Lipid nanoparticles comprising coding RNA molecules for use in gene editing and as vaccines and therapeutic agents

Abstract

The present disclosure describes improved LNP-based RNA vaccines, nucleobase editing systems, and therapeutics for use in treating and / or immunization against disease. In particular, the disclosure describes improved LNPs, including novel and improved ionizable lipids for making LNPs, that enhance the targeted delivery of LNP-based RNA vaccines and therapeutics based on linear and / or circular mRNAs. The improved LNPs protect linear and / or circular mRNA payloads from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced vaccines and / or therapeutic agents.

Description

EDITING AND AS VACCINES AND THERAPEUTIC AGENTS TECHNICAL FIELD

[0001] The present disclosure generally relates to the field of nucleic acid lipid nanoparticle (LNP) compositions and delivery thereof for use as vaccines and / or therapeutics for the treatment of disease. The disclosure further relates to compositions comprising LNPs formulated with coding RNAs, including linear and / or circular mRNAs, for the delivery of encoded vaccine antigens and / or therapeutic proteins for the vaccination against infectious agents and / or treatment of disease, including infectious disease and cancer. BACKGROUND

[0002] There are many challenges associated with the delivery of nucleic acids to affect a desired response in a biological system, such as an immune response or the production of a therapeutically beneficial protein to treat a disease. Nucleic acid-based therapeutics and vaccines have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell or organism in order to realize this potential.

[0003] Nucleic acid-based therapeutics and vaccines are generally composed of DNA or RNA. DNA is known to be relatively stable and easy to handle, however, the use of DNA bears the risk of undesired insertion into a cell’s genome which potentially may produce mutagenic events. As a further concern, the delivery of DNA is associated with unwanted immunogenicity and the production of anti-DNA antibodies. Yet another concern in the use of DNA is the limited expression level of the encoded peptide or protein that is achievable due to the requirement that the administered DNA must first enter the nucleus to undergo transcription prior to translation into a desired protein product (e.g., antigen or therapeutic protein).

[0004] In contrast to DNA, the use of RNA is substantially safer because RNA does not involve the risk of being integrated into the genome of a transfected cell, thus eliminating the concern that the introduced genetic material will disrupt the normal functioning of an essential gene or cause a mutation. In addition, RNA-based agents do not require extraneous promoter sequences for effective expression of an encoded protein and are also less immunogenic than DNA-based agents, in part because RNA has a relatively short half-life unlike DNA. In addition, while DNA must enter the nuclease to perform its function, RNA performs its function outside of the nucleus and is therefore more efficient.

[0005] Despite the advantages of using RNA-based therapeutics and vaccines, the stability of RNA (e.g., mRNA) is far lower than DNA, especially when it reaches the cytoplasm of a cell and is exposed to RNA-degrading enzymes. In addition, the presence of a hydroxyl group on the second carbon of the sugar moiety in RNA causes steric hindrance that prevents the RNA from forming a more stable double helix structure like in the case of DNA, thus making RNA more prone to hydrolytic degradation than DNA.

[0006] To circumvent these challenges, delivery of RNA vaccines (e.g., mRNA vaccines) and therapeutics has recently focused on the use of lipid nanoparticles (LNPs). Indeed, LNPs have emerged as the most promising nonviral delivery vehicle for exogenous mRNA (see e.g., Guan et al., “Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems,” Gene Ther, 24 (2017), pp.133-143). The LNP is a complex nanostructured body that provides protection to payload RNA molecules encapsulated within from the harshly degrading nuclease environment in vivo while facilitating intracellular delivery. LNPs are formed through self-assembly by combining the RNA payload with several lipid components, including an ionizable lipid that plays a central role in delivery efficacy (e.g., Miao et al., “Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation,” Nat. Biotechnol., 27 (2019), pp.1174-1185). Entrapment of RNA is achieved by mixing RNA with lipids at an acidic pH at which the ionizable lipid is positively charged, thus ensuring a charge-driven interaction with the negatively charged RNA molecules (e.g., Mindy et al., “Mechanism of macromolecular structure evolution in self-assembled lipid nanoparticles for siRNA delivery," Langmuir, 20 (2014), pp.4613-4622). The pH is then adjusted to above the pKa of the ionizable lipid, which results in a near-neutral surface charge desirable for clinical administration (see Id.). In addition, the incorporation of a pegylated lipid into the mixture achieves a sterically stabilized core shell nanoparticle useful for clinical applications as vaccines and / or therapeutics.

[0007] In spite of the development of LNPs, the delivery of RNA payloads to cells in vivo in a targeted manner that also allows for sufficient levels of protein production (e.g., production of vaccine antigens or therapeutic proteins) remains an important and significant challenge.

[0008] Genome editing tools encompass a diverse set of technologies that can make many types of genomic alterations in various contexts. These technologies have evolved over the last couple of decades to provide a range of user-programmable editing tools that include ZFN (zinc finger) nuclease editing systems, meganuclease editing systems, and TALENS (transcription activator-like effector nucleases). The past decade has seen an explosive growth in a new generation of genome editing systems based on components from bacterial immune pathways, including CRISPR (clustered regularly interspaced short palindromic repeats) and the associated CRISPR-associated proteins (e.g., CRISPR-Cas9) (Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, Vol.337 (6096), pp.816-821), meganuclease editors (Boissel et al., “megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering,” Nucleic Acids Research 42: pp.2591-2601) and bacterial retron systems (Schubert et al., “High-throughput functional variant screens via in vivo production of single-stranded DNA,” PNAS, April 27, 2021, Vol.118(18), pp.1-10). In particular, CRISPR-Cas9 has been derivatized in numerous ways to expand upon its guide RNA-based programmable double-strand cutting activity to form systems ranging from finding alternative CRISPR Cas nuclease enzymes having different PAM requirements and cutting properties (e.g., engineered Cas9 proteins and other naturally-occurring Cas9 homologs,including, but not limited to, Cas12a, Cas12f, Cas13a, and Cas13b, and their engineered variants) to base editing (Komor et al., “Programmable editing of a target base in genomic DNA without double- stranded DNA cleavage,” Nature, May 19, 2016, 533 (7603); pp.420-424 [cytosine base editors or CBEs] and Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature, Vol.551, pp.464-471 [adenine base editors or ABEs]) to prime editing (Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, Dec 2019, 576 (7789): pp.149-157) to twin prime editing (Anzalone et al., “Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing,” Nature Biotechnology, Dec 9, 2021, vol.40, pp.731-740) to epigenetic editing (Kungulovski and Jeltsch, “Epigenome Editing: State of the Art, Concepts, and Perspective,” Trends in Genetics, Vol.32, 206, pp.101-113) to CRISPR-directed integrase editing (Yarnell et al., “Drag-and-drop genome insertion of large sequences without double-stranded DNA cleavage using CRISPR-directed integrases,” Nature Biotechnology, Nov 24, 2022, (“PASTE”)).

[0009] While the expansion of genome editing tools has exploded, the development of safe and effective gene editing tool delivery systems has lagged behind. There remain numerous challenges associated with the delivery of gene editing tools—including, but not limited to, CRISPR-Cas9 and alternative Cas nuclease editors, retron editors, base editors, prime editors, twin prime editors, epigenetic editors, and integrase editors—to achieve safe and effective therapeutic application of such tools in cells and patients for treating disease and / or otherwise modifying the nucleotide sequence of a target nucleic acid molecule (e.g., a gene or genome) particularly as it relates to delivery in vivo. That said, the use of lipid nanoparticles (LNPs) has emerged as a leading delivery option for the safe, effective, and targeted delivery of gene editing tools to target tissues and cells. However, there remains a need for improved LNPs, including better performing ionizable lipids, that will enhance the targeted delivery of LNP-based gene editing tools. Preferably, such improved LNPs would protect payloads from degradation and clearance while achieving targeted delivery, be suitable for systemic or local delivery, and provide delivery of RNA cargo, including those relating to a wide variety of gene editing tools, such as those mentioned above. In addition, such improved LNP-based therapeutics should exhibit low toxicity and provide an adequate therapeutic index, such that patient treatment at an effective dose of the LNP minimizes risk to the patient while maximizing therapeutic benefit.

