 4 to about 50 amino acids. Peptides may be obtained via any method known to those skilled in the art. In some embodiments, peptides may be expressed in culture. In some embodiments, peptides may be obtained via chemical synthesis (e.g., solid phase peptide synthesis).
  • the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a simple protein which upon hydrolysis yields the amino acids and occasionally small carbohydrate compounds.
  • simple proteins include albumins, albuminoids, globulins, glutelins, histones and protamines.
  • the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or pay load which is a conjugated protein which may be a simple protein associated with a non-protein.
  • conjugated proteins include, glycoproteins, hemoglobins, lecithoproteins, nucleoproteins, and phosphoproteins.
  • the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a derived protein which is a protein that is derived from a simple or conjugated protein by chemical or physical means.
  • a derived protein which is a protein that is derived from a simple or conjugated protein by chemical or physical means.
  • Non-limiting examples of derived proteins include denatured proteins and peptides.
  • the polypeptide, protein or peptide may be unmodified.
  • the polypeptide, protein or peptide may be modified.
  • Types of modifications include, but are not limited to, Phosphorylation, Glycosylation, Acetylation, Ubiquitylation/Sumoylation, Methylation, Palmitoylation, Quinone, Amidation, Myristoylation, Pyrrolidone carboxylic acid, Hydroxylation, Phosphopantetheine, Prenylation, GPI anchoring, Oxidation, ADP-ribosylation, Sulfation, S-nitrosylation, Citrullination, Nitration, Gamma-carboxyglutamic acid, Formylation, Hypusine, Topaquinone (TPQ), Bromination, Lysine topaquinone (LTQ), Tryptophan tryptophylquinone (TTQ), Iodination, and Cysteine tryptophylquinone (CTQ).
  • the polypeptide, protein or peptide may be modified by
  • the polypeptide, protein or peptide may be modified using phosphorylation.
  • Phosphorylation or the addition of a phosphate group to serine, threonine, or tyrosine residues, is one of most common forms of protein modification.
  • Protein phosphorylation plays an important role in fine tuning the signal in the intracellular signaling cascades.
  • the polypeptide, protein or peptide may be modified using ubiquitination which is the covalent attachment of ubiquitin to target proteins.
  • Ubiquitination- mediated protein turnover has been shown to play a role in driving the cell cycle as well as in protein-degradation-independent intracellular signaling pathways.
  • the polypeptide, protein or peptide may be modified using acetylation and methylation which can play a role in regulating gene expression.
  • the acetylation and methylation could mediate the formation of chromatin domains (e.g., euchromatin and heterochromatin) which could have an impact on mediating gene silencing.
  • the polypeptide, protein or peptide may be modified using glycosylation.
  • Glycosylation is the atachment of one of a large number of glycan groups and is a modification that occurs in about half of all proteins and plays a role in biological processes including, but not limited to, embryonic development, cell division, and regulation of protein structure.
  • the two main types of protein glycosylation are N-glycosylation and O- glycosylation.
  • N-glycosylation the glycan is atached to an asparagine
  • O- glycosylation the glycan is atached to a serine or threonine.
  • the polypeptide, protein or peptide may be modified using Sumoylation.
  • Sumoylation is the addition of SUMOs (small ubiquitin-like modifiers) to proteins and is a post-translational modification similar to ubiquitination.
  • an “antibody” is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”).
  • Antibodies are primarily amino acid based molecules which are monomeric or multimeric polypeptides which comprise at least one amino acid region derived from a known or parental antibody sequence and at least one amino acid region derived from a non-antibody sequence.
  • the antibodies may comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.).
  • an “antibody” may comprise a heavy and light variable domain as well as an Fc region.
  • the cargo or payload may comprise or may encode polypeptides that form one or more functional antibodies.
  • the cargo or payload may comprise or may encode polypeptides that form or function as any antibody including, but not limited to, antibodies that are known in the art and/or antibodies that are commercially available which may be therapeutic, diagnostic, or for research purposes. Additionally, the cargo or payload may comprise or may encode fragments of such antibodies or antibodies such as, but not limited to, variable domains or complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the term "native antibody” refers to a usually heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding antibody heavy and light chains are known and segments making up each have been well characterized and described (Matsuda, F. et al., 1998. The Journal of Experimental Medicine. 188(11); 2151-62 and Li, A. et al., 2004. Blood. 103(12: 4602-9, the content of each of which are herein incorporated by reference in their entirety).
  • Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • the term "light chain” refers to a component of an antibody from any vertebrate species assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • variable domain refers to specific antibody domains found on both the antibody heavy and light chains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • Variable domains comprise hypervariable regions.
  • hypervariable region refers to a region within a variable domain comprising amino acid residues responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining regions (CDRs) that become part of the antigen-binding site of the antibody.
  • CDR refers to a region of an antibody comprising a structure that is complimentary to its target antigen or epitope.
  • the antigen-binding site (also known as the antigen combining site or paratope) comprises the amino acid residues necessary to interact with a particular antigen.
  • the exact residues making up the antigen-binding site are typically elucidated by co- crystallography with bound antigen, however computational assessments can also be used based on comparisons with other antibodies (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety).
  • Determining residues making up CDRs may include the use of numbering schemes including, but not limited to, those taught by Kabat [Wu, T.T. et al., 1970, JEM, 132(2):211-50 and Johnson, G. et al., 2000, Nucleic Acids Res. 28(1): 214-8, the contents of each of which are herein incorporated by reference in their entirety], Chothia [Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et al., Nature 342, 877 (1989) and Al-Lazikani, B. et al., 1997, J. Mol. Biol.
  • VH and VL domains each have three CDRs.
  • VL CDRs are referred to herein as CDR- Ll, CDR-L2 and CDR-L3, in order of occurrence when moving from N- to C- terminus along the variable domain polypeptide.
  • VH CDRs are referred to herein as CDR-H1, CDR-H2, and CDR-H3, in order of occurrence when moving from N- to C-terminus along the variable domain polypeptide.
  • Each of CDRs have favored canonical structures with the exception of the CDR- H3, which comprises amino acid sequences that may be highly variable in sequence and length between antibodies resulting in a variety of three-dimensional structures in antigen-binding domains. In some cases, CDR-H3s may be analyzed among a panel of related antibodies to assess antibody diversity.
  • Kabat CDRs and comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain.
  • Chothia and coworkers found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883, the contents of each of which is herein incorporated by reference in its entirety).
  • CDRs can be referred to as “Chothia CDRs,” “Chothia numbering,” or “numbered according to Chothia,” and comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-32 (CDR1), 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain.
  • CDR1 residues 24-34
  • CDR2 50-56
  • CDR3 89-97
  • CDR3 26-32
  • CDR1, 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain.
  • MacCallum also referred to as “numbered according to MacCallum,” or “MacCallum numbering” comprises about residues 30-36 (CDR1), 46-55 (CDR2) and 89-96 (CDR3) in the light chain variable domain, and 30-35 (CDR1), 47-58 (CDR2) and 93-101 (CDR3) in the heavy chain variable domain.
  • MacCallum et al. ((1996) J. Mol. Biol. 262(5):732-745), the contents of which is herein incorporated by reference in its entirety).
  • AbM The system described by AbM, also referred to as “numbering according to AbM,” or “AbM numbering” comprises about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26- 35 (CDR1), 50-58 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain.
  • IMGT INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM
  • numbering of variable regions can also be used, which is the numbering of the residues in an immunoglobulin variable heavy or light chain according to the methods of the IIMGT (Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains", The Immunologist, 7, 132-136 (1999), and is herein incorporated by reference in its entirety by reference).
  • IMGT sequence numbering or “numbered according to IMTG,” refers to numbering of the sequence encoding a variable region according to the IMGT.
  • the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3.
  • the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3.
  • the cargo or payload may comprise or may encode antibodies which have been produced using methods known in the art such as, but are not limited to immunization and display technologies (e.g., phage display, yeast display, and ribosomal display), hybridoma technology, heavy and light chain variable region cDNA sequences selected from hybridomas or from other sources,
  • immunization and display technologies e.g., phage display, yeast display, and ribosomal display
  • hybridoma technology e.g., heavy and light chain variable region cDNA sequences selected from hybridomas or from other sources
  • the cargo or payload may comprise or may encode antibodies which were developed using any naturally occurring or synthetic antigen.
  • an “antigen” is an entity which induces or evokes an immune response in an organism.
  • An immune response is characterized by the reaction of the cells, tissues and/or organs of an organism to the presence of a foreign entity. Such an immune response typically leads to the production by the organism of one or more antibodies against the foreign entity, e.g., antigen or a portion of the antigen.
  • antigens also refer to binding partners for specific antibodies or binding agents in a display library.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts.
  • each monoclonal antibody is directed against a single determinant on the antigen
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
  • humanized antibody refers to a chimeric antibody comprising a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • the cargo or payload may comprise or may encode antibody mimetics.
  • antibody mimetic refers to any molecule which mimics the function or effect of an antibody, and which binds specifically and with high affinity to their molecular targets.
  • antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold.
  • antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINSTM, fynomers, Kunitz domains, and domain peptides.
  • antibody mimetics may include one or more non-peptide regions.
  • the cargo or payload may comprise or may encode antibody fragments which comprise antigen binding regions from full-length antibodies.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. Also produced is a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • Compounds and/or compositions of the present disclosure may comprise one or more of these fragments.
  • the Fc region may be a modified Fc region wherein the Fc region may have a single amino acid substitution as compared to the corresponding sequence for the wild-type Fc region, wherein the single amino acid substitution yields an Fc region with preferred properties to those of the wild-type Fc region.
  • Fc properties that may be altered by the single amino acid substitution include bind properties or response to pH conditions
  • Fv refers to an antibody fragment comprising the minimum fragment on an antibody needed to form a complete antigen binding site. These regions consist of a dimer of one heavy chain and one light chain variable domain in tight, non- covalent association. Fv fragments can be generated by proteolytic cleavage, but are largely unstable. Recombinant methods are known in the art for generating stable Fv fragments, typically through insertion of a flexible linker between the light chain variable domain and the heavy chain variable domain to form a single chain Fv (scFv) or through the introduction of a disulfide bridge between heavy and light chain variable domains.
  • scFv single chain Fv
  • single chain Fv refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain by a flexible peptide linker.
  • the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding.
  • scFvs are utilized in conjunction with phage display, yeast display or other display methods where they may be expressed in association with a surface member (e.g., phage coat protein) and used in the identification of high affinity peptides for a given antigen.
  • antibody variant refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and/or function (e.g., an antibody mimetic).
  • Antibody variants may be altered in their amino acid sequence, composition, or structure as compared to a native antibody.
  • Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.
  • the cargo or payload may be or may encode antibodies that bind more than one epitope.
  • the terms “multibody” or “multispecific antibody” refer to an antibody wherein two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets.
  • a multispecific antibody is a "bispecific antibody,” which recognizes two different epitopes on the same or different antigens.
  • multi-specific antibodies may be prepared by the methods used by BIOATLA® and described in International Patent publication WO201109726, the contents of which are herein incorporated by reference in their entirety. First a library of homologous, naturally occurring antibodies is generated by any method known in the art (i.e., mammalian cell surface display), then screened by FACSAria or another screening method, for multi- specific antibodies that specifically bind to two or more target antigens. In some embodiments, the identified multi-specific antibodies are further evolved by any method known in the art, to produce a set of modified multi-specific antibodies. These modified multi-specific antibodies are screened for binding to the target antigens. In some embodiments, the multi-specific antibody may be further optimized by screening the evolved modified multi-specific antibodies for optimized or desired characteristics.
  • multi-specific antibodies may be prepared by the methods used by BIOATLA® and described in Unites States Publication No. US20150252119, the contents of which are herein incorporated by reference in their entirety.
  • the variable domains of two parent antibodies, wherein the parent antibodies are monoclonal antibodies are evolved using any method known in the art in a manner that allows a single light chain to functionally complement heavy chains of two different parent antibodies.
  • Another approach requires evolving the heavy chain of a single parent antibody to recognize a second target antigen.
  • a third approach involves evolving the light chain of a parent antibody so as to recognize a second target antigen.
  • the cargo or payload may be or may encode bispecific antibodies.
  • the term “bispecific antibody” refers to an antibody capable of binding two different antigens. Such antibodies typically comprise regions from at least two different antibodies. Such antibodies typically comprise antigen-binding regions from at least two different antibodies.
  • a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies, thus allowing the BsAb to bind to two different types of antigen.
  • the cargo or payload may be or may encode bispecific antibodies comprising antigen-binding regions from two different anti-tau antibodies.
  • bispecific antibodies may comprise binding regions from two different antibodies
  • Bispecific antibody frameworks may include any of those described in Riethmuller, G., 2012. Cancer Immunity. 12:12-18; Marvin, J.S. et al., 2005. Acta Pharmacologica Sinica. 26(6):649-58; and Schaefer, W. et al., 2011. PNAS. 108(27): 11187-92, the contents of each of which are herein incorporated by reference in their entirety.
  • BsMAb new generations of BsMAb, called “trifunctional bispecific” antibodies, have been developed. These consist of two heavy and two light chains, one each from two different antibodies, where the two Fab regions (the arms) are directed against two antigens, and the Fc region (the foot) comprises the two heavy chains and forms the third binding site.
  • the Fc region may additionally bind to a cell that expresses Fc receptors, like a macrophage, a natural killer (NK) cell or a dendritic cell.
  • NK natural killer
  • the targeted cell is connected to one or two cells of the immune system, which subsequently destroy it.
  • bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity and side-effects caused by cytokine liberation. They include chemically linked Fabs, consisting only of the Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs), fusion proteins mimicking the variable domains of two antibodies.
  • scFvs single-chain variable fragments
  • the furthest developed of these newer formats are the bi- specific T-cell engagers (BiTEs) and mAb2's, antibodies engineered to contain an Fcab antigen-binding fragment instead of the Fc constant region.
  • tascFv tandem scFv
  • TascFvs have been found to be poorly soluble and require refolding when produced in bacteria, or they may be manufactured in mammalian cell culture systems, which avoids refolding requirements but may result in poor yields. Construction of a tascFv with genes for two different scFvs yields a “bispecific single-chain variable fragments” (bis-scFvs).
  • Blinatumomab is an anti-CD19/anti-CD3 bispecific tascFv that potentiates T-cell responses to B-cell non-Hodgkin lymphoma in Phase 2.
  • MT110 is an anti-EP-CAM/anti- CD3 bispecific tascFv that potentiates T-cell responses to solid tumors in Phase 1.
  • Bispecific, tetravalent “TandAbs” are also being researched by Affimed.
  • the cargo or payload may be or may encode antibodies comprising a single antigen-binding domain. These molecules are extremely small, with molecular weights approximately one-tenth of those observed for full-sized mAbs. Further antibodies may include “nanobodies” derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, which lack light chains.
  • VHHs antigen-binding variable heavy chain regions
  • the cargo or payload may be or may encode tetravalent bispecific antibodies (TetBiAbs as disclosed and claimed in PCT Publication WO2014144357, the contents of which are herein incorporated in its entirety).
  • TetBiAbs feature a second pair of Fab fragments with a second antigen specificity attached to the C-terminus of an antibody, thus providing a molecule that is bivalent for each of the two antigen specificities.
  • the tetravalent antibody is produced by genetic engineering methods, by linking an antibody heavy chain covalently to a Fab light chain, which associates with its cognate, co-expressed Fab heavy chain.
  • the cargo or payload may be or may encode biosynthetic antibodies as described in U.S. Patent No. 5,091,513 (the contents of which are herein incorporated by reference in their entirety).
  • Such antibody may include one or more sequences of amino acids constituting a region which behaves as a biosynthetic antibody binding site (BABS).
  • the sites comprise 1) non-covalently associated or disulfide bonded synthetic VH and VL dimers, 2) VH-VL or VL-VH single chains wherein the VH and VL are attached by a polypeptide linker, or 3) individuals VH or VL domains.
  • the binding domains comprise linked CDR and FR regions, which may be derived from separate immunoglobulins.
  • the biosynthetic antibodies may also include other polypeptide sequences which function, e.g., as an enzyme, toxin, binding site, or site of attachment to an immobilization media or radioactive atom. Methods are disclosed for producing the biosynthetic antibodies, for designing BABS having any specificity that can be elicited by in vivo generation of antibody, and for producing analogs thereof.
  • the cargo or payload may be or may encode antibodies with antibody acceptor frameworks taught in U.S. Patent No. 8,399,625.
  • antibody acceptor frameworks may be particularly well suited accepting CDRs from an antibody of interest.
  • CDRs from anti-tau antibodies known in the art or developed according to the methods presented herein may be used.
  • the cargo or payload may be or may encode a “miniaturized” antibody.
  • mAb miniaturization are the small modular immunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains, all connected by linker domains. Presumably, such a molecule might offer the advantages of increased tissue or tumor penetration claimed by fragments while retaining the immune effector functions conferred by constant domains. At least three “miniaturized” SMIPs have entered clinical development.
  • TRU-015 an anti-CD20 SMIP developed in collaboration with Wyeth, is the most advanced project, having progressed to Phase 2 for rheumatoid arthritis (RA). Earlier attempts in systemic lupus erythrematosus (SLE) and B cell lymphomas were ultimately discontinued. Trubion and Facet Biotechnology are collaborating in the development of TRU- 016, an anti-CD37 SMIP, for the treatment of CLL and other lymphoid neoplasias, a project that has reached Phase 2. Wyeth has licensed the anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE, and possibly multiple sclerosis, although these projects remain in the earliest stages of clinical testing.
  • the cargo or payload may be or may encode diabodies.
  • the term "diabody” refers to a small antibody fragment with two antigen-binding sites. Diabodies comprise a heavy chain variable domain VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are functional bispecific single-chain antibodies (bscAb). These bivalent antigen-binding molecules are composed of non-covalent dimers of scFvs, and can be produced in mammalian cells using recombinant methods. (See, e.g., Mack et al, Proc. Natl. Acad. Sci., 92: 7021-7025, 1995). Few diabodies have entered clinical development.
  • the cargo or payload may be or may encode a “unibody,” in which the hinge region has been removed from IgG4 molecules. While IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with one another, deletion of the hinge region prevents heavy chain-heavy chain pairing entirely, leaving highly specific monovalent light/heavy heterodimers, while retaining the Fc region to ensure stability and half- life in vivo. This configuration may minimize the risk of immune activation or oncogenic growth, as IgG4 interacts poorly with FcRs and monovalent unibodies fail to promote intracellular signaling complex formation. These contentions are, however, largely supported by laboratory, rather than clinical, evidence. Other antibodies may be “miniaturized” antibodies, which are compacted 100 kDa antibodies.
  • the cargo or payload may be or may encode intrabodies.
  • intrabodies refers to a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling, and cell division.
  • methods of the present disclosure may include intrabody-based therapies.
  • variable domain sequences and/or CDR sequences disclosed herein may be incorporated into one or more constructs for intrabody-based therapy.
  • intrabodies may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.
  • intracellular antibodies against intracellular targets were first described (Biocca, Neuberger and Cattaneo EMBO J. 9: 101-108, 1990, the contents of which are herein incorporated by reference in their entirety).
  • the intracellular expression of intrabodies in different compartments of mammalian cells allows blocking or modulation of the function of endogenous molecules (Biocca, et al., EMBO J. 9: 101-108, 1990; Colby et al., Proc. Natl. Acad. Sci. U.S.A.
  • Intrabodies can alter protein folding, protein- protein, protein-DNA, protein-RNA interactions and protein modification. They can induce a phenotypic knockout and work as neutralizing agents by direct binding to the target antigen, by diverting its intracellular trafficking or by inhibiting its association with binding partners. They have been largely employed as research tools and are emerging as therapeutic molecules for the treatment of human diseases such as viral pathologies, cancer and misfolding diseases.
  • the fast-growing bio-market of recombinant antibodies provides intrabodies with enhanced binding specificity, stability, and solubility, together with lower immunogenicity, for their use in therapy.
  • intrabodies have advantages over interfering RNA (iRNA); for example, iRNA has been shown to exert multiple non-specific effects, whereas intrabodies have been shown to have high specificity and affinity to target antigens. Furthermore, as proteins, intrabodies possess a much longer active half-life than iRNA. Thus, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA may be slow to yield an effect, whereas the effects of intrabody expression can be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others.
  • iRNA interfering RNA
  • Intrabodies are often single chain variable fragments (scFvs) expressed from a recombinant nucleic acid molecule and engineered to be retained intracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum, or periplasm). Intrabodies may be used, for example, to ablate the function of a protein to which the intrabody binds. The expression of intrabodies may also be regulated through the use of inducible promoters in the nucleic acid expression vector comprising the intrabody. Intrabodies may be produced using methods known in the art, such as those disclosed and reviewed in: Marasco et al., 1993 Proc. Natl. Acad. Sci.
  • Intrabodies are often recombinantly expressed as single domain fragments such as isolated VH and VL domains or as a single chain variable fragment (scFv) antibody within the cell.
  • intrabodies are often expressed as a single polypeptide to form a single chain antibody comprising the variable domains of the heavy and light chains joined by a flexible linker polypeptide.
  • Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity.
  • Single chain antibodies can also be expressed as a single chain variable region fragment joined to the light chain constant region.
  • an intrabody can be engineered into recombinant polynucleotide vectors to encode sub-cellular trafficking signals at its N or C terminus to allow expression at high concentrations in the sub-cellular compartments where a target protein is located.
  • intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal.
  • Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol.
  • cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
  • Intrabodies may be promising therapeutic agents for the treatment of misfolding diseases, including Tauopathies, prion diseases, Alzheimer's, Parkinson's, and Huntington's, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be targeted to the potential sites of aggregation (both intra- and extracellular sites).
  • These molecules can work as neutralizing agents against amyloidogenic proteins by preventing their aggregation, and/or as molecular shunters of intracellular traffic by rerouting the protein from its potential aggregation site.
  • the cargo or payload may be or may encode a maxibody (bivalent scFV fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG.
  • the cargo or payload may be or may encode a chimeric antigen receptors (CARs) which when transduced into immune cells (e.g., T cells and NK cells), can re-direct the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR.
  • CARs chimeric antigen receptors
  • chimeric antigen receptor refers to a synthetic receptor that mimics TCR on the surface of T cells.
  • a CAR is composed of an extracellular targeting domain, a transmembrane domain/region and an intracellular signaling/activation domain.
  • the components: the extracellular targeting domain, transmembrane domain and intracellular signaling/activation domain are linearly constructed as a single fusion protein.
  • the extracellular region comprises a targeting domain/moiety (e.g., a scFv) that recognizes a specific tumor antigen or other tumor cell- surface molecules.
  • the intracellular region may contain a signaling domain of TCR complex (e.g., the signal region of CD3Q, and/or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD134).
  • a “first-generation CAR” only has the CD3 ⁇ signaling domain, whereas in an effort to augment T-cell persistence and proliferation, costimulatory intracellular domains are added, giving rise to second generation CARs having a CD3ijsignal domain plus one costimulatory signaling domain, and third generation CARs having CD3 ⁇ signal domain plus two or more costimulatory signaling domains.
  • a CAR when expressed by a T cell, endows the T cell with antigen specificity determined by the extracellular targeting moiety of the CAR.
  • one or more elements such as homing and suicide genes can be added to develop a more competent and safer architecture of CAR (so called the fourth generation CAR).
  • the extracellular targeting domain is joined through the hinge (also called space domain or spacer) and transmembrane regions to an intracellular signaling domain.
  • the hinge connects the extracellular targeting domain to the transmembrane domain which transverses the cell membrane and connects to the intracellular signaling domain.
  • the hinge may need to be varied to optimize the potency of CAR transformed cells toward cancer cells due to the size of the target protein where the targeting moiety binds, and the size and affinity of the targeting domain itself.
  • the intracellular signaling domain leads to an activation signal to the CAR T cell, which is further amplified by the “second signal” from one or more intracellular costimulatory domains.
  • the CAR T cell once activated, can destroy the target cell.
  • the CAR may be split into two parts, each part is linked a dimerizing domain, such that an input that triggers the dimerization promotes assembly of the intact functional receptor.
  • Wu and Lim reported a split CAR in which the extracellular CD19 binding domain and the intracellular signaling element are separated and linked to the FKBP domain and the FRB* (T2089L mutant of FKBP-rapamycin binding) domain that heterodimerize in the presence of the rapamycin analog AP21967.
  • the split receptor is assembled in the presence of AP21967 and together with the specific antigen binding, activates T cells (Wu et al., Science, 2015, 625(6258): aab4077, the contents of which are herein incorporated by reference in its entirety).
  • the CAR may be designed as an inducible CAR which has an incorporation of a Tet-On inducible system to a CD19 CAR construct.
  • the CD19 CAR is activated only in the presence of doxycycline (Dox).
  • Dox doxycycline
  • Sakemura reported that Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD 19+ cell lines and had equivalent cytokine production and proliferation upon CD 19 stimulation, compared with conventional CD19CAR T cells (Sakemura et al., Cancer Immuno. Res., 2016, Jun 21, Epub; the contents of which is herein incorporated by reference in its entirety).
  • the dual systems provide more flexibility to turn-on and off of the CAR expression in transduced T cells.
  • the cargo or payload may be or may encode a first generation CAR, or a second generation CAR, or a third generation CAR, or a fourth generation CAR.
  • the cargo or payload may be or may encode a full CAR construct composed of the extracellular domain, the hinge and transmembrane domain and the intracellular signaling region.
  • the cargo or payload may be or may encode a component of the full CAR construct including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more co- stimulatory domain, and other additional elements that improve CAR architecture and functionality including but not limited to a leader sequence, a homing element and a safety switch, or the combination of such components.
  • the cargo or payload may be or may encode a tunable CARs.
  • the reversible on-off switch mechanism allows management of acute toxicity caused by excessive CAR-T cell expansion.
  • the ligand conferred regulation of the CAR may be effective in offsetting tumor escape induced by antigen loss, avoiding functional exhaustion caused by tonic signaling due to chronic antigen exposure and improving the persistence of CAR expressing cells in vivo.
  • the tunable CAR may be utilized to down regulate CAR expression to limit on target on tissue toxicity caused by tumor lysis syndrome. Down regulating the expression of the CARs following anti -tumor efficacy may prevent (1) On target off tumor toxicity caused by antigen expression in normal tissue. (2) antigen independent activation in vivo.
  • the extracellular target moiety of a CAR may be any agent that recognizes and binds to a given target molecule, for example, a neoantigen on tumor cells, with high specificity and affinity.
  • the target moiety may be an antibody and variants thereof that specifically binds to a target molecule on tumor cells, or a peptide aptamer selected from a random sequence pool based on its ability to bind to the target molecule on tumor cells, or a variant or fragment thereof that can bind to the target molecule on tumor cells, or an antigen recognition domain from native T- cell receptor (TCR) (e.g.
  • TCR native T- cell receptor
  • the targeting domain of a CAR may be a Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, Fv, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a unibody, a nanobody, or an antigen binding region derived from an antibody that specifically recognizes a target molecule, for example a tumor specific antigen (TSA).
  • TSA tumor specific antigen
  • the targeting moiety is a scFv antibody.
  • the scFv domain when it is expressed on the surface of a CAR T cell and subsequently binds to a target protein on a cancer cell, is able to maintain the CAR T cell in proximity to the cancer cell and to trigger the activation of the T cell.
  • a scFv can be generated using routine recombinant DNA technology techniques and is discussed in the present disclosure.
  • the targeting moiety of a CAR construct may be an aptamer such as a peptide aptamer that specifically binds to a target molecule of interest.
  • the peptide aptamer may be selected from a random sequence pool based on its ability to bind to the target molecule of interest.
  • the targeting moiety of a CAR construct may be a natural ligand of the target molecule, or a variant and/or fragment thereof capable of binding the target molecule.
  • the targeting moiety of a CAR may be a receptor of the target molecule, for example, a full length human CD27, as a CD70 receptor, may be fused in frame to the signaling domain of CD3 forming a CD27 chimeric receptor as an immunotherapeutic agent for CD70-positive malignancies.
  • the targeting moiety of a CAR may recognize a tumor specific antigen (TSA), for example a cancer neoantigen which is restrictedly expressed on tumor cells.
  • TSA tumor specific antigen
  • the CAR of the present disclosure may comprise the extracellular targeting domain capable of binding to a tumor specific antigen selected from 5T4, 707-AP, A33, AFP (a -fetoprotein), AKAP-4 ( A kinase anchor protein 4), ALK, 0,5(31 - integrin, androgen receptor, annexin II, alpha- actinin-4, ART-4, Bl, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, beta-catenin, BKT-antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calretinin, CAIX (
  • the CAR of the present disclosure may comprise the extracellular targeting domain capable of binding to a tumor specific antigen such as Epidermal Growth Factor Receptor Variant III (EGFRvIII).
  • EGFRvIII Epidermal Growth Factor Receptor Variant III
  • the cargo is EGFRvIII.
  • Figure 31 describes the EGFRvIII and the Wt EGFR sequences.
  • 101 represents the extracellular domain (AAs 1-621)
  • 102 represent the transmembrane domain (AAs 622-644)
  • 103 represents the intracellular domain (AAs 645-1186).
  • 201 represents truncated extracellular domain (in-frame deletion of exons 2-7 / 268 amino acids deleted / novel glycine at exon junction)
  • 202 represents the insertion of novel Glycine residue.
  • the cargo or payload may be or may encode a CAR which comprises a universal immune receptor which has a targeting moiety capable of binding to a labelled antigen.
  • the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a pathogen antigen.
  • the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to non-protein molecules such as tumor- associated glycolipids and carbohydrates.
  • the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a component within the tumor microenvironment including proteins expressed in various tumor stroma cells including tumor associated macrophages (TAMs), immature monocytes, immature dendritic cells, immunosuppressive CD4+CD25+ regulatory T cells (Treg) and MDSCs.
  • TAMs tumor associated macrophages
  • Reg immunosuppressive CD4+CD25+ regulatory T cells
  • MDSCs immunosuppressive CD4+CD25+ regulatory T cells
  • the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a cell surface adhesion molecule, a surface molecule of an inflammatory cell that appears in an autoimmune disease, or a TCR causing autoimmunity.
  • a CAR which comprises a targeting moiety capable of binding to a cell surface adhesion molecule, a surface molecule of an inflammatory cell that appears in an autoimmune disease, or a TCR causing autoimmunity.
  • the targeting moiety of the present disclosure may be a scFv antibody that recognizes a tumor specific antigen (TSA), for example scFvs of antibodies SS, SSI and HN1 that specifically recognize and bind to human mesothelin, scFv of antibody of GD2, a CD 19 antigen binding domain, aNKG2D ligand binding domain, human anti-mesothelin scFvs, an anti-CSl binding agent, an anti-BCMA binding domain, anti-CD19 scFv antibody, GFR alpha 4 antigen binding fragments, anti-CLL-1 (C-type lectin-like molecule 1) binding domains, CD33 binding domains, a GPC3 (glypican-3) binding domain, a GFR alpha4 (Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a -receptor 4 cell- surface receptor) binding domain, CD123 binding domains, an TSA tumor specific anti
  • the intracellular domain of a CAR fusion polypeptide after binding to its target molecule, transmits a signal to the immune effector cell, activating at least one of the normal effector functions of immune effector cells, including cytolytic activity (e.g., cytokine secretion) or helper activity. Therefore, the intracellular domain comprises an “intracellular signaling domain" of a T cell receptor (TCR).