[0010] Thus, improved LNPs that enhance the delivery of LNP-based RNA vaccines and therapeutics to cells, tissues, and bodily sites and which are more protective of RNA payloads would advance the art. Preferably, such improved LNPs would protect RNA payloads from degradation and clearance while achieving delivery, be suitable for ex vivo or in vivo delivery , and provide delivery of any target, including RNA in linear and / or circular and / or modified form.. In addition, such improved LNP-based RNA vaccines and therapeutics should exhibit low toxicity and provide an adequate therapeutic index, such that patient treatment at an effective dose of the LNP minimizes risk to thepatient while maximizes therapeutic benefit. The present disclosure provides these and related advantages. SUMMARY

[0011] Described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA medicines (e.g., vaccines and gene-editing therapeutics). In particular, described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA medicines (e.g., vaccines and gene editing therapeutics) for the delivery of one or more coding and / or non-coding RNA molecules. In various embodiments, the non-coding RNAs may comprise one or more guide RNAs relating to a gene editing system, such as one based on CRISPR- Cas9 or CRISPR-Cas12a, each of which require complexing with a guide RNA that facilitates the localizing the protein-RNA complex to a target sequence having an enzyme-specific PAM site (protospacer adjacent motif – recognized by the CRISPR enzyme) and a target nucleotide sequence (i.e., the protospacer) that is complementary to a portion of the guide RNA (i.e., to the spacer region). In other embodiments, the coding RNA may encode any protein component of LNP-based RNA medicine, such as, but limited to a virus antigen (e.g., a viral envelope spike protein), a therapeutic protein (e.g., a functional version of a defective protein), or one or more gene editing components (e.g., a programmable nuclease or other effector protein, such as a deaminase or reverse transcriptase). Further described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA medicines (e.g., vaccines and / or gene editing therapeutics) for the delivery of one or more RNA molecules, e.g., a coding RNA that codes for one or more therapeutic proteins for the prophylactic and / or therapeutic treatment of one or more diseases or a symptom thereof, or a non-coding RNA, such as, but not limited to a guide RNA for a gene editing system. In various embodiments, the RNA molecule delivered by the herein disclosed LNPs can be a linear mRNA. In other embodiments, the RNA molecule delivered by the herein disclosed LNPs can be a circular mRNA. In still other embodiments, the RNA molecule delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA. In further embodiments, the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and / or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures). In various other embodiments, the disclosure provides novel lipid components of the herein disclosed LNPs, including, but not limited to, novel ionizable lipids.

[0012] The present disclosure describes improved LNP-based RNA medicines (e.g., vaccines and therapeutics) for use in treating and / or immunization against disease. In particular, the disclosure describes improved LNPs, including better performing ionizable lipids, that enhance the targeteddelivery of LNP-based RNA vaccines and therapeutics based on linear and / or circular mRNAs. The improved LNPs protect linear and / or circular mRNA cargos (i.e., the circular and / or linear mRNA molecules encapsulated by the LNPs) from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced vaccines and / or therapeutic agents.

[0013] In an aspect of the disclosure, provided herein is a compound having a structure of Formula (TL) or a pharmaceutically acceptable salt thereof, or any lipid in Table (I) or a salt, solvate, stereoisomer, or enantiomer thereof, see below, collectively referred to as "Lipids of the Disclosure" and each individually referred to as a "Lipid of the Disclosure."

[0014] In an aspect of the disclosure, provided herein is a pharmaceutical composition comprising a compound as disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0015] In an aspect, provided herein is a pharmaceutical composition comprising: a) at least one lipid nanoparticle comprising at least one compound having a structure of any of Formula (TL) or a pharmaceutically acceptable salt thereof, or any lipid in Table (I) or a salt, solvate, stereoisomer, or enantiomer thereof; and b) at least one nucleobase editing system.

[0016] In an aspect, provided herein is a method of delivering a nucleobase editing system to a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition disclosed herein.

[0017] In an aspect of the disclosure, provided herein is a lipid nanoparticle (LNP) comprising a compound having a structure of any of Formula (TL) or a pharmaceutically acceptable salt thereof, or any lipid in Table (I) or a salt, solvate, stereoisomer, or enantiomer thereof.

[0018] In another aspect of the disclosure, provided herein is a method for delivering a nucleic acid to a cell comprising contacting the cell with a LNP disclosed herein or a pharmaceutical composition disclosed herein.

[0019] In another aspect of the disclosure, provided herein is a method for treating a disease characterized by a deficiency of a functional protein, the method comprising administering to a subject having the disease, a LNP formulation comprising a LNP disclosed herein, wherein the mRNA encodes the functional protein or a protein having the same biological activity as the functional protein.

[0020] In another aspect of the disclosure, provided herein is a method for treating a disease characterized by overexpression of a polypeptide, comprising administering to a subject having the disease a LNP formulation comprising a LNP disclosed herein and a siRNA, wherein the siRNA targets expression of the overexpressed polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG.1 is a diagram illustrating the LNP-based RNA vaccines and therapeutics disclosed herein which are encapsulated with RNA payloads (e.g., linear and / or circular mRNAs).

[0022] FIG.2 is a diagram illustrating an originator polynucleotide construct of the present disclosure which may be linear or circular. DETAILED DESCRIPTION I. Introduction

[0023] The instant specification describes compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA medicines (e.g., vaccines, gene therapies, or gene-editing therapeutics). In various embodiments, the LNP-based RNA medicines comprise an LNP delivery system (as described in detail herein) and an encapsulated cargo / payload (e.g., RNA in the case of RNA medicines).

[0024] In various embodiments and as described further herein, the LNP delivery vehicle is a complex nanostructured body that provides protection to an encapsulated RNA payload (i.e., one or more RNA molecules) environmental damage (e.g., an intracellular environment). LNPs are formed through self- assembly of multiple lipid components, including (i) an ionizable lipid (e.g., ALC-0315 as in COMIRNATY® (Pfizer-BioNTech), SM-102 as in SPIKEVAX® (Moderna), or MC3 as in ONPATTRO® (Alnylam), or those ionizable lipids described herein), (ii) a helper lipid (such as, but not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), (iii) a sterol (e.g., cholesterol), and (iv) a PEG-lipid (e.g., PEG-DSPE).

[0025] In various embodiments and as described further herein, the RNA payload in the herein described LNP-based medicines may comprise coding and / or non-coding RNA, and / or mixtures thereof. The particular RNA payload constituents will generally reflect the medicine. For example, an LNP-based vaccine or therapeutic may comprise only coding RNA for expressing a vaccine antigen or a therapeutic protein, respectively. However, an LNP-based gene editing medicine may comprise a combination of coding RNA (e.g., encoding a CRISPR nuclease) and non-coding RNAs (e.g., guide RNAs). In various embodiments, the RNA molecule delivered by the herein disclosed LNPs can be a linear mRNA. In other embodiments, the RNA molecule delivered by the herein disclosed LNPs can be a circular mRNA. In still other embodiments, the RNA molecule delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA. In further embodiments, the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and / or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures). A. LNP-Based RNA Vaccines

[0026] Described herein in certain aspects are improved LNP-based RNA vaccines for use in immunization against disease. In various aspects, the disclosure describes improved LNPs, including better performing ionizable lipids, that enhance the targeted delivery of LNP-based RNA vaccines andtherapeutics based on linear and / or circular mRNAs. The improved LNPs protect linear and / or circular mRNA cargos (i.e., the circular and / or linear mRNA molecules encapsulated by the LNPs) from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced vaccines.

[0027] The instant specification describes compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA vaccines. In particular, as described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA vaccines for the delivery of an RNA molecule that codes for one or more immunogenic viral antigens for use as vaccine and / or immunogenic compositions. In various embodiments, the RNA molecules delivered by the herein disclosed LNPs can be linear mRNA. In other embodiments, the RNA molecules delivered by the herein disclosed LNPs can be circular mRNA. In still other embodiments, the RNA molecule delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA. In further embodiments, the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and / or structural modification (e.g., secondary- folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures). In various other embodiments, the disclosure provides novel lipid components of the herein disclosed LNPs, including, but not limited to, novel ionizable lipids. B. LNP-Based RNA Therapeutics

[0028] Described herein in certain aspects are improved LNP-based RNA therapeutics for use in treating disease or a symptom thereof. In various aspects, the disclosure describes improved LNPs, including better performing ionizable lipids, that enhance the targeted delivery of LNP-based RNA therapeutics based on linear and / or circular mRNAs. The improved LNPs protect linear and / or circular mRNA cargos (i.e., the circular and / or linear mRNA molecules encapsulated by the LNPs) from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced therapeutic agents.

[0029] The instant specification describes compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA therapeutics. In particular, described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA therapeutics for the delivery of an RNA molecule that codes for one or more therapeutic proteins for use treating a disease or a symptom thereof. Further described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA therapeutics for the administration of an RNA molecule that codes for one or more therapeutic proteins for the prophylactic and / or therapeutic treatment of one or more diseases or a symptomthereof. In various embodiments, the RNA molecules delivered by the herein disclosed LNPs can be linear mRNA. In other embodiments, the RNA molecules delivered by the herein disclosed LNPs can be circular mRNA. In still other embodiments, the RNA molecules delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA. In further embodiments, the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and / or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem- loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures). In various other embodiments, the disclosure provides novel lipid components of the herein disclosed LNPs, including, but not limited to, novel ionizable lipids. C. LNP-Based Gene Editing Therapeutics

[0030] Also described herein are LNP compositions comprising gene editing systems for use in treating disease and / or otherwise modifying the sequence and / or expression of target nucleotide sequences. The disclosure provides LNPs capable of delivering a gene editing system to a target organ, tissue, and / or cell. The gene editing systems may be delivered to cells under in vitro or ex vivo conditions and to organs, tissues, or cells under in vivo conditions (e.g., administered to a subject in an effective amount).

[0031] The disclosure also provides in various aspects therapeutic or pharmaceutical compositions comprising LNPs comprising gene editing systems or one or more components thereof. The gene editing systems may comprise DNA components, RNA components, protein components, nucleoprotein components, polysaccharide components, or combinations thereof. In other aspects, the disclosure provides nucleic acid molecules (e.g., RNA or DNA) that encode and / or constitute various componentry of the deliverable gene editing systems contemplated herein. In addition, other aspects of the disclosure provide nucleic acid molecules as components of the herein contemplated gene editing systems, such as, but not limited to plasmids or vectors encoding one or more components of a gene editing system, RNAs encoding one or more components of a gene editing system (e.g., mRNAs coding for a nuclease domain of a gene editing system), and non-coding RNAs (e.g., guide RNAs capable of complexing with and targeting a nucleic acid-programmable DNA binding domain to a specific target nucleotide sequence or a retron ncRNAs).

[0032] In further embodiments, the nucleic acid components (e.g., RNA) may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and / or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures).

[0033] The disclosure, in other aspects, describes various protein components (which may be encoded by the nucleic acid components described herein) of the various gene editing systems contemplated herein, including, but not limited to, user-programmable DNA binding proteins and various effector proteins, such as nucleases, polymerases, reverse transcriptases, recombinases, integrases, endonucleases, exonucleases, transposases, and deaminases.

[0034] The disclosure also describes nucleoprotein components of the gene editing systems contemplated herein, such as, but not limited to nuclease-guide RNA complexes. The disclosure also provides methods of modifying the sequence and / or expression level of a target nucleic acid molecule through the delivery and / or administration of an LNP described herein that comprises a gene editing system or components thereof. Still further, the disclosure provides methods of treating a disease by administering a therapeutically effective amount of an LNP-based gene editing system that results in the modification in the sequence and / or expression level of a target nucleic acid molecule (e.g., a disease-associated gene or regulatory sequence, such as a promoter, transcription factor binding site, or gene enhancer site).

[0035] The gene editing systems deliverable by the herein disclosed LNPs can be any type of gene editing system. Without limitation, the gene editing systems contemplated herein can include (A) nucleobase gene editing systems which result in one or more the changes to the sequence of a target nucleic acid molecule (e.g., a gene or gene regulatory sequence) (sequence modifications may include, but are not limited to, an insertion of one or more base pairs, a deletion of one of more base pairs, a substitution or one or more base pairs, a conversion of a base pair to another base pair (e.g., a G:C pair converted to an A:T pair), an inversion, or a translocation), (B) an epigenetic editing system which results in one or more modifications to the epigenome to bring about an effect on gene expression without altering the sequence of a nucleic acid molecule, and (C) gene editing systems that combine the features of nucleobase editing systems and epigenetic editing systems (e.g., combining components from both types of systems to change the sequence and an epigenomic component with one system).

[0036] Nucleobase editing systems include a wide array of configurations with various combinations of protein functionalities and / or nucleic acid molecule components, all of which are contemplated herein. In general, nucleobase editing systems comprise at least a (i) DNA binding domain that is user-programmable to target a specific sequence in a nucleic acid molecule and optionally (ii) one or more effector domains that facilitate the modification of the sequence of the nucleic acid molecule. User-programmability may comprise amino acid sequence-programmable DNA binding domains (e.g., TALENS, zinc finger-binding domains, meganucleases (or homing endonucleases)) or nucleic acid sequence-programmable DNA binding domains or proteins (“naspDBP”) (e.g., CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas12f, CRISPR-Cas13a, CRISPR-Cas13b, or TnpB).

[0037] Similarly, epigenetic editing systems comprise at least a (i) DNA binding domain that targets a specific sequence in a nucleic acid molecule and (ii) one or more effector domains that facilitates the modification of one or more epigenomic features of the nucleic acid molecule.

[0038] Gene editing systems may comprise one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and / or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.

[0039] In addition, gene editing systems that utilize a nucleic acid sequence-programmable DNA binding domain or protein (naspDBP) may also comprise one or more non-coding nucleic acids, such as, one or more guide RNAs which complex with the nucleic acid programmable DNA binding protein (naspDBP) and target the complex to a specific nucleotide sequence. In the case of prime editing, the guide RNA may be a prime editing guide RNA (“pegRNA”) which comprises a specialized RNA template molecule that provides a template or coding sequence for a reverse transcriptase of the prime editing system. In some embodiments, the RNA template molecule may be coupled to a guide RNA as an extension arm at the 5’ or 3’ end of the guide RNA. In other embodiments, the RNA template molecule may be provided in trans as a separate molecule in a manner such that the RNA template molecule may itself become localized and associated with the target sequence and / or the gene editing system at the site of editing. In some embodiments, co- localization of an in trans RNA template molecule may be achieved with an aptamer or other RNA structure which binds to a binding partner that is coupled to, integrated with, or otherwise associated with the editing complex.