  • TCR T cell receptor
  • the entire intracellular signaling domain can be employed.
  • a truncated portion of the intracellular signaling domain may be used in place of the intact chain as long as it transduces the effector function signal.
  • the intracellular signaling domain may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Examples of IT AM containing cytoplasmic signaling sequences include those derived from TCR CD3zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain is a CD3 zeta (CD3Q signaling domain.
  • the intracellular region further comprises one or more costimulatory signaling domains which provide additional signals to the immune effector cells.
  • costimulatory signaling domains in combination with the signaling domain can further improve expansion, activation, memory, persistence, and tumor-eradicating efficiency of CAR engineered immune cells (e.g., CAR T cells).
  • the costimulatory signaling region contains 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and /or costimulatory molecules.
  • the costimulatory signaling domain may be the intracellular/cytoplasmic domain of a costimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, ICOS (CD278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function- associated antigen- 1), LIGHT, NKG2C, B7-H3.
  • the costimulatory signaling domain is derived from the cytoplasmic domain of CD28.
  • the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137).
  • the co-stimulatory signaling domain may be an intracellular domain of GITR as taught in U.S. Pat. NO.: 9, 175, 308; the contents of which are incorporated herein by reference in its entirety.
  • the intracellular region may comprise a functional signaling domain from a protein selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation protein (SLAM) such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, SLAMF7, an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44,
  • SLAM signaling lympho
  • the intracellular signaling domain of the present disclosure may contain signaling domains derived from JAK-STAT.
  • the intracellular signaling domain of the present disclosure may contain signaling domains derived from DAP-12 (Death associated protein 12) (Topfer et al., Immunol., 2015, 194: 3201-3212; and Wang et al., Cancer Immunol., 2015, 3: 815-826).
  • DAP-12 is a key signal transduction receptor in NK cells. The activating signals mediated by DAP-12 play important roles in triggering NK cell cytotoxicity responses toward certain tumor cells and virally infected cells.
  • the cytoplasmic domain of DAP 12 contains an Immunoreceptor Tyrosine-based Activation Motif (IT AM). Accordingly, a CAR containing a DAP12-derived signaling domain may be used for adoptive transfer of NK cells.
  • the CAR may comprise a transmembrane domain.
  • Transmembrane domain refers broadly to an amino acid sequence of about 15 residues in length which spans the plasma membrane.
  • the transmembrane domain may include at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane.
  • the transmembrane domain may be derived either from a natural or from a synthetic source.
  • the transmembrane domain of a CAR may be derived from any naturally membrane-bound or transmembrane protein.
  • the transmembrane region may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.
  • the transmembrane domain of the present disclosure may be synthetic.
  • the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain may be selected from the group consisting of a CD8a transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human IgG4 Fc region.
  • the CAR may comprise an optional hinge region (also called spacer).
  • a hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain that moves the target binding domain away from the effector cell surface to enable proper cell/ cell contact, target binding and effector cell activation.
  • the hinge sequence may be positioned between the targeting moiety and the transmembrane domain.
  • the hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.
  • the hinge sequence may be derived from all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge, the extracellular regions of type 1 membrane proteins such as CD8a CD4, CD28 and CD7, which may be a wild-type sequence or a derivative.
  • Some hinge regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain.
  • the hinge region may be modified from an IgGl, IgG2, IgG3, or IgG4 that includes one or more amino acid residues, for example, 1, 2, 3, 4 or 5 residues, substituted with an amino acid residue different from that present in an unmodified hinge.
  • the CAR may comprise one or more linkers between any of the domains of the CAR.
  • the linker may be between 1-30 amino acids long.
  • the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  • the linker may be flexible.
  • the components including the targeting moiety, transmembrane domain and intracellular signaling domains may be constructed in a single fusion polypeptide.
  • the fusion polypeptide may be the payload.
  • the cargo or payload may be or may encode a CD 19 specific CAR targeting different B cell malignancies and HER2-specific CAR targeting sarcoma, glioblastoma, and advanced Her2 -positive lung malignancy. Tandem CAR (TanCAR).
  • the CAR may be a tandem chimeric antigen receptor (TanCAR) which is able to target two, three, four, or more tumor specific antigens.
  • the CAR is a bispecific TanCAR including two targeting domains which recognize two different TSAs on tumor cells.
  • the bispecific TanCAR may be further defined as comprising an extracellular region comprising a targeting domain (e.g., an antigen recognition domain) specific for a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) specific for a second tumor antigen.
  • the CAR is a multispecific TanCAR that includes three or more targeting domains configured in a tandem arrangement.
  • the space between the targeting domains in the TanCAR may be between about 5 and about 30 amino acids in length, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids.
  • the CAR components including the targeting moiety, transmembrane domain and intracellular signaling domains may be split into two or more parts such that it is dependent on multiple inputs that promote assembly of the intact functional receptor.
  • the split CAR consists of two parts that assemble in a small molecule-dependent manner; one part of the receptor features an extracellular antigen binding domain (e.g., scFv) and the other part has the intracellular signaling domains, such as the CD3 ⁇ intracellular domain.
  • the split parts of the CAR system can be further modified to increase signal.
  • the second part of cytoplasmic fragment may be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., CD8a transmembrane domain) to the construct.
  • An additional extracellular domain may also be added to the second part of the CAR system, for instance an extracellular domain that mediates homo- dimerization. These modifications may increase receptor output activity, i.e., T cell activation.
  • the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of a heterodimerizing small molecule. As such, the receptor components are assembled in the presence of the small molecule, to form an intact system which can then be activated by antigen engagement.
  • any known heterodimerizing components can be incorporated into a split CAR system.
  • Other small molecule dependent heterodimerization domains may also be used, including, but not limited to, gibberellin-induced dimerization system (GID 1 -GAI), trimethoprim-SLF induced ecDHFR and FKBP dimerization and ABA (abscisic acid) induced dimerization of PP2C and PYL domains.
  • GID 1 -GAI gibberellin-induced dimerization system
  • trimethoprim-SLF induced ecDHFR and FKBP dimerization abcisic acid induced dimerization of PP2C and PYL domains.
  • ABA abcisic acid induced dimerization of PP2C and PYL domains.
  • the dual regulation using inducible assembly (e.g., ligand dependent dimerization) and degradation (e.g., destabilizing domain induced CAR degradation) of the split CAR system may
  • the CAR may be a switchable CAR which is a controllable CARs that can be transiently switched on in response to a stimulus (e.g., a small molecule).
  • a system is directly integrated in the hinge domain that separate the scFv domain from the cell membrane domain in the CAR.
  • Such system is possible to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture.
  • This integrated system can switch the scFv and antigen interaction between on/off states controlled by the absence/presence of the stimulus.
  • the CAR may be a reversible CAR system.
  • a LID domain ligand-induced degradation
  • the CAR can be temporarily down-regulated by adding a ligand of the LID domain.
  • the CAR may be inhibitory CARs.
  • Inhibitory CAR refers to a bispecific CAR design wherein a negative signal is used to enhance the tumor specificity and limit normal tissue toxicity.
  • This design incorporates a second CAR having a surface antigen recognition domain combined with an inhibitory signal domain to limit T cell responsiveness even with concurrent engagement of an activating receptor.
  • This antigen recognition domain is directed towards a normal tissue specific antigen such that the T cell can be activated in the presence of first target protein, but if the second protein that binds to the iCAR is present, the T cell activation is inhibited.
  • iCARs against Prostate specific membrane antigen (PMSA) based on CTLA4 and PD1 inhibitory domains demonstrated the ability to selectively limit cytokine secretion, cytotoxicity and proliferation induced by T cell activation.
  • PMSA Prostate specific membrane antigen
  • the cargo or payload may be or may encode a chimeric switch receptors which can switch a negative signal to a positive signal.
  • chimeric switch receptor refers to a fusion protein comprising a first extracellular domain and a second transmembrane and intracellular domain, wherein the first domain includes a negative signal region, and the second domain includes a positive intracellular signaling region.
  • the fusion protein is a chimeric switch receptor that contains the extracellular domain of an inhibitory receptor on T cell fused to the transmembrane and cytoplasmic domain of a co-stimulatory receptor. This chimeric switch receptor may convert a T cell inhibitory signal into a T cell stimulatory signal.
  • the chimeric switch receptor may comprise the extracellular domain of PD-1 fused to the transmembrane and cytoplasmic domain of CD28.
  • Extracellular domains of other inhibitory receptors such as CTLA-4, LAG-3, TIM-3, KIRs and BTLA may also be fused to the transmembrane and cytoplasmic domain derived from costimulatory receptors such as CD28, 4-1BB, CD27, 0X40, CD40, GTIR and ICOS.
  • chimeric switch receptors may include recombinant receptors comprising the extracellular cytokine-binding domain of an inhibitory cytokine receptor (e.g., IL-13 receptor a (IL-13R ⁇ l), IL-10R, and IL-4R ⁇ ) fused to an intracellular signaling domain of a stimulatory cytokine receptor such as IL-2R (IL-2R ⁇ , IL-2R0 and IL-2Rgamma) and IL- 7R ⁇ .
  • an inhibitory cytokine receptor e.g., IL-13 receptor a (IL-13R ⁇ l), IL-10R, and IL-4R ⁇
  • IL-2R IL-2R ⁇ , IL-2R0 and IL-2Rgamma
  • the chimeric switch receptor may be a chimeric TGF ⁇ receptor.
  • the chimeric TGF ⁇ receptor may comprise an extracellular domain derived from a TGF ⁇ receptor such as TGF ⁇ receptor 1, TGF ⁇ receptor 2, TGF ⁇ receptor 3, or any other TGF ⁇ receptor or variant thereof; and a non- TGF ⁇ receptor intracellular domain.
  • the non- TGF ⁇ receptor intracellular domain may be the intracellular domain or fragment thereof derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CD28, 4-1BB (CD137), 0X40 (CD134), CD3zeta, CD40, CD27, or a combination thereof
  • the cargo or payload may be or may encode an activation- conditional chimeric antigen receptor, which is only expressed in an activated immune cell.
  • the expression of the CAR may be coupled to activation conditional control region which refers to one or more nucleic acid sequences that induce the transcription and/or expression of a sequence e.g., a CAR under its control.
  • activation conditional control regions may be promoters of genes that are upregulated during the activation of the immune effector cell e.g., IL2 promoter or NF AT binding sites.
  • the cargo or payload may be or may encode a CAR that targets specific types of cancer cells.
  • Human cancer cells and metastasis may express unique and otherwise abnormal proteoglycans, such as polysaccharide chains (e.g., chondroitin sulfate (CS), dermatan sulfate (DS or CSB), heparan sulfate (HS) and heparin).
  • the CAR may be fused with a binding moiety that recognizes cancer associated proteoglycans.
  • a CAR may be fused with VAR2CSA polypeptide (VAR2-CAR) that binds with high affinity to a specific type of chondroitin sulfate A (CSA) attached to proteoglycans.
  • VAR2-CAR VAR2CSA polypeptide
  • the extracellular ScFv portion of the CAR may be substituted with VAR2CSA variants comprising at least the minimal CSA binding domain, generating CARs specific to chondroitin sulfate A (CSA) modifications.
  • the CAR may be fused with a split-protein binding system to generate a spy-CAR, in which the scFv portion of the CAR is substituted with one portion of a split-protein binding system such as SpyTag and Spy-catcher and the cancer-recognition molecules (e.g., scFv and or VAR2-CSA) are attached to the CAR through the split-protein binding system.
  • a split-protein binding system such as SpyTag and Spy-catcher and the cancer-recognition molecules (e.g., scFv and or VAR2-CSA) are attached to the CAR through the split-protein binding system.
  • the lipid nanoparticles of the present disclosure may comprise a payload region (which may also be referred to as a cargo region) which is a nucleic acid.
  • a payload region which may also be referred to as a cargo region
  • nucleic acid includes any compound and/or substance that comprise a polymer of nucleotides which may be referred to as polynucleotides.
  • Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof.
  • the payload region comprises nucleic acid sequences encoding more than one cargo or payload.
  • the payload region may be or encode a coding nucleic acid sequence.
  • the payload region may be or encode a non-coding nucleic acid sequence.
  • the payload region may be or encode both a coding and a non- coding nucleic acid sequence.
  • Deoxyribonucleic acid is a molecule that carries genetic information for all living things and consists of two strands that wind around one another to form a shape known as a double helix. Each strand has a backbone made of alternating sugar (deoxyribose) and phosphate groups. Attached to each sugar is one of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T). The two strands are held together by bonds between adenine and thymine or cytosine and guanine. The sequence of the bases along the backbones serves as instructions for assembling protein and RNA molecules.
  • the payload region may be or encode a coding DNA.
  • the payload region may be or encode a non-coding DNA.
  • the payload region may be or encode both a coding and a non- coding DNA.
  • the DNA may be modified.
  • Types of modifications include, but are not limited to, methylation, acetylation, phosphorylation, ubiquitination, and sumoylation.
  • the originator constructs and/or benchmark constructs described herein can be or be encoded by vectors such as plasmids or viral vectors.
  • the originator constructs and/or benchmark constructs are or are encoded by viral vectors.
  • Viral vectors may be, but are not limited to, Herpesvirus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, and the like.
  • the viral vectors are AAV vectors.
  • the viral vectors are lentiviral vectors.
  • the viral vectors are retroviral vectors.
  • the viral vectors are adenoviral vectors.
  • AAVs Adeno-Associated Viral
  • Viruses of the Parvoviridae family are small non-enveloped icosahedral capsid viruses characterized by a single stranded DNA genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Due to its relatively simple structure, easily manipulated using standard molecular biology techniques, this virus family is useful as a biological tool.
  • the genome of the virus may be modified to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to express or deliver a desired payload, which may be delivered to a target cell, tissue, organ, or organism.
  • the Parvoviridae family comprises the Dependovirus genus which includes adeno- associated viruses (AAV) capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.
  • AAV adeno- associated viruses
  • the AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nts) in length.
  • the AAV vector genome can comprise a payload region and at least one inverted terminal repeat (ITR) or ITR region. ITRs traditionally flank the coding nucleotide sequences for the non-structural proteins (encoded by Rep genes) and the structural proteins (encoded by capsid genes or Cap genes). While not wishing to be bound by theory, an AAV vector genome typically comprises two ITR sequences.
  • the AAV vector genome comprises a characteristic T-shaped hairpin structure defined by the self- complementary terminal 145 nucleotides of the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region.
  • the double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.
  • AAV vector genomes may comprise, in whole or in part, of any naturally occurring and/or recombinant AAV serotype nucleotide sequence or variant.
  • AAV variants may have sequences of significant homology at the nucleic acid (genome or capsid) and amino acid levels (capsids), to produce constructs which are generally physical and functional equivalents, replicate by similar mechanisms, and assemble by similar mechanisms. Chiorini et al., J. Vir. 71: 6823-33(1997); Srivastava et al., J. Vir. 45:555- 64 (1983); Chiorini et al., J. Vir.
  • the AAV vector genome comprises at least one control element which provides for the replication, transcription, and translation of a coding sequence encoded therein. Not all of the control elements need always be present as long as the coding sequence is capable of being replicated, transcribed, and/or translated in an appropriate host cell.
  • expression control elements include sequences for transcription initiation and/or termination, promoter and/or enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficacy (e.g., Kozak consensus sequence), sequences that enhance protein stability, and/or sequences that enhance protein processing and/or secretion.
  • AAV vector genomes may be produced recombinantly and may be based on adeno- associated virus (AAV) parent or reference sequences.
  • AAV adeno- associated virus
  • a “vector genome” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.
  • scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.
  • the AAV vector genome is an scAAV.
  • the AAV vector genome is an ssAAV.
  • the AAV vector genome may be part of an AAV particles where the serotype of the capsid may be, but is not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV
  • AAVhu.29R AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39,
  • AAVhu.44R3 AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2,
  • AAVhu.48R3 AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56,
  • the AAV vector genomes may comprise at least one ITR region and a payload region.
  • the vector genome has two ITRs. These two ITRs flank the payload region at the 5’ and 3’ ends.
  • the ITRs function as origins of replication comprising recognition sites for replication.
  • ITRs comprise sequence regions which can be complementary and symmetrically arranged.
  • ITRs incorporated into vector genomes may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.
  • the ITRs may be derived from the same serotype as the capsid or a derivative thereof.
  • the ITR may be of a different serotype than the capsid.
  • the AAV particle has more than one ITR.
  • the AAV particle has a vector genome comprising two ITRs.
  • the ITRs are of the same serotype as one another.
  • the ITRs are of different serotypes.
  • Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid.
  • both ITRs of the vector genome of the AAV particle are AAV2 ITRs.
  • each ITR may be about 100 to about 150 nucleotides in length.
  • An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146-150 nucleotides in length.
  • the ITRs are 140-142 nucleotides in length.
  • Non-limiting examples of ITR length are 102, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity thereto.
  • the pay load region of the vector genome comprises at least one element to enhance the transgene target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety).
  • elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.
  • the promoter is efficient when it drives expression of the polypeptide(s) encoded in the pay load region of the vector genome of the AAV particle. [0425] In some embodiments, the promoter is deemed to be efficient when it drives expression in the cell being targeted.
  • the promoter drives expression of the payload for a period of time in targeted tissues.
  • Expression driven by a promoter may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,
  • Expression may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.
  • the promoter drives expression of the payload for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years.
  • Promoters may be naturally occurring or non-naturally occurring.
  • Non-limiting examples of promoters include viral promoters, plant promoters and mammalian promoters.
  • the promoters may be human promoters.
  • the promoter may be truncated.
  • Promoters which drive or promote expression in most tissues include, but are not limited to, human elongation factor la-subunit (EFla), cytomegalovirus (CMV) immediate- early enhancer and/or promoter, chicken [3-actin (CBA) and its derivative CAG, f> glucuronidase (GUSB), or ubiquitin C (UBC).
  • EFla human elongation factor la-subunit
  • CMV cytomegalovirus
  • CBA chicken [3-actin
  • GUSB glucuronidase
  • UPC ubiquitin C
  • Tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, muscle specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.
  • muscle specific promoters such as, but not limited to, muscle specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.
  • Non-limiting examples of muscle-specific promoters include mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, mammalian troponin I (TNNI2) promoter, and mammalian skeletal alpha-actin (ASKA) promoter (see, e.g., U.S. Patent Publication US20110212529, the contents of which are herein incorporated by reference in their entirety)
  • Non-limiting examples of tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-0), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+/calmoduhn-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), 0-globin minigene nf>2.
  • preproenkephalin PPE
  • Enk excitatory amino acid transporter 2
  • EAAT2 excitatory amino acid transporter 2
  • tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters.
  • GFAP glial fibrillary acidic protein
  • EAAT2 EAAT2 promoters
  • a non-limiting example of a tissue-specific expression element for oligodendrocytes includes the myelin basic protein (MBP) promoter.
  • the promoter may be less than 1 kb.
  • the promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
  • the promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800.
  • the promoter may be a combination of two or more components of the same or different starting or parental promoters such as, but not limited to, CMV and CBA.
  • Each component may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386, 387,
  • Each component may have a length between
  • the promoter is a combination of a 382 nucleotide CMV- enhancer sequence and a 260 nucleotide CBA-promoter sequence.
  • the vector genome comprises a ubiquitous promoter.
  • ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc ), EF-la, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).
  • the promoter is not cell specific.
  • the vector genome comprises an engineered promoter.
  • the vector genome comprises a promoter from a naturally expressed protein.
  • wild type untranslated regions of a gene are transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.
  • UTRs features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production.
  • a 5’ UTR from mRNA normally expressed in the liver e.g., albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII
  • albumin serum amyloid A
  • Apolipoprotein A/B/E transferrin
  • alpha fetoprotein erythropoietin
  • Factor VIII Factor VIII
  • wild-type 5' untranslated regions include features which play roles in translation initiation.
  • Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs.
  • Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another 'G 1 .
  • the 5 ’UTR in the vector genome includes a Kozak sequence.
  • the 5 ’UTR in the vector genome does not include a Kozak sequence.
  • AU rich elements can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in its entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions.
  • Class II AREs such as, but not limited to, GM- CSF and TNF-a, possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers.
  • Class III ARES such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif.
  • Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules can lead to HuR binding and thus, stabilization of the message in vivo.
  • AREs 3' UTR AU rich elements
  • AREs 3' UTR AU rich elements
  • one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
  • the 3' UTR of the vector genome may include an oligo(dT) sequence for templated addition of a poly -A tail.
  • the vector genome may include at least one miRNA seed, binding site or full sequence.
  • microRNAs are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
  • a microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence of the nucleic acid.
  • the vector genome may be engineered to include, alter or remove at least one miRNA binding site, sequence, or seed region.
  • any UTR from any gene known in the art may be incorporated into the vector genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected, or they may be altered in orientation or location.
  • the UTR used in the vector genome of the AAV particle may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs known in the art.
  • the term “altered” as it relates to a UTR means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • the vector genome of the AAV particle comprises at least one artificial UTRs which is not a variant of a wild-type UTR.
  • the vector genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature or property.
  • the vector genome comprises at least one polyadenylation sequence between the 3’ end of the payload coding sequence and the 5’ end of the 3TTR.
  • the polyadenylation (poly-A) sequence may range from absent to about 500 nucleotides in length.
  • the polyadenylation sequence may be, but is not limited to,
  • the polyadenylation sequence is 50-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-160 nucleotides in length.
  • the polyadenylation sequence is 60-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-160 nucleotides in length.
  • the polyadenylation sequence is 80-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-200 nucleotides in length.
  • Vector genomes may be engineered with one or more spacer or linker regions to separate coding or non-coding regions.
  • the payload region of the vector genome may optionally encode one or more linker sequences.
  • the linker may be a peptide linker that may be used to connect the polypeptides encoded by the payload region (i.e., light and heavy antibody chains during expression). Some peptide linkers may be cleaved after expression to separate heavy and light chain domains, allowing assembly of mature antibodies or antibody fragments. Linker cleavage may be enzymatic. In some cases, linkers comprise an enzymatic cleavage site to facilitate intracellular or extracellular cleavage. Some payload regions encode linkers that interrupt polypeptide synthesis during translation of the linker sequence from an mRNA transcript. Such linkers may facilitate the translation of separate protein domains from a single transcript. In some cases, two or more linkers are encoded by a payload region of the vector genome.
  • IRES Internal ribosomal entry site
  • 2A peptides are small “self-cleaving” peptides (18-22 amino acids) derived from viruses such as foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A), Thoseaasigna virus (T2A), or equine rhinitis A virus (E2A).
  • the 2A designation refers specifically to a region of picomavirus polyproteins that lead to a ribosomal skip at the glycyl- prolyl bond in the C-terminus of the 2A peptide (Kim, J.H. et al., 2011. PLoS One 6(4): el 8556; the contents of which are herein incorporated by reference in its entirety).
  • 2A peptides generate stoichiometric expression of proteins flanking the 2A peptide and their shorter length can be advantageous in generating viral expression vectors.
  • Some payload regions encode linkers comprising furin cleavage sites.
  • Furin is a calcium dependent serine endoprotease that cleaves proteins just downstream of a basic amino acid target sequence (Arg-X-(ArgZLys)-Arg) (Thomas, G., 2002. Nature Reviews Molecular Cell Biology 3(10): 753-66; the contents of which are herein incorporated by reference in its entirety).
  • Furin is enriched in the trans-golgi network where it is involved in processing cellular precursor proteins.
  • Furin also plays a role in activating a number of pathogens. This activity can be taken advantage of for expression of polypeptides.
  • the payload region may encode one or more linkers comprising cathepsin, matrix metalloproteinases or legumain cleavage sites.
  • linkers are described e.g., by Cizeau and Macdonald in International Publication No. W02008052322, the contents of which are herein incorporated in their entirety.
  • Cathepsins are a family of proteases with unique mechanisms to cleave specific proteins.
  • Cathepsin B is a cysteine protease and cathepsin D is an aspartyl protease.
  • Matrix metalloproteinases are a family of calcium- dependent and zinc-containing endopeptidases.
  • Legumain is an enzyme catalyzing the hydrolysis of (-Asn-Xaa-) bonds of proteins and small molecule substrates.
  • payload regions may encode linkers that are not cleaved.
  • Such linkers may include a simple amino acid sequence, such as a glycine rich sequence.
  • linkers may comprise flexible peptide linkers comprising glycine and serine residues.
  • the linker may be (G4S)5 (Gly-Gly-Gly-Gly-Ser)5.
  • payload regions may encode small and unbranched serine-rich peptide linkers, such as those described by Huston et al. in US Patent No. US5525491, the contents of which are herein incorporated in their entirety.
  • Polypeptides encoded by the payload region, linked by serine-rich linkers, have increased solubility.
  • payload regions may encode artificial linkers, such as those described by Whitlow and Filpula in US Patent No. US5856456 and Ladner et al. in US Patent No. US 4946778, the contents of each of which are herein incorporated by their entirety.
  • the payload region comprises at least one element to enhance the expression such as one or more introns or portions thereof.
  • introns include, MVM (67-97 bps), F.IX truncated intron 1 (300 bps), [3-globin SD/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor/immunoglobin splice acceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S) (180 bps) and hybrid adenovirus splice donor/IgG splice acceptor (230 bps).
  • the intron or intron portion may be 100-500 nucleotides in length.
  • the intron may have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500.
  • the intron may have a length between 80-100, 80-120, 80-140, 80-160, 80-180, 80- 200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500.
  • Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells because their viral shell can pass through the intact membrane of the nucleus of the target cell. Lentiviral vectors have the ability to deliver transgenes in tissues that had long appeared irremediably refractory to stable genetic manipulation. Lentivectors have also opened fresh perspectives for the genetic treatment of a wide array of hereditary as well as acquired disorders, and a real proposal for their clinical use seems imminent.
  • RNA Ribonucleic acid
  • the nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C).
  • A adenine
  • G guanine
  • U uracil
  • C cytosine
  • RNA mostly exists in the single-stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA.
  • RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., IncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., IncRNA) sequence.
  • the payload region may be or encode a coding RNA.
  • the payload region may be or encode a non-coding RNA.
  • the payload region may be or encode both a coding and a non- coding RNA.
  • the payload region comprises nucleic acid sequences encoding more than one cargo or payload.
  • the payload region comprises a nucleic acid sequence to enhance the expression of a gene.
  • the nucleic acid sequence is a messenger RNA (mRNA).
  • the nucleic acid sequence is a circular RNA (oRNA).
  • the payload region comprises a nucleic acid sequence to reduce or inhibit the expression of a gene.
  • the nucleic acid sequence is a small interfering RNA (siRNA) or a microRNA (miRNA).
  • siRNA small interfering RNA
  • miRNA microRNA
  • mRNA Messenger RNA
  • the originator constructs and/or benchmark constructs may be mRNA.
  • mRNA messenger RNA
  • the term "messenger RNA” refers to any polynucleotide which encodes a target of interest and which is capable of being translated to produce the encoded target of interest in vitro, in vivo, in situ or ex vivo.
  • an mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail.
  • one or more structural and/or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced.
  • a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a nucleic acid without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications can result in a different sequence of nucleotides. For example, the polynucleotide "ATCG” may be chemically modified to "AT-5meC-G".
  • the shortest length of a region of the originator constructs and/or benchmark constructs can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.
  • the length may be sufficient to encode a peptide of 2-30 amino acids, e.g., 5-30, 10-30, 2-25, 5-25, 10-25, or 10- 20 amino acids.
  • the length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g., no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.
  • the length of the region of the mRNA encoding a target of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).
  • the mRNA includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to
  • the region or regions flanking the region encoding the target of interest may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
  • 15-1,000 nucleotides in length e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides.
  • the mRNA comprises a tailing sequence which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides).
  • the tailing region is a polyA tail
  • the length may be determined in units of or as a function of polyA Binding Protein binding.
  • the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein.
  • PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.
  • the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap.
  • the capping sequence may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length.
  • the caping sequence is absent.
  • the mRNA comprises a region comprising a start codon.
  • the region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA comprises a region comprising a stop codon.
  • the region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA comprises a region comprising a restriction sequence.
  • the region comprising the restriction sequence may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA comprises at least one untranslated region (UTR) which flanks the region encoding the target of interest.
  • UTRs are transcribed by not translated.
  • the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3 'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, the UTRs may have a regulatory role in terms of translation and stability of the nucleic acid.
  • Natural 5' UTRs usually include features which have a role in translation initiation as they tend to include Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5'UTR also have been known to form secondary structures which are involved in elongation factor binding.
  • AU rich elements can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined.
  • one or more copies of an ARE can be introduced to make mRNA less stable and thereby curtail translation and decrease production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
  • the introduction of features often expressed in genes of target organs the stability and protein production of the mRNA can be enhanced in a specific organ and/or tissue.
  • the feature can be a UTR.
  • the feature can be introns or portions of introns sequences.
  • the 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5' proximal introns removal during mRNA splicing.
  • Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0-methylated.
  • 5'- decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • Modifications to mRNA may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5 ' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.
  • a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.
  • Additional modified guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and/or 5 '-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring.
  • Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule.
  • the Anti -Reverse Cap Analog (ARC A) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5 '-guanosine (m7G- 3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G).
  • the 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA).
  • the N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA).
  • mCAP which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm- PPP-G).
  • cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
  • mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures.
  • more authentic refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
  • Non-limiting examples of more authentic 5 'cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '- triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation, and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-methyl.
  • Capl structure Such a structure is termed the Capl structure.
  • Cap structures include, but are not limited to, 7mG(5*)ppp(5*)N,pN2p (cap 0), 7mG(5*)ppp(5*)NlmpNp (cap 1), and 7mG(5*)-ppp(5')NlmpN2mp (cap 2).
  • the 5' terminal caps may include endogenous caps or cap analogs.
  • a 5' terminal cap may comprise a guanine analog.
  • Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido-guanosine.
  • the mRNA may contain an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • An IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure.
  • An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
  • An mRNA that contains more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes.
  • IRES sequences that can be used include without limitation, those from picomaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
  • picomaviruses e.g., FMDV
  • CFFV pest viruses
  • PV polio viruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot-and-mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV murine leukemia virus
  • SIV simian immune deficiency viruses
  • CrPV cricket paralysis viruses
  • a long chain of adenine nucleotides may be added to a polynucleotide such as an mRNA molecule in order to increase stability.
  • a polynucleotide such as an mRNA molecule
  • the 3' end of the transcript may be cleaved to free a 3' hydroxyl.
  • poly-A polymerase adds a chain of adenine nucleotides to the R A.
  • the process called polyadenylation, adds a poly-A tail of a certain length.
  • the length of a poly-A tail is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).
  • the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700,
  • the mRNA includes a poly-A tail from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1 ,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to
  • the poly -A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the region coding for a target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the ultimate product expressed from the mRNA.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the mRNA or feature thereof.
  • the poly-A tail may also be designed as a fraction of mRNA to which it belongs.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail.
  • engineered binding sites and conjugation of mRNA for poly-A binding protein may enhance expression.
  • multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3'-end using modified nucleotides at the 3 '-terminus of the poly- A tail.
  • Transfection experiments can be conducted in relevant cell lines at, and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.
  • the mRNA are designed to include a polyA-G quartet.
  • the G- quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G- rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail.