[0040] In the case of editing systems comprising a nucleic acid sequence-programmable DNA binding protein (naspDBP), such as a CRISPR-Cas9 or CRISPR-Cas12a nuclease, appropriate guides may be designed and synthesized using methods, software, and commercial sources which are well known to those having ordinary skill in the art such that guide RNAs for any given naspDBP may be obtained without undue experimentation.

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PMID: 31694550; PMCID: PMC6836449; (4) Pliatsika V, Rigoutsos I. "Off-Spotter": very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR / Cas guide RNAs. Biol Direct.2015 Jan 29;10:4. doi: 10.1186 / s13062-015- 0035-z. PMID: 25630343; PMCID: PMC4326336; (5) Hoof JB, Nødvig CS, Mortensen UH. Genome Editing: CRISPR-Cas9. Methods Mol Biol.2018;1775:119-132. doi: 10.1007 / 978-1-4939-7804-5_11. PMID: 29876814; (6) Labun K, Krause M, Torres Cleuren Y, Valen E. CRISPR Genome Editing Made Easy Through the CHOPCHOP Website. Curr Protoc.2021 Apr;1(4):e46. doi: 10.1002 / cpz1.46. PMID: 33905612; (7) Lee CM, Davis TH, Bao G. Examination of CRISPR / Cas9 design tools and the effect of target site accessibility on Cas9 activity. Exp Physiol.2018 Apr 1;103(4):456-460. doi: 10.1113 / EP086043. Epub 2017 Apr 12. PMID: 28303677; PMCID: PMC7266697; (8) Ma S, Lv J, Feng Z, Rong Z, Lin Y. Get ready for the CRISPR / Cas system: A beginner's guide to the engineering and design of guide RNAs. J Gene Med.2021 Nov;23(11):e3377. doi: 10.1002 / jgm.3377. Epub 2021 Jul 28. PMID: 34270141; (9) Hiranniramol K, Chen Y, Wang X. CRISPR / Cas9 Guide RNA Design Rules for Predicting Activity. Methods Mol Biol.2020;2115:351- 364. doi: 10.1007 / 978-1-0716-0290-4_19. PMID: 32006410; (10) Wiles MV, Qin W, Cheng AW, Wang H. CRISPR-Cas9-mediated genome editing and guide RNA design. Mamm Genome.2015 Oct;26(9-10):501-10. doi: 10.1007 / s00335-015-9565-z. Epub 2015 May 20. PMID: 25991564; PMCID: PMC4602062; (11) Creutzburg SCA, Wu WY, Mohanraju P, Swartjes T, Alkan F, Gorodkin J, Staals RHJ, van der Oost J. Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a. Nucleic Acids Res.2020 Apr 6;48(6):3228-3243. doi: 10.1093 / nar / gkz1240. PMID: 31989168; PMCID: PMC7102956; (12) Heigwer F, Boutros M. Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments. Methods Mol Biol.2021;2162:3-22. doi: 10.1007 / 978-1-0716-0687-2_1. PMID: 32926374; (13) Dronina J, Samukaite-Bubniene U, Ramanavicius A. Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review). J Nanobiotechnology.2022 Jan 21;20(1):41. doi: 10.1186 / s12951-022- 01246-7. PMID: 35062978; PMCID: PMC8777428; (14) Krysler AR, Cromwell CR, Tu T, Jovel J, Hubbard BP. Guide RNAs containing universal bases enable Cas9 / Cas12a recognition of polymorphic sequences. Nat Commun.2022 Mar 25;13(1):1617. doi: 10.1038 / s41467-022-29202-x. PMID: 35338140; PMCID: PMC8956631; (15) Shin HR, Kweon J, Kim Y. Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a. Methods Mol Biol.2021;2162:185-193. doi: 10.1007 / 978- 1-0716-0687-2_10. PMID: 32926383; (16) Schubert MS, Thommandru B, Woodley J, Turk R, Yan S, Kurgan G, McNeill MS, Rettig GR. Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair. Sci Rep.2021 Sep 30;11(1):19482. doi: 10.1038 / s41598-021-98965-y. PMID: 34593942; PMCID: PMC8484621; (17) Crone MA, MacDonald JT, Freemont PS, Siciliano V. gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells. NPJ Syst Biol Appl.2022 Sep 16;8(1):34. doi: 10.1038 / s41540-022-00241-w.PMID: 36114193; PMCID: PMC9481559; (18) Konstantakos V, Nentidis A, Krithara A, Paliouras G. CRISPR-Cas9 gRNA efficiency prediction: an overview of predictive tools and the role of deep learning. Nucleic Acids Res.2022 Apr 22;50(7):3616-3637. doi: 10.1093 / nar / gkac192. PMID: 35349718; PMCID: PMC9023298; (19) Wang J, Zhang X, Cheng L, Luo Y. An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools. RNA Biol.2020 Jan;17(1):13-22. doi: 10.1080 / 15476286.2019.1669406. Epub 2019 Sep 27. PMID: 31533522; PMCID: PMC6948960; and (20) Cram D, Kulkarni M, Buchwaldt M, Rajagopalan N, Bhowmik P, Rozwadowski K, Parkin IAP, Sharpe AG, Kagale S. WheatCRISPR: a web-based guide RNA design tool for CRISPR / Cas9-mediated genome editing in wheat. BMC Plant Biol.2019 Nov 6;19(1):474. doi: 10.1186 / s12870-019-2097-z. PMID: 31694550; PMCID: PMC6836449; each of which are incorporated herein by reference in their entireties.

[0042] In the case of prime editing, in particular, further reference may be made to the following references providing information and tools for the design, synthesis, modification, and structural configuration of pegRNAs: (1) Hsu JY, Grünewald J, Szalay R, Shih J, Anzalone AV, Lam KC, Shen MW, Petri K, Liu DR, Joung JK, Pinello L. PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat Commun.2021 Feb 15;12(1):1034. doi: 10.1038 / s41467-021-21337- 7. PMID: 33589617; PMCID: PMC7884779; (2) Li Y, Chen J, Tsai SQ, Cheng Y. Easy-Prime: a machine learning-based prime editor design tool. Genome Biol.2021 Aug 19;22(1):235. doi: 10.1186 / s13059-021-02458-0. PMID: 34412673; PMCID: PMC8377858; (3) Zhang W, Petri K, Ma J, Lee H, Tsai CL, Joung JK, Yeh JJ. Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions. bioRxiv [Preprint].2023 Aug 15:2023.08.14.553324. doi: 10.1101 / 2023.08.14.553324. PMID: 37645936; PMCID: PMC10462064; (4) Jin S, Lin Q, Gao Q, Gao C. Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs). Nat Protoc.2023 Mar;18(3):831-853. doi: 10.1038 / s41596-022-00773-9. Epub 2022 Nov 25. PMID: 36434096; (5) Lin Q, Jin S, Zong Y, Yu H, Zhu Z, Liu G, Kou L, Wang Y, Qiu JL, Li J, Gao C. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat Biotechnol.2021 Aug;39(8):923-927. doi: 10.1038 / s41587-021-00868-w. Epub 2021 Mar 25. PMID: 33767395; (6) Standage-Beier K, Tekel SJ, Brafman DA, Wang X. Prime Editing Guide RNA Design Automation Using PINE-CONE. ACS Synth Biol.2021 Feb 19;10(2):422-427. doi: 10.1021 / acssynbio.0c00445. Epub 2021 Jan 19. PMID: 33464043; PMCID: PMC7901017; (7) Zhang W, Petri K, Ma J, Lee H, Tsai CL, Joung JK, Yeh JJ. Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions. bioRxiv [Preprint].2023 Aug 15:2023.08.14.553324. doi: 10.1101 / 2023.08.14.553324. PMID: 37645936; PMCID: PMC10462064; (8) Chow RD, Chen JS, Shen J, Chen S. A web tool for the design of prime-editing guide RNAs. Nat Biomed Eng.2021 Feb;5(2):190-194. doi: 10.1038 / s41551-020-00622-8. Epub 2020 Sep 28. PMID: 32989284; PMCID: PMC7882013; each of which are incorporated herein by reference in their entireties.