  • the mRNA may include one stop codon. In some embodiments, the mRNA may include two stop codons. In some embodiments, the mRNA may include three stop codons. In some embodiments, the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As non-limiting examples, the stop codon may be selected from TGA, TAA and TAG. [0509] In some embodiments, the mRNA includes the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA.
  • the originator construct and/or the benchmark construct is a circular RNA (oRNA).
  • oRNA circular RNA
  • the terms "oRNA”, “circRNA” or “circular RNA” are used interchangeably and can refer to an RNA that forms a circular structure through covalent or non-covalent bonds.
  • the oRNA may be non-immunogenic in a mammal (e.g., a human, non-human primate, rabbit, rat, and mouse).
  • a mammal e.g., a human, non-human primate, rabbit, rat, and mouse.
  • the oRNA may be capable of replicating or replicates in a cell from an aquaculture animal (e.g., fish, crabs, shrimp, oysters etc.), a mammalian cell, a cell from a pet or zoo animal (e.g., cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (e.g., horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof.
  • an aquaculture animal e.g., fish, crabs, shrimp, oysters etc.
  • a mammalian cell e.g., a cell from a
  • the oRNA has a half-life of at least that of a linear counterpart. In some embodiments, the oRNA has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater.
  • the oRNA has a half-life or persistence in a cell for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.
  • the oRNA has a half-life or persistence in a cell for no more than about 10 mins to about 7 days, or no more than about 1 hour, 2 hours,
  • the oRNA has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the oRNA has a half-life or persistence in a cell post division. In certain embodiments, the oRNA has a half-life or persistence in a dividing cell for greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days,
  • the oRNA modulates a cellular function, e.g., transiently or long term.
  • the cellular function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer.
  • the cellular function is transiently altered, e.g., such as a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.
  • a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours,
  • the oRNA is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 8,000 nucleo
  • the maximum size of the oRNA may be limited by the ability of packaging and delivering the RNA to a target.
  • the size of the oRNA is a length sufficient to encode polypeptides, and thus, lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be useful.
  • the oRNA comprises one or more elements described elsewhere herein.
  • the elements may be separated from one another by a spacer sequence or linker.
  • the elements may be separated from one another by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000
  • one or more elements are contiguous with one another, e.g., lacking a spacer element.
  • one or more elements is conformationally flexible.
  • the conformational flexibility is due to the sequence being substantially free of a secondary structure.
  • the oRNA comprises a secondary or tertiary structure that accommodates a binding site for a ribosome, translation, or rolling circle translation.
  • the oRNA comprises particular sequence characteristics.
  • the oRNA may comprise a particular nucleotide composition.
  • the oRNA may include one or more purine rich regions (adenine or guanosine).
  • the oRNA may include one or more purine rich regions (adenine or guanosine).
  • the oRNA may include one or more AU rich regions or elements (AREs).
  • the oRNA may include one or more adenine rich regions.
  • the oRNA comprises one or more modifications described elsewhere herein.
  • the oRNA comprises one or more expression sequences and is configured for persistent expression in a cell of a subject in vivo.
  • the oRNA is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point.
  • the expression of the one or more expression sequences can be either maintained at a relatively stable level or can increase over time. The expression of the expression sequences can be relatively stable for an extended period of time.
  • the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.
  • the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.
  • the oRNA comprises a regulatory element.
  • a “regulatory element” is a sequence that modifies expression of an expression sequence.
  • the regulatory element may include a sequence that is located adj acent to a payload or cargo region.
  • the regulatory element may be operatively linked operatively to a payload or cargo region.
  • a regulatory element may increase an amount of payload or cargo expressed as compared to an amount expressed when no regulatory element exists.
  • one regulatory element can increase an amount of payloads or cargos expressed for multiple payload or cargo sequences attached in tandem.
  • a regulatory element may comprise a sequence to selectively initiates or activates translation of a payload or cargo.
  • a regulatory element may comprise a sequence to initiate degradation of the oRNA or the payload or cargo.
  • Non-limiting examples of the sequence to initiate degradation includes, but is not limited to, riboswitch aptazymes and miRNA binding sites.
  • a regulatory element can modulate translation of the payload or cargo in the oRNA.
  • the modulation can create an increase (enhancer) or decrease (suppressor) in the payload or cargo.
  • the regulatory element may be located adjacent to the payload or cargo (e.g., on one side or both sides of the payload or cargo).
  • a translation initiation sequence functions as a regulatory element.
  • the translation initiation sequence comprises an AUG/ATG codon.
  • a translation initiation sequence comprises any eukaryotic start codon such as, but not limited to, AUG/ATG, CUG/CTG, GUG/GTG, UUG/TTG, ACG, AUC/ATC, AUU, AAG, AU A/ ATA, or AGG.
  • a translation initiation sequence comprises a Kozak sequence.
  • translation begins at an alternative translation initiation sequence, e.g., translation initiation sequence other than AUG/ATG codon, under selective conditions, e.g., stress induced conditions.
  • the translation of the circular polyribonucleotide may begin at alternative translation initiation sequence, such as ACG.
  • the circular polyribonucleotide translation may begin at alternative translation initiation sequence, CUG/CTG.
  • the translation may begin at alternative translation initiation sequence, GUG/GTG.
  • the translation may begin at a repeat-associated non- AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC, CAG, CTG.
  • RAN repeat-associated non- AUG
  • Masking any of the nucleotides flanking a codon that initiates translation may be used to alter the position of translation initiation, translation efficiency, length and/or structure of the oRNA.
  • a masking agent may be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon.
  • Non-limiting examples of masking agents include antisense locked nucleic acids (LNA) oligonucleotides and exon junction complexes (EJCs).
  • a masking agent may be used to mask a start codon of the oRNA in order to increase the likelihood that translation initiate at an alternative start codon.
  • the oRNA encodes a polypeptide or peptide and may comprise a translation initiation sequence.
  • the translation initiation sequence may comprise, but is not limited to a start codon, a non-coding start codon, a Kozak sequence or a Shine-Dalgamo sequence.
  • the translation initiation sequence may be located adjacent to the payload or cargo (e.g., on one side or both sides of the payload or cargo).
  • the translation initiation sequence provides conformational flexibility to the oRNA. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the oRNA.
  • the oRNA may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon.
  • the oRNA may initiate at a codon which is not the first start codon, e.g., AUG.
  • Translation of the circular polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CUG/CTG, GUG/GTG, AU A/ ATA, AUU/ATT, UUG/TTG.
  • translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions.
  • the translation of the oRNA may begin at alternative translation initiation sequence, such as ACG.
  • the oRNA translation may begin at alternative translation initiation sequence, CUG/CTG.
  • the oRNA translation may begin at alternative translation initiation sequence, GTG/GUG.
  • the oRNA may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC, CAG, CTG.
  • RAN repeat-associated non-AUG
  • the oRNA described herein comprises an internal ribosome entry site (IRES) element capable of engaging a eukaryotic ribosome.
  • IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides.
  • the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, and a Drosophila.
  • viral DNA may be derived from, but is not limited to, picomavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA.
  • cDNA picomavirus complementary DNA
  • EMCV encephalomyocarditis virus
  • Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.
  • the IRES element is at least partially derived from a virus, for instance, it can be derived from a viral IRES element, such as ABPV IGRpred, AEV, ALPV IGRpred, BQCV IGRpred, BVDV1 1-385, BVDV1 29-391, CrPV 5NCR, CrPV IGR, crTMV IREScp, crTMV_IRESmp75, crTMV_IRESmp228, crTMV IREScp, crTMV IREScp, CSFV, CVB3, DCV IGR, EMCV-R, EoPV_5NTR, ERAV 245-961, ERBV 162-920, EV71_l-748, FeLV-Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy _env, gypsyD5, gypsy D2, HAV_HM175, HCV_type_la, HiPV
  • the IRES element is at least partially derived from a cellular IRES, such as AML1/RUNX1, Antp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP4, ATIR varl, ATlR_var2, ATlR_var3, ATlR_var4, BAGl_p36delta236 nt, BAGl_p36, BCL2, BiP_-222_-3, c-IAPl_285-1399, C-IAP1 1313-1462, c-jun, c-myc, Cat-1224, CCND1, DAPS, eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A, FMRI, Gtx-133-141, Gtx-1-166, Gtx-1-120, Gtx-1-196, hairless, HAP4, HIFla, hSNMl, Hsp
  • the oRNA includes one or more cargo or payload sequences (also referred to as expression sequences) and each cargo or payload sequence may or may not have a termination element.
  • the oRNA includes one or more cargo or payload sequences and the sequences lack a termination element, such that the oRNA is continuously translated. Exclusion of a termination element may result in rolling circle translation or continuous expression of the encoded peptides or polypeptides as the ribosome cannot stalling or fall-off. In such an embodiment, rolling circle translation expresses a continuous expression through each cargo or payload sequence.
  • one or more cargo or payload sequences in the oRNA comprise a termination element.
  • not all of the cargo or payload sequences in the oRNA comprise a termination element.
  • the cargo or payload may fall off the ribosome when the ribosome encounters the termination element and terminates translation.
  • translation is terminated while at least one region of the ribosome remains in contact with the oRNA.
  • the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least one round of translation of the oRNA.
  • the oRNA as described herein is competent for rolling circle translation.
  • the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9 rounds, at least 10 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds, at least 15 rounds, at least 20 rounds, at least 30 rounds, at least 40 rounds, at least 50 rounds, at least 60 rounds, at least 70 rounds, at least 80 rounds, at least 90 rounds, at least 100 rounds, at least 150 rounds, at least 200 rounds, at least 250 rounds, at least 500 rounds, at least 1000 rounds, at least 1500 rounds, at least 2000 rounds, at least 5000 rounds, at least 10000 rounds, at least 10.sup.5 rounds, or at least 10.sup.6 rounds of translation of the oRNA.
  • the rolling circle translation of the oRNA leads to generation of polypeptide that is translated from more than one round of translation of the oRNA.
  • the oRNA comprises a stagger element, and rolling circle translation of the oRNA leads to generation of polypeptide product that is generated from a single round of translation or less than a single round of translation of the oRNA.
  • a linear RNA may be cyclized, or concatemerized. In some embodiments, the linear RNA may be cyclized in vitro prior to formulation and/or delivery. In some embodiments, the linear RNA may be cyclized within a cell.
  • the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed.
  • the newly formed 5'-/3'-linkage may be intramolecular or intermolecular.
  • the 5'-end and the 3 '-end of the nucleic acid contain chemically reactive groups that, when close together, form anew covalent linkage between the 5 '-end and the 3 '-end of the molecule.
  • the 5 '-end may contain an NHS-ester reactive group and the 3 '- end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino- terminated nucleotide on the 3 '-end of a synthetic mRNA molecule can undergo a nucleophilic attack on the 5 '-NHS-ester moiety forming a new 5 '-/3 '-amide bond.
  • T4 RNA ligase may be used to enzymatically link a 5'- phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid forming anew phosphorodiester linkage.
  • a g of a nucleic acid molecule is incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol.
  • the ligation reaction may occur in the presence of a split oligonucleotide capable of base-pairing with both the 5'- and 3'-region in juxtaposition to assist the enzymatic ligation reaction.
  • either the 5 '-or 3 '-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5 '-end of a nucleic acid molecule to the 3 '-end of a nucleic acid molecule.
  • the ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • the ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.
  • the oRNA is made via circularization of a linear RNA.
  • the linear RNA is cyclized, or concatemerized using a chemical method to form an oRNA.
  • the 5'-end and the 3'-end of the nucleic acid e.g., alinear RNA
  • the 5'-end and the 3'-end of the nucleic acid includes chemically reactive groups that, when close together, may form anew covalent linkage between the 5'-end and the 3'-end of the molecule.
  • the 5'-end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a linear RNA can undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'-/3'-amide bond.
  • a DNA or RNA ligase may be used to enzymatically link a 5'- phosphorylated nucleic acid molecule (e.g., a linear RNA) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) forming a new phosphorodiester linkage.
  • a linear RNA is incubated at 37C for 1 hour with 1-10 units of T4 RNA ligase according to the manufacturer's protocol.
  • the ligation reaction may occur in the presence of a linear nucleic acid capable of base-pairing with both the 5'- and 3'-region in juxtaposition to assist the enzymatic ligation reaction.
  • the ligation is splint ligation where a single stranded polynucleotide (splint), like a single stranded RNA, can be designed to hybridize with both termini of a linear RNA, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint.
  • Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear RNA, generating an oRNA.
  • a DNA or RNA ligase may be used in the synthesis of the oRNA.
  • the ligase may be a circ ligase or circular ligase.
  • either the 5'- or 3'-end of the linear RNA can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear RNA includes an active ribozyme sequence capable of ligating the 5'-end of the linear RNA to the 3'-end of the linear RNA.
  • the ligase ribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).
  • a linear RNA may be cyclized or concatemerized by using at least one non-nucleic acid moiety.
  • the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus and/or near the 3' terminus of the linear RNA in order to cyclize or concatermerize the linear RNA.
  • the at least one non- nucleic acid moiety may be located in or linked to or near the 5' terminus and/or the 3' terminus of the linear RNA.
  • the non-nucleic acid moieties contemplated may be homologous or heterologous.
  • the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage and/or a cleavable linkage.
  • the non-nucleic acid moiety is a ligation moiety.
  • the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.
  • a linear RNA may be cyclized or concatemerized due to a non- nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near or linked to the 5' and 3' ends of the linear RNA.
  • one or more linear RNA may be cyclized or concatemerized by intermolecular forces or intramolecular forces.
  • intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, Van der Waals forces, and London dispersion forces.
  • intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibonding.
  • the linear RNA may comprise a ribozyme RNA sequence near the 5' terminus and near the 3' terminus.
  • the ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme.
  • the peptides covalently linked to the ribozyme RNA sequence near the 5' terminus and the 3' terminus may associate with each other causing a linear RNA to cyclize or concatemerize.
  • the peptides covalently linked to the ribozyme RNA near the 5' terminus and the 3' terminus may cause the linear RNA to cyclize or concatemerize after being subjected to ligated using various methods known in the art such as, but not limited to, protein ligation.
  • the linear RNA may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase).
  • RppH RNA 5' pyrophosphohydrolase
  • apyrase ATP diphosphohydrolase
  • converting the 5' triphosphate of the linear RNA into a 5' monophosphate may occur by a two- step reaction comprising: (a) contacting the 5' nucleotide of the linear RNA with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.
  • a phosphatase e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase
  • a kinase e.g., Polynucleotide Kinase
  • RNA may be circularized using the methods described in WO2017222911 and WO2016197121, the contents of each of which are herein incorporated by reference in their entirety.
  • RNA may be circularized, for example, by backsplicing of a non-mammalian exogenous intron or splint ligation of the 5' and 3 ' ends of a linear RNA.
  • the circular RNA is produced from a recombinant nucleic acid encoding the target RNA to be made circular.
  • the method comprises: a) producing a recombinant nucleic acid encoding the target RNA to be made circular, wherein the recombinant nucleic acid comprises in 5' to 3 ' order: i) a 3 ' portion of an exogenous intron comprising a 3' splice site, ii) a nucleic acid sequence encoding the target RNA, and iii) a 5 ' portion of an exogenous intron comprising a 5 ' splice site; b) performing transcription, whereby RNA is produced from the recombinant nucleic acid; and c) performing splicing of the RNA, whereby the RNA circularizes to produce a oRNA.
  • circular RNAs generated with exogenous introns are recognized by the immune system as "non-self’ and trigger an innate immune response.
  • circular RNAs generated with endogenous introns are recognized by the immune system as "self’ and generally do not provoke an innate immune response, even if carrying an exon comprising foreign RNA.
  • circular RNAs can be generated with either an endogenous or exogenous intron to control immunological self/nonself discrimination as desired.
  • intron sequences from a wide variety of organisms and viruses are known and include sequences derived from genes encoding proteins, ribosomal RNA (rRNA), or transfer RNA (tRNA).
  • Circular RNAs can be produced from linear RNAs in a number of ways.
  • circular RNAs are produced from a linear RNA by backsplicing of a downstream 5' splice site (splice donor) to an upstream 3' splice site (splice acceptor).
  • Circular RNAs can be generated in this manner by any nonmammalian splicing method.
  • linear RNAs containing various types of introns including self-splicing group I introns, self-splicing group II introns, spliceosomal introns, and tRNA introns can be circularized.
  • group I and group II introns have the advantage that they can be readily used for production of circular RNAs in vitro as well as in vivo because of their ability to undergo self-splicing due to their autocatalytic ribozyme activity.
  • circular RNAs can be produced in vitro from a linear RNA by chemical or enzymatic ligation of the 5' and 3' ends of the RNA.
  • Chemical ligation can be performed, for example, using cyanogen bromide (BrCN) or ethyl-3-(3'- dimethylaminopropyl) carbodiimide (EDC) for activation of a nucleotide phosphomonoester group to allow phosphodiester bond formation.
  • cyanogen bromide BrCN
  • EDC ethyl-3-(3'- dimethylaminopropyl) carbodiimide
  • enzymatic ligation can be used to circularize RNA.
  • exemplary ligases that can be used include T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2).
  • splint ligation using an oligonucleotide splint that hybridizes with the two ends of a linear RNA can be used to bring the ends of the linear RNA together for ligation.
  • Hybridization of the splint which can be either a DNA or an RNA, orientates the 5 '- phosphate and 3' -OH of the RNA ends for ligation.
  • Subsequent ligation can be performed using either chemical or enzymatic techniques, as described above.
  • Enzymatic ligation can be performed, for example, with T4 DNA ligase (DNA splint required), T4 RNA ligase 1 (RNA splint required) or T4 RNA ligase 2 (DNA or RNA splint).
  • Chemical ligation, such as with BrCN or EDC, in some cases is more efficient than enzymatic ligation if the structure of the hybridized splint-RNA complex interferes with enzymatic activity.
  • the oRNA may further comprise an internal ribosome entry site (IRES) operably linked to an RNA sequence encoding a polypeptide.
  • IRES internal ribosome entry site
  • Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA.
  • the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al, Biochem. Biophys. Res. Comm.
  • the circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%.
  • the oRNA includes at least one splicing element.
  • the splicing element can be a complete splicing element that can mediate splicing of the oRNA, or the spicing element can be a residual splicing element from a completed splicing event.
  • a splicing element of a linear RNA can mediate a splicing event that results in circularization of the linear RNA, thereby the resultant oRNA comprises a residual splicing element from such splicing-mediated circularization event.
  • the residual splicing element is not able to mediate any splicing.
  • the residual splicing element can still mediate splicing under certain circumstances.
  • the splicing element is adjacent to at least one expression sequence.
  • the oRNA includes a splicing element adjacent each expression sequence.
  • the splicing element is on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s).
  • the oRNA includes an internal splicing element that when replicated the spliced ends are joined together.
  • Some examples may include miniature introns ( ⁇ 100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in (suptable4 enriched motifs) cis-sequence elements proximal to backsplice events such as sequences in the 200 bp preceding (upstream of) or following (downstream from) a backsplice site with flanking exons.
  • the oRNA includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repetitive nucleotide sequence may include repeated sequences from the Alu family of introns.
  • the oRNA may include canonical splice sites that flank head- to-tail junctions of the oRNA.
  • the oRNA may include a bulge-helix-bulge motif, comprising a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl group and 2', 3'-cyclic phosphate. Circularization proceeds by nucleophilic atack of the 5'-OH group onto the 2', 3'- cyclic phosphate of the same molecule forming a 3', 5'-phosphodi ester bridge.
  • the oRNA may include a sequence that mediates self-ligation.
  • sequences that can mediate self-ligation include a self-circularizing intron, e.g., a 5' and 3' slice junction, or a self-circularizing catalytic intron such as a Group I, Group II or Group III Introns.
  • group I intron self-splicing sequences may include self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena.
  • linear RNA may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns.
  • the oRNA includes a repetitive nucleic acid sequence.
  • the repetitive nucleotide sequence includes poly CA or poly UG sequences.
  • the oRNA includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the oRNA, with the hybridized segment forming an internal double strand.
  • the complementary sequences are found at the 5' and 3' ends of the linear RNA.
  • the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.
  • chemical methods of circularization may be used to generate the oRNA.
  • Such methods may include, but are not limited to click chemistry (e.g., alkyne and azide based methods, or clickable bases), olefin metathesis, phosphorami date ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.
  • enzymatic methods of circularization may be used to generate the oRNA.
  • a ligation enzyme e.g., DNA or RNA ligase, may be used to generate a template of the oRNA or complement, a complementary strand of the oRNA, or the oRNA.
  • siRNAs Small Interfering RNAs
  • the payload region may be or encode an RNA interference (RNAi) sequence which can be used to reduce or inhibit the expression of a gene.
  • RNAi also known as post-transcriptional gene silencing (PTGS), quelling, or co-suppression
  • PTGS post-transcriptional gene silencing
  • co-suppression is a post- transcriptional gene silencing process in which RNA molecules, in a sequence specific manner, reduce or inhibit gene expression, typically by causing the destruction of specific mRNA molecules.
  • RNAi short/small double stranded RNAs
  • siRNAs small interfering RNAs
  • 15-30 nucleotides e.g., 19 to 25, 19 to 24 or 19-21 nucleotides
  • 2 nucleotide 3’ overhangs and that match the nucleic acid sequence of the target gene.
  • These short RNA species may be naturally produced in vivo by Dicer-mediated cleavage of larger dsRNAs, and they are functional in mammalian cells.
  • miRNAs Naturally expressed small RNA molecules, named microRNAs (miRNAs), elicit gene silencing by regulating the expression of mRNAs.
  • the miRNAs -containing RNA Induced Silencing Complex (RISC) targets mRNAs presenting a perfect sequence complementarity with nucleotides 2-7 in the 5 ’region of the miRNA which is called the seed region, and other base pairs with its 3 ’region.
  • miRNA-mediated down-regulation of gene expression may be caused by cleavage of the target mRNAs, translational inhibition of the target mRNAs, or mRNA decay.
  • miRNA targeting sequences are usually located in the 3’-UTR of the target mRNAs.
  • a single miRNA may target more than 100 transcripts from various genes, and one mRNA may be targeted by different miRNAs.
  • siRNA duplexes or dsRNA targeting a specific mRNA may be designed and synthesized in vitro and introduced into cells for activating RNAi processes. It has been previously shown that 21 -nucleotide siRNA duplexes (termed small interfering RNAs) were capable of effecting potent and specific gene knockdown without inducing immune response in mammalian cells. Now post-transcriptional gene silencing by siRNAs has quickly emerged as a powerful tool for genetic analysis in mammalian cells and has the potential to produce novel therapeutics.
  • siRNA sequences may be introduced into cells in order to activate RNAi.
  • An exogenous siRNA duplex when it is introduced into cells, similar to the endogenous dsRNAs, can be assembled to form the RNA Induced Silencing Complex (RISC), a multiunit complex that interacts with RNA sequences that are complementary to one of the two strands of the siRNA duplex (i.e., the antisense strand).
  • RISC RNA Induced Silencing Complex
  • the sense strand (or passenger strand) of the siRNA is tost from the complex, while the antisense strand (or guide strand) of the siRNA is matched with its complementary RNA.
  • the targets of siRNA containing RISC complexes are mRNAs presenting a perfect sequence complementarity. Then, siRNA mediated gene silencing occurs by cleaving, releasing and degrading the target.
  • the siRNA duplex comprised of a sense strand homologous to the target mRNA and an antisense strand that is complementary to the target mRNA offers much more advantage in terms of efficiency for target RNA destruction compared to the use of the single strand (ss)-siRNAs (e.g., antisense strand RNA or antisense oligonucleotides). In many cases, it requires higher concentration of the ss-siRNA to achieve the effective gene silencing potency of the corresponding duplex.
  • ss-siRNAs single strand
  • siRNA sequence preference include, but are not limited to, (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nucleotides in length.
  • highly effective siRNA constructs essential for suppressing mammalian target gene expression may be readily designed.
  • siRNA constructs e.g., siRNA duplexes or encoded dsRNA
  • Such siRNA constructs can specifically, suppress gene expression and protein production.
  • the siRNA constructs are designed and used to selectively “knock out” gene variants in cells, i.e., mutated transcripts that are identified in patients or that are the cause of various diseases and/or disorders.
  • the siRNA constructs are designed and used to selectively “knock down” variants of the gene in cells.
  • the siRNA constructs are able to inhibit or suppress both the wild type and mutated versions of the gene.
  • an siRNA sequence comprises a sense strand and a complementary antisense strand in which both strands are hybridized together to form a duplex structure.
  • the antisense strand has sufficient complementarity to the mRNA sequence to direct target-specific RNAi, i.e., the siRNA sequence has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • an siRNA sequence comprises a sense strand and a complementary antisense strand in which both strands are hybridized together to form a duplex structure and where the start site of the hybridization to the mRNA is between nucleotide 100 and 10,000 on the mRNA sequence.
  • the start site may be between nucleotide 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500- 550, 550-600, 600-650, 650-700, 700-70, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350- 1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850, 1850-1900, 1900-1950, 1950-2000, 2000-2050, 2050-2100, 2100- 2150, 2150-2200, 2200-2250, 2250-2300, 2300-2350, 2350-2400, 2400-2450, 2450-2500, 2500-2550, 2550-2600, 2600
  • the antisense strand and target mRNA sequences have 100% complementary.
  • the antisense strand may be complementary to any part of the target mRNA sequence.
  • the antisense strand and target mRNA sequences comprise at least one mismatch.
  • the antisense strand and the target mRNA sequence have at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or at least 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-99%, 30-40%, 30-
  • the siRNA sequence has a length from about 10-50 or more nucleotides, i.e., each strand comprising 10-50 nucleotides (or nucleotide analogs).
  • the siRNA sequence has a length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementarity to a target region.
  • the siRNA sequence has a length from about 19 to 25, 19 to 24 or 19 to 21 nucleotides.
  • the siRNA sequences can be synthetic RNA duplexes comprising about 19 nucleotides to about 25 nucleotides, and two overhanging nucleotides at the 3'-end.
  • the siRNA constructs may be unmodified RNA molecules.
  • the siRNA constructs may contain at least one modified nucleotide, such as base, sugar or backbone modifications.
  • the siRNA sequences can be encoded in plasmid vectors, viral vectors or other nucleic acid expression vectors for delivery to a cell.
  • DNA expression plasmids can be used to stably express the siRNA duplexes or dsRNA in cells and achieve long-term inhibition of the target gene expression.
  • the sense and antisense strands of a siRNA duplex are typically linked by a short spacer sequence leading to the expression of a stem-loop structure termed short hairpin RNA (shRNA). The hairpin is recognized and cleaved by Dicer, thus generating mature siRNA constructs.
  • shRNA short hairpin RNA
  • the sense and antisense strands of a siRNA duplex may be linked by a short spacer sequence, which may optionally be linked to additional flanking sequence, leading to the expression of a flanking arm-stem-loop structure termed primary microRNA (pri-miRNA).
  • pri-miRNA flanking arm-stem-loop structure
  • the pri-miRNA may be recognized and cleaved by Drosha and Dicer, and thus generate mature siRNA constructs.
  • the siRNA duplexes or encoded dsRNA suppress (or degrade) target mRNA. Accordingly, the siRNA duplexes or encoded dsRNA can be used to substantially inhibit gene expression in a cell.
  • the inhibition of gene expression refers to an inhibition by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95- 100%.
  • the protein product of the targeted gene may be inhibited by at least about 20%, preferably by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100%.
  • the siRNA constructs comprise a miRNA seed match for the target located in the guide strand. In another embodiment, the siRNA constructs comprise a miRNA seed match for the target located in the passenger strand. In yet another embodiment, the siRNA duplexes or encoded dsRNA targeting gene do not comprise a seed match for the target located in the guide or passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting the gene may have almost no significant full-length off targets for the guide strand. In another embodiment, the siRNA duplexes or encoded dsRNA targeting the gene may have almost no significant full- length off target effects for the passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting the gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 1-5%, 2-6%, 3-7%, 4-8%, 5- 9%, 5-10%, 6-10%, 5-15%, 5-20%, 5-25% 5-30%, 10-20%, 10-30%, 10-40%, 10-50%, 15- 30%, 15-40%, 15-45%, 20-40%, 20-50%, 25-50%, 30-40%, 30-50%, 35-50%, 40-50%, 45- 50% full-length off target effects for the passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting the gene may have almost no significant full-length off targets for the guide strand or the passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting the gene may have less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 1-5%, 2-6%, 3-7%, 4-8%, 5-9%, 5- 10%, 6-10%, 5-15%, 5-20%, 5-25% 5-30%, 10-20%, 10-30%, 10-40%, 10-50%, 15-30%, 15- 40%, 15-45%, 20-40%, 20-50%, 25-50%, 30-40%, 30-50%, 35-50%, 40-50%, 45-50% full- length off target effects for the guide or passenger strand.
  • the siRNA duplexes or encoded dsRNA targeting the gene may have high activity in vitro.
  • the siRNA constructs may have low activity in vitro.
  • the siRNA duplexes or dsRNA targeting the gene may have high guide strand activity and low passenger strand activity in vitro.
  • the siRNA constructs have a high guide strand activity and low passenger strand activity in vitro.
  • the target knock-down (KD) by the guide strand may be at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% or 100%.
  • the target knock-down by the guide strand may be 40-50%, 45-50%, 50-55%, 50-60%, 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-99.5%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%, 65-99.5%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%, 70-99.5%, 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-99.5%, 75-100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-99.5%, 80-100%, 85- 90%, 85-95%, 85-99%, 85-99.5%, 85-100%, 90-95%, 90-99%, 90-99.5%, 90-100%, 95-99%, 95-99.5%, 95-100%, 99-
  • the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is 1 : 10, 1 :9, 1 :8, 1 :7, 1:6, 1 :5, 1 :4, 1:3, 1 :2, 1 ;1, 2: 10, 2:9, 2:8,
  • the guide to passenger ratio refers to the ratio of the guide strands to the passenger strands after the intracellular processing of the pri-microRNA. For example, an 80:20 guide-to-passenger ratio would have 8 guide strands to every 2 passenger strands processed from the precursor.
  • the guide-to-passenger strand ratio is 8:2 in vitro.
  • the guide-to-passenger strand ratio is 8:2 in vivo.
  • the guide-to- passenger strand ratio is 9: 1 in vitro.
  • the guide-to-passenger strand ratio is 9:1 in vivo.
  • the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is greater than 1. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is greater than 2. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is greater than 5. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is greater than 10. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is greater than 20.
  • the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is greater than 50. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is at least 3: 1. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is at least 5: 1. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is at least 10: 1. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is at least 20: 1. In some embodiments, the guide to passenger (G:P) (also referred to as the antisense to sense) strand ratio expressed is at least 50: 1.
  • the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is 1: 10, 1:9, 1 :8, 1 :7, 1 :6, 1 :5, 1:4, 1:3, 1:2, l;l, 2: 10, 2:9, 2:8,
  • the passenger to guide ratio refers to the ratio of the passenger strands to the guide strands after the excision of the guide strand.
  • an 80:20 passenger to guide ratio would have 8 passenger strands to every 2 guide strands processed from the precursor.
  • the passenger-to-guide strand ratio is 80:20 in vitro.
  • the passenger-to-guide strand ratio is 80:20 in vivo.
  • the passenger-to-guide strand ratio is 8:2 in vitro.
  • the passenger-to- guide strand ratio is 8:2 in vivo.
  • the passenger-to-guide strand ratio is 9: 1 in vitro.