[0043] Reference may also be made to the following commercial vendors which sell guide RNAs for CRISPR editing applications (including base editing and prime editing) and provide various tools and instruction for the ordering, design, synthesis, modification, and structural configuration of guide RNAs: GENSCRIPT, SYNTHEGO, TAKARA BIO, INTEGRATED DNA TECHNOLOGIES, LC SCIENCES, HORIZON DISCOVERY; SIGMA-ALDRICH; ORIGENE, and TWIST BIOSCIENCES, among others.

[0044] In addition, guide RNA may be modified with chemical modifications and / or structural modifications for enhancing various properties thereof, including specificity, stability, and limiting off-target activity. One of ordinary skill in the art will be able to modify a guide RNA with any known modification without undue experimentation. Guide modifications are discussed in the following references: (1) Ke Y, Ghalandari B, Huang S, Li S, Huang C, Zhi X, Cui D, Ding X.2'-O- Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR-Cas12a systems. Chem Sci.2022 Feb 1;13(7):2050-2061. doi: 10.1039 / d1sc06832f. PMID: 35308857; PMCID: PMC8848812; (2) Allen D, Rosenberg M, Hendel A. Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells. Front Genome Ed.2021 Jan 28;2:617910. doi: 10.3389 / fgeed.2020.617910. PMID: 34713240; PMCID: PMC8525374; (3) Basila M, Kelley ML, Smith AVB. Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity. PLoS One.2017 Nov 27;12(11):e0188593. doi: 10.1371 / journal.pone.0188593. PMID: 29176845; PMCID: PMC5703482; (4) Sakovina L, Vokhtantsev I, Vorobyeva M, Vorobyev P, Novopashina D. Improving Stability and Specificity of CRISPR / Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides. Int J Mol Sci.2022 Nov 3;23(21):13460. doi: 10.3390 / ijms232113460. PMID: 36362256; PMCID: PMC9655745; (5) Shapiro J, Tovin A, Iancu O, Allen D, Hendel A. Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells. Methods Mol Biol.2021;2162:37-48. doi: 10.1007 / 978-1-0716-0687-2_3. PMID: 32926376; (6) Filippova J, Matveeva A, Zhuravlev E, Stepanov G. Guide RNA modification as a way to improve CRISPR / Cas9-based genome-editing systems. Biochimie.2019 Dec;167:49-60. doi: 10.1016 / j.biochi.2019.09.003. Epub 2019 Sep 4. PMID: 31493470; (7) Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol.2015 Sep;33(9):985-989. doi: 10.1038 / nbt.3290. Epub 2015 Jun 29. PMID: 26121415; PMCID: PMC4729442; (8)_ Ryan DE, Taussig D, Steinfeld I, Phadnis SM, Lunstad BD, Singh M, Vuong X, Okochi KD, McCaffrey R, Olesiak M, Roy S, Yung CW, Curry B, Sampson JR, Bruhn L, Dellinger DJ. Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Res.2018 Jan 25;46(2):792-803. doi: 10.1093 / nar / gkx1199. Erratum in: Nucleic AcidsRes.2022 Mar 21;50(5):2986. PMID: 29216382; PMCID: PMC5778453; (9) Palumbo CM, Gutierrez-Bujari JM, O'Geen H, Segal DJ, Beal PA. Versatile 3' Functionalization of CRISPR Single Guide RNA. Chembiochem.2020 Jun 2;21(11):1633-1640. doi: 10.1002 / cbic.201900736. Epub 2020 Mar 5. PMID: 31943634; PMCID: PMC7323579; (10) Mullally G, van Aelst K, Naqvi MM, Diffin FM, Karvelis T, Gasiunas G, Siksnys V, Szczelkun MD.5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage. Nucleic Acids Res.2020 Jul 9;48(12):6811-6823. doi: 10.1093 / nar / gkaa477. PMID: 32496535; PMCID: PMC7337959; (12) Lu S, Zhang Y, Yin H. Chimeric DNA-RNA Guide RNA Designs. Methods Mol Biol.2021;2162:79-85. doi: 10.1007 / 978-1-0716-0687-2_6. PMID: 32926379; each of which are incorporated by reference herein in their entireties.

[0045] In the specific case of prime editing, pegRNAs may be modified with chemical modifications and / or structural modifications for enhancing various properties thereof, including specificity, stability, and limiting off-target activity. One of ordinary skill in the art will be able to modify a pegRNA for prime editing with any known modification without undue experimentation. pegRNA modifications are discussed in the following references: (1) Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, An M, Newby GA, Chen JC, Hsu A, Liu DR. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol.2022 Mar;40(3):402-410. doi: 10.1038 / s41587-021-01039-7. Epub 2021 Oct 4. Erratum in: Nat Biotechnol.2021 Dec 8;: PMID: 34608327; PMCID: PMC8930418; (2) Liu B, Dong X, Cheng H, Zheng C, Chen Z, Rodríguez TC, Liang SQ, Xue W, Sontheimer EJ. A split prime editor with untethered reverse transcriptase and circular RNA template. Nat Biotechnol.2022 Sep;40(9):1388-1393. doi: 10.1038 / s41587-022-01255- 9. Epub 2022 Apr 4. PMID: 35379962; each of which are incorporated by reference herein in their entireties.

[0046] Other specialized guide RNAs may be included depending upon the requirements and / or nature of the gene editing system and the cognate nucleic acid programmable proteins. For example, TnpB enzymes require a specialized guide RNA referred to as reRNA. Also, guide RNAs have different characteristics (e.g., PAM preferences, the spacer length, and the scaffold portion that binds to the nuclease protein) depending upon the programmable nuclease requirements.

[0047] The gene editing systems contemplated here may introduce a wide variety of changes, including (A) a change in the sequence of the target nucleic acid molecule, such as, but not limited to, (i) a nucleobase substitution (e.g., a purine to a pyrimidine), (ii) a deletion of one or more nucleobases, (iii) an insertion of one or more nucleobases, (iv) a combination of a deletion and insertion of one or more nucleobases, (v) an inversion of a nucleobase sequence, a (vi) translocation of a nucleobase sequence, and (vii) a combination or two or more such modifications, and (B) one or more modifications to the epigenome to bring about an effect on gene expression without altering the sequence of a nucleic acid molecule wherein said epigenetic change results in altered gene expression through altered chromatin structure or accessibility.

[0048] The LNP compositions and / or gene editing systems described herein may include a variety of coding RNA molecules that code for the various components of gene editors. In various aspects, the coding RNA may be linear mRNA. In other embodiments, the coding RNA may be circular mRNA. In various aspects, the improved LNPs protect linear and / or circular mRNA cargos from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced gene editing platforms and / or therapeutic agents.

[0049] In various other aspects, the LNP compositions and / or gene editing systems described herein may also include a repair template, e.g., an homology-directed repair (HDR)-dependent repair template (or HDR template). Such HDR templates are well-known in the art and can include single- strand or double-stranded DNA (e.g., oligos) or RNA. Further information regarding HDR and HDR templates for use in editing systems for various applications, such as gene knock-in, may be found in Fu YW, Dai XY, Wang WT, Yang ZX, Zhao JJ, Zhang JP, Wen W, Zhang F, Oberg KC, Zhang L, Cheng T, Zhang XB. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res.2021 Jan 25;49(2):969-985. doi: 10.1093 / nar / gkaa1251. PMID: 33398341; PMCID: PMC7826255; Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LJ, Lee J, Liu P, Luk K, Mintzer E, Guo D, Soares de Brito J, Emerson CP Jr, Zamore PD, Sontheimer EJ, Wolfe SA. Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors. CRISPR J.2022 Oct;5(5):685-701. doi: 10.1089 / crispr.2022.0058. Epub 2022 Sep 7. PMID: 36070530; PMCID: PMC9595650; and Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol.2016 Mar;34(3):339-44. doi: 10.1038 / nbt.3481. Epub 2016 Jan 20. PMID: 26789497, each of which are incorporated herein by reference in their entireties.

[0050] Accordingly, the instant specification describes compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based gene editing systems as therapeutic compositions. Further described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based gene editing therapeutics for the prophylactic and / or therapeutic treatment of one or more diseases or a symptom thereof. The components capable of being encapsulated by or otherwise incorporated by the LNPs described herein may be referred to as LNP “payloads” and may include all of the biological materials described above, including DNA molecules, RNA molecules (coding and / or non-coding), proteins, and nucleoproteins (e.g., Cas / guide RNA complexes). II. LNP delivery systems

[0051] The RNA payloads (e.g., linear and circular mRNAs) described herein may be encapsulated and delivered by lipid nanoparticles (LNPs) and compositions and / or formulations comprising RNA- encapsulated LNPs.

[0052] Below describes LNPs that may be used as the RNA payload delivery vehicles contemplated herein, as well as the various ionizable lipids, structural lipids, PEGylated lipids, and phospholipids that may be used to make the herein LNPs for delivery RNA payloads to cells. In addition, below describes additional LNP components that are contemplated, such as targeting moieties and other lipid components. A. Lipid Nanoparticle Compositions

[0053] In one aspect, the present disclosure further provides delivery systems for delivery of a therapeutic payload (e.g., the RNA payloads described herein which may encode a polypeptid of interest, e.g., an antigen or a therapeutic protein) disclosed herein. In some embodiments, a delivery system suitable for delivery of the therapeutic payload disclosed herein comprises a lipid nanoparticle (LNP) formulation.

[0054] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid. In alternative embodiments, an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid. In some embodiments, an LNP further comprises a 5th lipid, besides any of the aforementioned lipid components. In some embodiments, the LNP encapsulates one or more elements of the active agent of the present disclosure. In some embodiments, an LNP further comprises a targeting moiety covalently or non-covalently bound to the outer surface of the LNP. In some embodiments, the targeting moiety is a targeting moiety that binds to, or otherwise facilitates uptake by, cells of a particular organ system.

[0055] In some embodiments, an LNP has a diameter of at least about 20nm, 30 nm, 40nm, 50nm, 60nm, 70nm, 80nm, or 90nm. In some embodiments, an LNP has a diameter of less than about 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, or 160nm. In some embodiments, an LNP has a diameter of less than about 120 nm. In some embodiments, an LNP has a diameter of less than about 100nm. In some embodiments, an LNP has a diameter of less than about 90nm. In some embodiments, an LNP has a diameter of less than about 80nm. In some embodiments, an LNP has a diameter of about 60-100nm. In some embodiments, an LNP has a diameter of about 50-120nm. In some embodiments, an LNP has a diameter of about 75-80nm.

[0056] In some embodiments, the lipid nanoparticle compositions of the present disclosure are described according to the respective molar ratios of the component lipids in the formulation. As a non-limiting example, the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol- %. As a non-limiting example, the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 40 mol-%. As a non-limiting example, the mol-% of the ionizablelipid may be from about 25 mol-% to about 35 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 10 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 15 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 20 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 25 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 30 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 33 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 35 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 40 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 45 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 55 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 60 mol-%.

[0057] In some embodiments, the mol-% of the phospholipid may be from about 1 mol-% to about 50 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 3 mol-% to about 40 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol- % to about 30 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid is from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the phospholipid is from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the phospholipid is from about 35 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid is about 10 mol-%. In some embodiments, the mol-% of the phospholipid is about 15 mol-%. In some embodiments, the mol-% of the phospholipid is about 20 mol-%. In some embodiments, the mol-% of the phospholipid is about 25 mol-%. In some embodiments, the mol-% of the phospholipid is about 30 mol-%. In some embodiments, the mol-% of the phospholipid is about 35 mol-%. In some embodiments, the mol-% of the phospholipid is about 40 mol-%. In some embodiments, the mol-% of the phospholipid is about 45 mol-%. In some embodiments, the mol-% of the phospholipid is about 55 mol-%. In some embodiments, the mol-% of the phospholipid is about 60 mol-%.

[0058] In some embodiments, the mol-% of the phospholipid as described above comprises two or more phospholipids at an individual mol-% that totals to an aforementioned amount. In certain embodiments, the mol-% of the phospholipid is about 20 mol-% each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 15 mol-% each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 25 mol-% each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 30 mol-% each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 15 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 30 mol-% of a first phospholipid and about 10 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 25 mol-% of a first phospholipid and about 10mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 25 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 15 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid.

[0059] In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol- % to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%.

[0060] In some embodiments, the mol-% of the PEG lipid may be from about 0.1 mol-% to about 10 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 2.5 mol-%.

[0061] Where reference is made above to “mol-%” or “mol %”, the amount of the noted LNP component is intended to be the mol% of the specific component as compared to the total lipid component content of the lipid nanoparticle. i. Ionizable lipids

[0062] In some embodiments, an LNP disclosed herein comprises an ionizable lipid. In some embodiments, an LNP comprises two or more ionizable lipids.

[0063] Described below are a number of exemplary ionizable Lipids of the Disclosure. Formula (TL’’)

[0064] The present disclosure, in some embodiments, provides compounds of Formula (TL’’)or a pharmaceutically acceptable salt thereof, wherein: A1is selected from CH and N; A2is selected from CH and N;L1is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; , ,, wherein the bond marked with an "*" is attached to L1;of -O-, -NR-, and -S-; Z3is selected from the group consisting of a bond, -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -NRC(O)-, - C(O)NR-,-NRC(O)O-, or -OC(O)NR-; each Z4is independently selected from =CR- and =N-; L2is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; R1is selected from the group consisting of -OH, -OAc, -NR2,,a or RZis NR2or OH; X1and XAare each independently a bond or optionally substituted C1-C6 aliphatic; Y1is selected from the group consisting of or, and bond; wherein the bond marked with an "*" is attached to X1;and X6is independently a bond or optionally substituted C1-C12 aliphatic; each of Y2, Y3, and Y4is independently selected from the group consisting of or; wherein the bond marked with an "*" is attached to X2, X3, of X6, as appropriate;and X7is independently optionally substituted C1-C6 aliphatic;R2is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14aliphatic-R10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R3is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14aliphatic-R11, wherein one or more methylene linkages of R3are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R12is -CH(OR13)(OR14), -CH(SR13)(SR14), -CH(R13)(R14), -R15, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R15, wherein one or more methylene linkages of R12are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R6and R7are each independently optionally substituted -C1-C14aliphatic, -R10, or optionally substituted -C1-C14aliphatic-R10; wherein one or more methylene linkages of R6and R7are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R8and R9are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R13and R14are each independently optionally substituted -C1-C14aliphatic, -R15, or optionally substituted -C1-C14aliphatic-R15; wherein one or more methylene linkages of R13and R14are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each of R10, R11, and R15is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl. Formula (TL’)