  • the passenger-to-guide strand ratio is 9: 1 in vivo.
  • the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is greater than 1. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is greater than 2. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is greater than 5. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is greater than 10. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is greater than 20.
  • the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is greater than 50. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is at least 3: 1. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is at least 5: 1. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is at least 10:1. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is at least 20: 1. In some embodiments, the passenger to guide (P:G) (also referred to as the sense to antisense) strand ratio expressed is at least 50: 1.
  • a passenger-guide strand duplex is considered effective when the pri- or pre-microRNAs demonstrate, but methods known in the art and described herein, greater than 2-fold guide to passenger strand ratio when processing is measured.
  • the pri- or pre-microRNAs demonstrate great than 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 2 to 5-fold, 2 to 10-fold, 2 to 15-fold, 3 to 5-fold, 3 to 10-fold, 3 to 15-fold, 4 to 5-fold, 4 to 10- fold, 4 to 15 -fold, 5 to 10-fold, 5 to 15 -fold, 6 to 10-fold, 6 to 15 -fold, 7 to 10-fold, 7 to 15- fold, 8 to 10-fold, 8 to 15-fold, 9 to 10-fold, 9 to 15-fold, 10 to 15-fold, 11 to 15-fold, 12 to 15- fold
  • the vector genome encoding the dsRNA comprises a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% of the full length of the construct.
  • the vector genome comprises a sequence which is at least 80% of the full length sequence of the construct.
  • the siRNA constructs may be used to silence a wild type or mutant gene by targeting at least one exon on the sequence.
  • the siRNA constructs when not delivered as a precursor or DNA, may be chemically modified to modulate some features of RNA molecules, such as, but not limited to, increasing the stability of siRNAs in vivo.
  • the chemically modified siRNA constructs can be used in human therapeutic applications, and are improved without compromising the RNAi activity of the siRNA constructs.
  • the siRNA constructs modified at both the 3' and the 5' end of both the sense strand and the antisense strand.
  • the modified nucleotides may be on just the sense strand. [0604] In some embodiments, the modified nucleotides may be on just the antisense strand. [0605] In some embodiments, the modified nucleotides may be in both the sense and antisense strands.
  • the chemically modified nucleotide does not affect the ability of the antisense strand to pair with the target mRNA sequence.
  • microRNA (miR) Scaffolds
  • the siRNA constructs may be encoded in a polynucleotide sequence which also comprises a microRNA (miR) scaffold construct.
  • a “microRNA (miR) scaffold construct” is a framework or starting molecule that forms the sequence or structural basis against which to design or make a subsequent molecule.
  • the miR scaffold construct comprises at least one 5’ flanking region.
  • the 5’ flanking region may comprise a 5’ flanking sequence which may be of any length and may be derived in whole or in part from wild type microRNA sequence or be a completely artificial sequence.
  • the miR scaffold construct comprises at least one 3’ flanking region.
  • the 3’ flanking region may comprise a 3’ flanking sequence which may be of any length and may be derived in whole or in part from wild type microRNA sequence or be a completely artificial sequence.
  • the miR scaffold construct comprises at least one loop motif region.
  • the loop motif region may comprise a sequence which may be of any length.
  • the miR scaffold construct comprises a 5’ flanking region, a loop motif region and/or a 3’ flanking region.
  • At least one payload may be encoded by a polynucleotide which may also comprise at least one miR scaffold construct.
  • the miR scaffold construct may comprise a 5 ’ flanking sequence which may be of any length and may be derived in whole or in part from wild type microRNA sequence or be completely artificial.
  • the 3’ flanking sequence may mirror the 5’ flanking sequence and/or a 3’ flanking sequence in size and origin. Either flanking sequence may be absent.
  • the 3’ flanking sequence may optionally contain one or more CNNC motifs, where “N” represents any nucleotide.
  • the 5’ arm of the stem loop structure of the polynucleotide comprising or encoding the miR scaffold construct comprises a sequence encoding a sense sequence.
  • the 3’ arm of the stem loop of the polynucleotide comprising or encoding the miR scaffold construct comprises a sequence encoding an antisense sequence.
  • the antisense sequence in some instances, comprises a “G” nucleotide at the 5’ most end.
  • the sense sequence may reside on the 3’ arm while the antisense sequence resides on the 5’ arm of the stem of the stem loop structure of the polynucleotide comprising or encoding the miR scaffold construct.
  • the sense and antisense sequences may be completely complementary across a substantial portion of their length. In other embodiments the sense sequence and antisense sequence may be at least 70, 80, 90, 95 or 99% complementarity across independently at least 50, 60, 70, 80, 85, 90, 95, or 99 % of the length of the strands.
  • separating the sense and antisense sequence of the stem loop structure of the polynucleotide is a loop sequence (also known as a loop motif, linker or linker motif).
  • the loop sequence may be of any length, between 4-30 nucleotides, between 4-20 nucleotides, between 4-15 nucleotides, between 5-15 nucleotides, between 6-12 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, and/or 15 nucleotides.
  • the loop sequence comprises a nucleic acid sequence encoding at least one UGUG motif. In some embodiments, the nucleic acid sequence encoding the UGUG motif is located at the 5’ terminus of the loop sequence.
  • spacer regions may be present in the polynucleotide to separate one or more modules (e.g., 5’ flanking region, loop motif region, 3’ flanking region, sense sequence, antisense sequence) from one another. There may be one or more such spacer regions present.
  • modules e.g., 5’ flanking region, loop motif region, 3’ flanking region, sense sequence, antisense sequence
  • a spacer region of between 8-20, i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present between the sense sequence and a flanking region sequence.
  • the length of the spacer region is 13 nucleotides and is located between the 5’ terminus of the sense sequence and the 3’ terminus of the flanking sequence. In some embodiments, a spacer is of sufficient length to form approximately one helical turn of the sequence.
  • a spacer region of between 8-20, i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides may be present between the antisense sequence and a flanking sequence.
  • the spacer sequence is between 10-13, i.e., 10, 11, 12 or 13 nucleotides and is located between the 3’ terminus of the antisense sequence and the 5’ terminus of a flanking sequence. In some embodiments, a spacer is of sufficient length to form approximately one helical turn of the sequence.
  • the polynucleotide comprises in the 5’ to 3’ direction, a 5’ flanking sequence, a 5’ arm, a loop motif, a 3’ arm and a 3’ flanking sequence.
  • the 5’ arm may comprise a sense sequence and the 3’ arm comprises the antisense sequence.
  • the 5’ arm comprises the antisense sequence and the 3’ arm comprises the sense sequence.
  • the 5’ arm, payload (e.g., sense and/or antisense sequence), loop motif and/or 3’ arm sequence may be altered (e.g., substituting 1 or more nucleotides, adding nucleotides and/or deleting nucleotides).
  • the alteration may cause a beneficial change in the function of the construct (e.g., increase knock-down of the target sequence, reduce degradation of the construct, reduce off target effect, increase efficiency of the payload, and reduce degradation of the pay load).
  • the miR scaffold construct of the polynucleotides is aligned in order to have the rate of excision of the guide strand be greater than the rate of excision of the passenger strand.
  • the rate of excision of the guide or passenger strand may be, independently, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%.
  • the rate of excision of the guide strand is at least 80%.
  • the rate of excision of the guide strand is at least 90%.
  • the rate of excision of the guide strand is greater than the rate of excision of the passenger strand.
  • the rate of excision of the guide strand may be at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99% greater than the passenger strand.
  • the efficiency of excision of the guide strand is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more than 99%. As a non-limiting example, the efficiency of the excision of the guide strand is greater than 80%. [0630] In some embodiments, the efficiency of the excision of the guide strand is greater than the excision of the passenger strand from the miR scaffold construct. The excision of the guide strand may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times more efficient than the excision of the passenger strand from the miR scaffold construct.
  • the miR scaffold construct comprises a dual-function targeting polynucleotide.
  • a “dual-function targeting” polynucleotide is a polynucleotide where both the guide and passenger strands knock down the same target or the guide and passenger strands knock down different targets.
  • the miR scaffold construct of the polynucleotides described herein may comprise a 5’ flanking region, a loop motif region and a 3’ flanking region.
  • the polynucleotide is designed using at least one of the following properties: loop variant, seed mismatch/bulge/wobble variant, stem mismatch, loop variant and vassal stem mismatch variant, seed mismatch and basal stem mismatch variant, stem mismatch and basal stem mismatch variant, seed wobble and basal stem wobble variant, or a stem sequence variant.
  • the miR scaffold construct may be a natural pri-miRNA scaffold.
  • the selection of a miR scaffold construct is determined by a method of comparing polynucleotides in pri-miRNA.
  • the selection of a miR scaffold construct is determined by a method of comparing polynucleotides in natural pri-miRNA and synthetic pri-miRNA.
  • tRNA Transfer RNA
  • Transfer RNAs are RNA molecules that translate mRNA into proteins.
  • tRNA include a cloverleaf structure that comprise a 3’ acceptor site, 5’ terminal phosphate, D arm, T arm, and anticodon arm.
  • the main purpose of a tRNA is to carry amino acids on its 3’ acceptor site to a ribosome complex with the help of aminoacyl-tRNA synthetases which are enzymes that load the appropriate amino acid onto a free tRNA to synthesize proteins.
  • aminoacyl-tRNA synthetases are enzymes that load the appropriate amino acid onto a free tRNA to synthesize proteins.
  • the anticodon arm of the tRNA is the site of the anticodon, which is complementary to an mRNA codon and dictates which amino acid to carry.
  • tRNAs are also known to have a role in the regulation of apoptosis by acting as a cytochrome c scavenger.
  • the originator construct and/or the benchmark construct comprises or encodes a tRNA.
  • rRNA Ribosomal RNA
  • Ribosomal RNAs are RNA which form ribosomes. Ribosomes are essential to protein synthesis and contain a large and small ribosomal subunit. In prokaryotes, a small 30S and large 50S ribosomal subunit make up a 70S ribosome. In eukaryotes, the 40S and 60S subunit form an 80S ribosome. In order to bind aminoacyl-tRNAs and link amino acids together to create polypeptides, the ribosome contains 3 sites: an exit site (E), a peptidyl site (P), and acceptor site (A).
  • E exit site
  • P a peptidyl site
  • A acceptor site
  • the originator construct and/or the benchmark construct comprises or encodes a rRNA.
  • microRNA miRNA
  • microRNAs are 19-25 nucleotide long noncoding RNAs that bind to the 3'UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
  • the originator constructs and/or benchmark constructs may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds.
  • a microRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence.
  • a microRNA seed may comprise positions 2- 8 or 2-7 of the mature microRNA.
  • a microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1.
  • a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1.
  • the bases of the microRNA seed have complete complementarity with the target sequence.
  • microRNA site refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that “binding” may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.
  • Non-limiting examples of tissues where microRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let- 7, miR-133, miR-126).
  • MicroRNA can also regulate complex biological processes such as angiogenesis (miR-132).
  • miR-122 a microRNA abundant in liver, can inhibit the expression of the gene of interest if one or multiple target sites of miR-122 are engineered into the 3' UTR of the mRNA.
  • Introduction of one or multiple binding sites for different microRNA can be engineered to further decrease the longevity, stability, and protein translation of a mRNA.
  • microRNA binding sites can be engineered out of (i.e., removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues.
  • miR-122 binding sites may be removed to improve protein expression in the liver. Regulation of expression in multiple tissues can be accomplished through introduction or removal or one or several microRNA binding sites.
  • IncRNAs Long non-coding RNAs
  • the IncRNA designation is generally restricted to non-coding transcripts longer than about 200 nucleotides.
  • the length designation differentiates IncRNA from small regulatory RNAs such as short interfering RNA (siRNA) and micro RNA (miRNA).
  • siRNA short interfering RNA
  • miRNA micro RNA
  • the number of IncRNA species is thought to greatly exceed the number of protein-coding species.
  • IncRNAs drive biologic complexity observed in vertebrates compared to invertebrates. Evidence of this complexity is seen in many cellular compartments of a vertebrate organism such as the T lymphocyte compartment of the adaptive immune system. Differences in expression and function of IncRNA can be major contributors to human disease.
  • the originator constructs and/or the benchmark constructs comprise IncRNAs.
  • the originator constructs or benchmark constructs may contain one or more modified nucleotides such as, but not limited to, sugar modified nucleotides, nucleobase modifications and/or backbone modifications. In some aspects, the originator constructs or benchmark constructs may contain combined modifications, for example, combined nucleobase and backbone modifications.
  • the modified nucleotide may be a sugar-modified nucleotide.
  • Sugar modified nucleotides include, but are not limited to 2'-fluoro, 2'-amino and 2'-thio modified ribonucleotides, e.g., 2'-fluoro modified ribonucleotides.
  • Modified nucleotides may be modified on the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
  • the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.
  • the modified nucleotide may be a nucleobase-modified nucleotide.
  • the modified nucleotide may be a backbone-modified nucleotide.
  • the originator constructs or benchmark constructs may further comprise other modifications on the backbone.
  • a normal “backbone”, as used herein, refers to the repeating alternating sugar-phosphate sequences in a DNA or RNA molecule. The deoxyribose/ribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester,” bonds/linker (PO linkage). The PO backbones may be modified as “phosphorothioate backbone (PS linkage).
  • the natural phosphodiester bonds may be replaced by amide bonds but the four atoms between two sugar units are kept.
  • Such amide modifications can facilitate the solid phase synthesis of oligonucleotides and increase the thermodynamic stability of a duplex formed with siRNA complement.
  • Modified bases refer to nucleotide bases such as, but not limited to, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
  • nucleobase moieties include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination.
  • More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6- methylguanine, N,N, -dimethyl adenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1- methylinosine, 3-methyluridine, 5-methylcytidine, 5 -methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5- halouridine, 4-acetylcytidine, 1 -methyladenosine, 2-methyladenosine, 3 -methylcytidine, 6- methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza- adenosine,
  • the originator constructs and/or benchmark constructs may include one or more substitutions, insertions and/or additions, deletions, and covalent modifications with respect to reference sequences, in particular, the parent RNA.
  • the originator constructs and/or benchmark constructs includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc).
  • the one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999).
  • the first isolated nucleic acid comprises messenger RNA (mRNA).
  • the originator constructs and/or benchmark constructs comprise at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2- thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2- thio-uridine, 1 -taurinomethyl-4-thio-uridine, 5-methyl-uridine,
  • the mRNA comprises at least one nucleoside selected from the group consisting of 5 -aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l- methyl-l-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5 -methyl -zebula
  • the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7-deaza- adenine, 7- deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbam
  • mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl- guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7- methyl-8-oxo-guanosine, 1 -methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • nucleoside selected from the group
  • the originator constructs and/or benchmark constructs may include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone).
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro).
  • modifications e.g., one or more modifications
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GAAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the originator constructs and/or benchmark constructs includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency.
  • the N(6)methyladenosine (m6A) modification can reduce immunogeneicity of the originator constructs and/or benchmark constructs.
  • the modification may include a chemical or cellular induced modification.
  • RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA- protein interactions" from Nat Reviews Mol Cell Biol, 2017, 18:202-210.
  • RNA may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), base modifications (e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases.
  • the modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine.
  • base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the RNA.
  • the modification includes a bi-orthogonal nucleotides, e.g., an unnatural base. See for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI: 10.1039/c7cc06661a, which is hereby incorporated by reference.
  • sugar modifications e.g., at the 2' position or 4' position
  • replacement of the sugar one or more RNA may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages.
  • modifications include modified backbones or no natural intemucleoside linkages such as intemucleoside modifications, including modification or replacement of the phosphodiester linkages.
  • RNA having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • the RNA can include ribonucleotides with a phosphorus atom in its intemucleoside backbone.
  • Modified RNA backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5'
  • the modified nucleotides can be modified on the intemucleoside linkage (e.g., phosphate backbone).