[0065] The present disclosure, in some embodiments, provides compounds of Formula (TL’)or aA1is selected from CH and N; A2is selected from CH and N; L1is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; , ,, wherein the bond marked with an "*" is attached to L1;of -O-, -NR-, and -S-; Z3is selected from the group consisting of a bond, -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -NRC(O)-, - C(O)NR-,-NRC(O)O-, or -OC(O)NR-;each Z4is independently selected from =CR- and =N-; L2is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; R1is selected from the group consisting of -OH, -OAc, -NR2, ,XZis a bond or optionally substituted C1-C6aliphatic; RZis NR2or OH; X1and XAare each independently a bond or optionally substituted C1-C6aliphatic; Y1is selected from the group consisting of or, and bond; wherein the bond marked with an "*" is attached to X1;and X6is independently a bond or optionally substituted C1-C12 aliphatic; each of Y2, Y3, and Y4is independently selected from the group consisting ofor; an as and X7is independently optionally substituted C1-C6aliphatic;- (OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14aliphatic-R10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R3is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14aliphatic-R11, wherein one or more methylene linkages of R3are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R12is -CH(OR13)(OR14), -CH(SR13)(SR14), -CH(R13)(R14), -R15, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14 aliphatic-R15, wherein one or more methylene linkages of R12are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R6and R7are each independently optionally substituted -C1-C14aliphatic, -R10, or optionally substituted -C1-C14aliphatic-R10; wherein one or more methylene linkages of R6and R7are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R8and R9are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R13and R14are each independently optionally substituted -C1-C14aliphatic, -R15, or optionally substituted -C1-C14aliphatic-R15; wherein one or more methylene linkages of R13and R14are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each of R10, R11, and R15is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.Formula (TL)

[0066] The present disclosure, in some embodiments, provides compounds of Formula (TL)or a A1is selected from CH and N; A2is selected from CH and N; L1is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; Z1is selected from the group consisting of , ,, wherein the bond marked with an "*" is attached to L1;of -O-, -NR-, and -S-; Z3is selected from the group consisting of a bond, -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -NRC(O)-, - C(O)NR-,-NRC(O)O-, or -OC(O)NR-;each Z4is independently selected from =CR- and =N-; L2is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; R1is selected from the group consisting of -OH, -OAc, -NR2, ,XZis a bond or optionally substituted C1-C6aliphatic; RZis NR2or OH; X1and XAare each independently a bond or optionally substituted C1-C6aliphatic; Y1is selected from the group consisting of or, and bond; wherein the bond marked with an "*" is attached to X1;and X6is independently a bond or optionally substituted C1-C12 aliphatic; each of Y2, Y3, and Y4is independently selected from the group consisting ofor; an as and X7is independently optionally substituted C1-C6aliphatic;- (OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14aliphatic-R10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R3is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14aliphatic-R11, wherein one or more methylene linkages of R3are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R12is -CH(OR13)(OR14), -CH(SR13)(SR14), -CH(R13)(R14), -R15, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14 aliphatic-R15, wherein one or more methylene linkages of R12are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R6and R7are each independently optionally substituted -C1-C14aliphatic, -R10, or optionally substituted -C1-C14aliphatic-R10; wherein one or more methylene linkages of R6and R7are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R8and R9are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R13and R14are each independently optionally substituted -C1-C14aliphatic, -R15, or optionally substituted -C1-C14aliphatic-R15; wherein one or more methylene linkages of R13and R14are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each of R10, R11, and R15is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.Formula (TL-A)

[0067] The present disclosure, in some embodiments, provides compounds of Formula (TL-A) or a pharmaceuticallyZ1, X2, X3, X4, X5, Y2, Y3, Y4, R2, R3, R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15are as described in Formula (TL) or as otherwise described in any embodiments below. Formula (TL-B)

[0068] The present disclosure, in some embodiments, provides compounds of Formula (TL-B)or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, L1, L2, X2, X3, X4, X5, Y2, Y3, Y4, R2, R3, R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15are as described in Formula (TL) or as otherwise described in any embodiments below. Formula (TL-B’)

[0069] The present disclosure, in some embodiments, provides compounds of Formula (TL-B’)or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, L1, L2, X2, X3, X4, X5, Y2, Y3, Y4, R2, R3, R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15are as described in Formula (TL) or as otherwise described in any embodiments below. Formula (TL-C)

[0070] The present disclosure, in some embodiments, provides compounds of Formula (TL-C)or a pharmaceuticallyX2, X3, X4, X5, Y2, Y3, Y4, R2, R3, R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15are as described in Formula (TL) or as otherwise described in any embodiments below.Formula (TL-D)

[0071] The present disclosure, in some embodiments, provides compounds of Formula (TL-D)or a pharmaceutically X4, X5, Y2, Y3, Y4, R2, R3, R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15are as described in Formula (TL) or asdescribed in any embodiments below. Formulae (TL-E), (TL-E’), and (TL-E’’)

[0072] The present disclosure, in some embodiments, provides compounds of Formula (TL-E), (TL- E’), and (TL-E’’) E),, , or a Y2, Y3, Y4, R6, R7, 89 10R , R , R , are as or as described inembodiments below. Formulae (TL-E-1), (TL-E-2), (TL-E’-1), (TL-E’-2), (TL-E’’-1), and (TL-E’’-2)

[0073] The present disclosure, in some embodiments, provides compounds of Formula (TL-E-1), (TL- E-2), (TL-E’-1), (TL-E’-2), (TL-E’’-1), and (TL-E’’-2) 1), 2),, , 1), 2), or aX5, Y2, Y3, R6, R7, R8, R9, R10, R11, R13, R14, and R15are as described in Formula (TL) or as otherwise described in any embodiments below.Formulae (TL-F), (TL-F’), and (TL-F’’)

[0074] The present disclosure, in some embodiments, provides compounds of Formula (TL-F), (TL- F’), and (TL-F’’) , , or a2 3 4 6 7Y , Y , Y , R , R , R8, R9, R10, R11, R13, R14, and R15are as described in Formula (TL) or as otherwise described in any embodiments below. Formulae (TL-F-1), (TL-F-2), (TL-F’-1), (TL-F’-2), (TL-F’’-1), and (TL-F’’-2)

[0075] The present disclosure, in some embodiments, provides compounds of Formula (TL-F-1), (TL- F-2), (TL-F’-1), (TL-F’-2), (TL-F’’-1), and (TL-F’’-2), , 1), 2),1), 2), or a X5, Y2, Y3, R6, R7, R8, 910R , R , R11, R13, are as or as in any embodiments below. Formulae (TL-G), (TL-G’), and (TL-G’’)

[0076] The present disclosure, in some embodiments, provides compounds of Formula (TL-G), (TL- G’), and (TL-G’’) , ,, or a Y2, Y3, Y4, R6, R7, R8, R9, R10, R11,described in any embodiments below. Formulae (TL-H), (TL-H’), and (TL-H’’)

[0077] The present disclosure, in some embodiments, provides compounds of Formula (TL-H), (TL- H’), and (TL-H’’) , ,or a pharmaceutically acceptable salt thereof, wherein R, X1, L1, L2, X2, X3, X4, X5, Y2, Y3, Y4, R6, R7, R8, R9, R10, R11, R13, R14, and R15are as described in Formula (TL) or as otherwise described in any embodiments below.