  • phosphate backbone e.g., phosphate backbone
  • backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another intemucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene- phosphonates).
  • the a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • Phosphorothioate linked to the RNA is expected to reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
  • a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5'- O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thiophosphate)- pseudouridine).
  • alpha-thio-nucleoside e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5'- O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thiophosphate)- pseudouridine).
  • the RNA may include one or more cytotoxic nucleosides.
  • cytotoxic nucleosides may be incorporated into RNA, such as bifunctional modification.
  • Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1 -(2-C-cy ano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine.
  • Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D- arabinofuranosylcytosine, N4-octadecyl-l-beta-D-arabinofuranosylcytosine, N4-palmitoyl-l- (2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5'- elaidic acid ester).
  • the RNA sequence includes or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5 -propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- o
  • the RNA may or may not be uniformly modified along the entire length of the molecule.
  • one or more or all types of nucleotide e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU
  • the RNA includes a pseudouridine.
  • the RNA includes an inosine, which may aid in the immune system characterizing the RNA as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation.
  • all nucleotides in the RNA are modified.
  • the modification may include an m6A, which may augment expression, an inosine, which may attenuate an immune response, pseudouridine, which may increase RNA stability, or translational readthrough (stagger element), an m5C, which may increase stability, and a 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization).
  • RNA modifications may exist at various positions in the RNA.
  • nucleotide analogs or other modification(s) may be located at any position(s) of the RNA, such that the function of the RNA is not substantially decreased.
  • a modification may also be a non-coding region modification.
  • the RNA may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
  • any one or more of A, G, U or C) or any intervening percentage e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 100%, and from 95% to 100%).
  • any intervening percentage e.g.
  • a nucleotide sequence of the originator construct and/or benchmark construct may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g.
  • Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
  • the ORF sequence is optimized using optimization algorithms.
  • the lipid nanoparticles comprising cargos/pay loads can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed expression of the cargo/payload; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein; and/or (7) allow for regulatable expression of the cargo/payload.
  • excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed expression of the cargo/payload; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein; and/or (7) allow for regulatable expression of the cargo/payload.
  • Formulations can include, in addition to the lipid nanoparticles of the present disclosure, without limitation, saline, liposomes, other lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • pharmaceutical composition refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.
  • such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
  • active ingredient generally refers either to an originator construct or benchmark construct with a payload region or cargo or payload as described herein.
  • Formulations of the lipid nanoparticles comprising payloads and pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use for humans and for veterinary use.
  • an excipient may be approved by United States Food and Drug Administration.
  • an excipient may be of pharmaceutical grade.
  • an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
  • the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, or at least 80% (w/w) active ingredient.
  • lipid nanoparticles comprising payloads and/or pharmaceutical compositions described herein may be administered by any delivery route which results in a therapeutically effective outcome.
  • lipid nanoparticles comprising payloads and/or pharmaceutical compositions described herein may be administered parenterally.
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzy
  • oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • surfactants are included such as hydroxypropylcellulose.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the rate of drug release can be controlled.
  • biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • Formulations may also be delivered to a patient via intranasal administration.
  • lipid nanoparticles comprising cargos/pay loads described herein may be used as a therapeutic to diagnose, prevent, treat and/or manage disease, disorders and conditions, or as a diagnostic.
  • the therapeutic may be used in personalized medicine, immuno-oncology, cancer, vaccines, gene editing (e.g., CRISPR).
  • the methods of use can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of disease progression, including stabilization, slowing down and complete arrest; (2) reduction in the number of disease episodes and/or symptoms; (3) inhibition (i.e., reduction, slowing down or complete stopping) of a disease cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e.
  • the methods, components and compositions of the present disclosure may be used to diagnose, prevent, treat and/or manage infectious diseases.
  • Infectious diseases also known as transmissible diseases or communicable diseases
  • Infection agents are species typically not present within the body and may be, but are not limited to, viruses, bacteria, prions, nematodes, fungus, parasites or arthropods.
  • an infection or symptoms associated with an infection may be caused by one or more toxins produced by such agents.
  • Humans, and other mammals react to infections with an innate immune system response, often involving an inflammation. The illnesses and symptoms involved with infections vary according to the infectious agent.
  • infections may be subclinical without presenting any definite or observable symptoms, whereas some infections cause severe symptoms, require hospitalization or may be life-threatening. Some infections are localized, whereas some may overcome the body through blood circulation or lymphatic vessels. Some infections have long-term effects on wellbeing of infected individuals.
  • infection agents may be transmitted to humans via different routes.
  • infection agents may be transmitted by direct contact with an infected human, an infected animal, or an infected surface.
  • Infections may be transmitted by direct contact with bodily fluids of an infected human or an animal, e.g., blood, saliva, sweat, tears, mucus, female ejaculate, semen, vomit or urine.
  • infection may be transmitted by a fecal-oral route, referring to an infected person shedding the virus in fecal particles which then enters to person’s mouth causing infection.
  • the fecal-oral route is especially common transmission route in environments with poor sanitation and hygiene.
  • agents transmitted by the fecal-oral route include bacteria, e.g., shigella, Salmonella typhii and Vibrio Cholerae, virus, e.g. norovirus, rotavirus, enteroviruses, and hepatitis A, fungi, e.g. Enlamadeba histolytica, parasites, tape worms, transmitted by contaminated food or beverage, leading to food poisoning or gastroenteritis.
  • Infections may be transmitted by a respiratory route, referring to agents that are spread through the air. Typical examples include agents spread as small droplets of liquid or as aerosols, e.g., respiratory droplets expelled from the mouth and nose while coughing and sneezing.
  • Typical examples of respiratory transmitted diseases include the common cold mostly implicated to rhinoviruses, influenza caused by influenza viruses, respiratory tract infections caused by e.g., respiratory syncytial virus (RSV).
  • Infections may be transmitted by a sexual transmission route.
  • Examples of common sexually transmitted infections include e.g., human immunodeficiency virus (HIV) causing acquired immune deficiency syndrome (AIDS), chlamydia caused by Neisseria gonorrhoeae bacteria, fungal infection Candidiasis caused by Candida yeast, and Herpes Simplex disease caused by herpes simplex virus.
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • chlamydia caused by Neisseria gonorrhoeae bacteria
  • fungal infection Candidiasis caused by Candida yeast
  • Herpes Simplex disease caused by herpes simplex virus.
  • Infections may be transmitted by an oral transmission route, e.g., by kissing or
  • a common infection transmitted by oral transmission is an infectious mononucleosis caused by Epstein-Barr virus.
  • Infections may be transmitted by a vertical transmission, also known as “mother-to-child transmission,” from mother to an embryo, fetus or infant during pregnancy or childbirth.
  • Examples of infection agents that may be transmitted vertically include HIV, chlamydia, rubella, Toxoplasma gondii, and herpes simplex virus.
  • Infections may be transmitted by an iatrogenic route, referring to a transmission by medical procedures such as injection (contaminated reused needles and syringes), or transplantation of infected material, blood transfusions, or infection occurring during surgery.
  • MRSA methicillin -resistant Staphylococcus aureus
  • Infections may also be transmitted by vector-bome transmission, where a vector may be an organism transferring the infection agents from one host to another.
  • vectors may be triatomine bugs, e.g., trypanosomes, parasites, animals, arthropods including e.g. mosquitos, flies, lice, flees, tick and mites or humans.
  • mosquito-borne infections include Dengue fever, West Nile virus related infections, yellow fever and Chikungunya fever.
  • Non-limiting examples of parasite-bome diseases include malaria, Human African trypanosomiasis and Lyme disease.
  • diseases spread by humans or mammals include HIV, Ebola hemorrhagic fever and Marburg fever.
  • Medical prevention, treatment and/or management of bacterial infections may include administration of antibiotics.
  • Antibiotics may inhibit the colonization of bacteria or kill the bacteria.
  • Antibiotics include e.g., penicillins, cephalosporins, macrolides, fluoroquinolones, sulfonamides, tetracyclines, and aminoglycosides.
  • Antibiotics may be specific to a certain bacteria or act against broad spectrum of bacteria. Some types of bacteria are especially susceptible to antibiotics, whereas some bacteria are more resistant. Development of bacterial strain mutations that are resistant to antibiotics is an increasing concern.
  • Methicillin-resistant Staphylococcus aureus MRSA
  • vancomycin-resistant Enterococcus VRE
  • multi-drug- resistant Mycobacterium tuberculosis MDR-TB
  • Klebsiella pneumoniae carbapenemase- producing bacteria KPC
  • Antiviral medications may be specific to a certain bacteria or act against a broad spectrum of viruses. Currently antiviral medications are available for e.g., HIV, influenza, hepatitis B and C.
  • Medical prevention, treatment and/or management of viral infections may include administration of antifungal medication.
  • Antifungal medication kills or prevents the growth of fungi.
  • Types of antifungal medications include e.g., imidazoles, triazoles and thiazoles, allylamines, and echinocandins.
  • Development of antifungal medication capable of targeting fungal cells without affecting human cells is a challenge due to the similarities of human and fungal cell on the molecular level.
  • medical treatment is combined with good supportive care, which includes provision of fluids, bed rest, medication to relieve pain and lower fever, supportive alternative medicine such as vitamins, antioxidants and other supplements important for wellbeing of patients.
  • Antibody therapies for infectious diseases have also been developed.
  • examples of commercial therapeutic antibodies include raxibacumab (developed by Cambridge Antibody Technology and Human Genome Sciences) which is an antibody for the prophylaxis and treatment of inhaled anthrax, SHIGAMABTM (developed by Bellus Health Inc.) is a monoclonal antibody for treatment of Shiga toxin induced hemolytic uremic syndrome, and actoxumab and bezlotoxumab (developed by Medarex Inc. and the University of Massachusetts Medical School) are commercial human monoclonal antibodies targeting C. difficile toxin A and toxin B, respectively.
  • Infectious diseases and/or infection related diseases, disorders, and/or conditions that may be treated by methods, components and compositions of the present disclosure include, but are not limited to, 14-day measles, 5-day fever, acne, acquired immunodeficiency syndrome (AIDS), acrodermatitis chronica atrophicans (ACA), acute hemorrhagic conjunctivitis, acute hemorrhagic cystitis, acute rhinosinusitis, adult T-cell leukemia- lymphoma (ATLL), African sleeping sickness, alveolar hydatid, amebiasis, amebic meningoencephalitis, anaplasmosis, anthrax, arboviral, ascariasis, aseptic meningitis, Athlete's foot, Australian tick typhus, avian Influenza, babesiosis, bacillary angiomatosis, bacterial meningitis, bacterial vaginosis, balanitis, bal
  • coli eastern equine encephalitis, Ebola hemorrhagic fever, Ebola virus disease (EVD), ectothrix, ehrlichiosis, encephalitis, endemic relapsing fever, endemic syphilis, endophthalmitis, endothrix, enterobiasis, enterotoxin - B poisoning (staph food poisoning), enterovirus, epidemic keratoconjunctivitis, epidemic relapsing fever, epidemic typhus, epiglottitis, epsilon toxin, erysipelis, erysipeloid, erysipelothricosis, erythema chronicum migrans, erythema infectiosum, erythema marginatum, erythema multiforme, erythema nodosum, erythema nodosum leprosum, erythrasma, espundia,
  • the infectious disease to be treated is elicited by an infectious agent which is Campylobacter jejuni, Clostridium difficile, entamoeba histolytica, enterotoxin B, Norwalk virus or norovirus, Helicobacter pylori, rotavirus, Candida yeast, coronavirus including SARS-CoV, SARS-CoV-2 and MERS-CoV, Enterovirus 71, Epstein-Barr virus, Gram-Negative Bacteria including Bordetella, Gram-Positive Bacteria including Clostridium Tetani, Francisella Tularensis, Streptococcus bacteria and Staphylococcus bacteria, and Hepatitis, Human Cytomegalovirus, Human Immunodeficiency Virus, Human Papilloma Virus, Influenza, John Cunningham Virus, Mycobacterium, Poxviruses, Pseudomonas Aeruginosa, Respiratory Sync
  • the lipid nanoparticles comprising cargos/payloads described herein may be used to prevent disease or stabilize the progression of disease.
  • the lipid nanoparticles comprising cargos/payloads described herein may be used as a prophylactic to prevent a disease or disorder in the future.
  • the lipid nanoparticles comprising cargos/payloads described herein may be used to halt further progression of a disease or disorder.
  • the lipid nanoparticles comprising cargos/payloads described herein may be used as, and/or in a manner similar to that of a vaccine.
  • a "vaccine” is a biological preparation that improves immunity to a particular disease or infectious agent.
  • the present disclosure thus provides vaccines comprising the lipid nanoparticles as defined herein and at least one cargo/payload, wherein the cargo/payload preferably comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide.
  • the cargo/payload comprises at least one nucleic acid such as mRNA.
  • the lipid nanoparticles comprising cargos/payloads s described herein may be used as, and/or in a manner similar to that of a vaccine for a therapeutic area such as, but not limited to, cardiovascular, CNS, dermatology, endocrinology, oncology, immunology, respiratory, and anti-infective.
  • the lipid nanoparticles comprising cargos/payloads described herein may be used as a vaccine to diagnose, prevent, treat and/or manage a foodbome illness.
  • the lipid nanoparticles comprising cargos/payloads described herein may be used as a vaccine to diagnose, prevent, treat and/or manage gastroenteritis.

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Abstract

L'invention concerne un composé lipidique diester de formule (I) ou un sel pharmaceutiquement acceptable de celui-ci, le composé pouvant être utilisé pour obtenir des nanoparticules lipidiques. Dans certains modes de réalisation, la nanoparticule lipidique peut comprendre (a) d'environ 40 à environ 100% en moles du composé de formule (I) ; (b) de 0 à environ 10% en moles d'un lipide neutre ; (c) de 0 à environ 50% en moles d'un lipide auxiliaire ; (d) de 0 à environ 5% en moles d'un lipide conjugué à un polymère ; et (e) de 0 à environ 5% en moles d'un composant hydrophobe ; les % en moles étant basés sur les lipides totaux présents dans la nanoparticule. (I)
PCT/CA2024/050922 2023-07-10 2024-07-10 Lipides diester, nanoparticule lipidique contenant des lipides diester et formulations associées Pending WO2025010504A1 (fr)

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WO2023115221A1 (fr) * 2021-12-22 2023-06-29 Providence Therapeutics Holdings Inc. Lipides de disulfure ionisables et nanoparticules lipidiques dérivées de ceux-ci

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Publication number Priority date Publication date Assignee Title
WO2023115221A1 (fr) * 2021-12-22 2023-06-29 Providence Therapeutics Holdings Inc. Lipides de disulfure ionisables et nanoparticules lipidiques dérivées de ceux-ci

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* Cited by examiner, † Cited by third party
Title
HALD ALBERTSEN CAMILLA; KULKARNI JAYESH A.; WITZIGMANN DOMINIK; LIND MARIANNE; PETERSSON KARSTEN; SIMONSEN JENS B.: "The role of lipid components in lipid nanoparticles for vaccines and gene therapy", ADVANCED DRUG DELIVERY REVIEWS, ELSEVIER, AMSTERDAM , NL, vol. 188, 3 July 2022 (2022-07-03), Amsterdam , NL , XP087150008, ISSN: 0169-409X, DOI: 10.1016/j.addr.2022.114416 *
HAN XUEXIANG, ZHANG HANWEN, BUTOWSKA KAMILA, SWINGLE KELSEY L., ALAMEH MOHAMAD-GABRIEL, WEISSMAN DREW, MITCHELL MICHAEL J.: "An ionizable lipid toolbox for RNA delivery", NATURE COMMUNICATIONS, vol. 12, no. 1, 1 December 2021 (2021-12-01), XP055938542, DOI: 10.1038/s41467-021-27493-0 *

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