[0078] In certain embodiments, the compound is of Formula (TL), (TL-A), (TL-B), (TL-B’), (TL-C), (TL-D), (TL-E), (TL-E’), (TL-E’’), (TL-F), (TL-F’), or (TL-F’’) wherein Y2, Y3, and Y4are each . the compound is of Formula (TL), (TL-A), (TL-B), (TL-B’), (TL-C),, , , (TL-E’’), (TL-F), (TL-F’), (TL-F’’), (TL-G), (TL-G’), (TL-G’’), (TL-H), (TL-H’), and (TL-H’’) wherein Y2, Y3, and Y4are . A1and A2

[0080] As disclosed in Formula (TL), in certain CH and N, and A2is selected from CH and N.

[0081] In certain embodiments, A1is CH. In certain embodiments, A1is N. In certain embodiments, A2is CH. In certain embodiments, A2is N. In certain embodiments, A1and A2are both CH. In certain embodiments, A1and A2are both N. In certain embodiments, A1is CH and A2is N. In certain embodiments, A1is N and A2is CH. L1

[0082] As disclosed in Formula (TL), in certain embodiments, L1is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-. In certain embodiments, L1is unsubstituted C1-C8 aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-. In certain embodiments, L1is unsubstituted C1-C8aliphatic, wherein one methylene linkage of L1is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- , or -C(O)O-. In certain embodiments, L1is unsubstituted C1-C8aliphatic, wherein one methylene linkage of L1is replaced with -C(O)-. In certain embodiments, L1is unsubstituted C1-C8 aliphatic. In certain embodiments, L1is unsubstituted C1-C6 aliphatic. In certain embodiments, L1is optionally substituted C1-C8 alkylene. In certain embodiments, L1is optionally substituted C1-C6 alkylene. In certain embodiments, L1is unsubstituted C1-C8alkylene. In certain embodiments, L1is unsubstituted C1-C6alkylene. In certain embodiments, L1is optionally substituted C1-C8alkylene, wherein one methylene linkage of L1is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- , or -C(O)O-. In certain embodiments, L1is optionally substituted C1-C8 alkylene, wherein one methylene linkage of L1is replaced with -C(O)-. In certain embodiments, L1is optionally substituted C1-C6 alkylene, wherein one methylene linkage of L1is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-. In certain embodiments, L1is optionally substituted C1- C6alkylene, wherein one methylene linkage of L1is replaced with -C(O)-. In certain embodiments, L1is optionally substituted C1-C4 alkylene. In certain embodiments, L1is unsubstituted C1-C4 alkylene. In certain embodiments, L1is optionally substituted C1-C4 alkylene, wherein one methylene linkage of L1is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or - C(O)O-. In certain embodiments, L1is optionally substituted C1-C4alkylene, wherein one methylene linkage of L1is replaced with -C(O)-. In certain embodiments, L1is optionally substituted C1-C2alkylene. In certain embodiments, L1is unsubstituted C1-C2alkylene. In certain embodiments, L1is unsubstituted C2-C6 alkylene. In certain embodiments, L1is optionally substituted methylene. In certain embodiments, L1is optionally substituted C2 alkylene. In certain embodiments, L1is optionally substituted C3 alkylene. In certain embodiments, L1is optionally substituted C4 alkylene. In certain embodiments, L1is optionally substituted C5 alkylene. In certain embodiments, L1is optionally substituted C6alkylene. In certain embodiments, L1is –(CH2)-. In certain embodiments, L1is –(CH2)2-. In certain embodiments, L1is –(CH2)3-. In certain embodiments, L1is –(CH2)4-. In certain embodiments, L1is –(CH2)5-. In certain embodiments, L1is –(CH2)6-.

[0083] In certain embodiments, L1is selected ,.from the group consisting of , ,, wherein the bond marked with an "*" is attached to L1; of -O-, -NR-, and -S-;of a bond, -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -NRC(O)-, - C(O)NR-, -NRC(O)O-, or -OC(O)NR-; each Z4is independently selected from =CR- and =N-;

[0086] In certain embodiments Z1is wherein the bond marked with an "*" is

[0087] In some embodiments, Z1 is selected ,wherein the bond marked with an "*" is

[0088] In certain embodiments Z1. In certain embodiments Z1. In certainembodiments Z1is . In certain embodiments Z1.

[0089] In someis -O-. In somesome embodiments, Z2is -S-. In some embodiments, Z3is a bond. In some embodiments, Z3is -O-. In some embodiments, Z3is -NR-. In some embodiments, Z3is -S-. In some embodiments, Z3is -OC(O)-. In some embodiments, Z3is -C(O)O-. In some embodiments, Z3is -NRC(O)-. In some embodiments, Z3is - C(O)NR-. In some embodiments, Z3is -NRC(O)O-. In some embodiments, Z3is -OC(O)NR-. In some embodiments, each Z4is =CR-. In some embodiments, one Z4is =N- and the remaining Z4are =CR-.

[0090] In some embodiments, Z1is . In some embodiments, Z1is . Insome embodiments, Z1 . In some embodiments, Z1 is..Inis

[0091] In some embodiments, Z1is selected ,**, wherein the bond marked with an "*" is attached to L1.L2

[0093] As disclosed in Formula (TL), in certain embodiments, L2is optionally substituted C1-C8 aliphatic, wherein one or more methylene linkages of L2are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-. In certain embodiments, L2is unsubstituted C1-C8aliphatic, wherein one or more methylene linkages of L2areeach optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-. In certain embodiments, L2is unsubstituted C1-C8aliphatic, wherein one methylene linkage of L2is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- , or -C(O)O-. In certain embodiments, L2is unsubstituted C1-C8 aliphatic, wherein one methylene linkage of L2is replaced with -C(O)-. In certain embodiments, L2is unsubstituted C1-C8 aliphatic. In certain embodiments, L2is unsubstituted C1-C6aliphatic. In certain embodiments, L2is optionally substituted C1-C8alkylene. In certain embodiments, L2is optionally substituted C1-C6alkylene. In certain embodiments, L2is unsubstituted C1-C8alkylene. In certain embodiments, L2is unsubstituted C1-C6 alkylene. In certain embodiments, L2is optionally substituted C1-C8 alkylene, wherein one methylene linkage of L2is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- , or -C(O)O-. In certain embodiments, L2is optionally substituted C1-C8 alkylene, wherein one methylene linkage of L2is replaced with -C(O)-. In certain embodiments, L2is optionally substituted C1-C6alkylene, wherein one methylene linkage of L2is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, - OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-. In certain embodiments, L2is optionally substituted C1- C6alkylene, wherein one methylene linkage of L2is replaced with -C(O)-. In certain embodiments, L2is optionally substituted C1-C4 alkylene. In certain embodiments, L2is unsubstituted C1-C4 alkylene. In certain embodiments, L2is optionally substituted C1-C4 alkylene, wherein one methylene linkage of L2is replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or - C(O)O-. In certain embodiments, L2is optionally substituted C1-C4alkylene, wherein one methylene linkage of L2is replaced with -C(O)-. In certain embodiments, L2is optionally substituted C1-C2alkylene. In certain embodiments, L2is unsubstituted C1-C2alkylene. In certain embodiments, L2is unsubstituted C2-C6alkylene. In certain embodiments, L2is optionally substituted methylene. In certain embodiments, L2is optionally substituted C2 alkylene. In certain embodiments, L2is optionally substituted C3 alkylene. In certain embodiments, L2is optionally substituted C4 alkylene. In certain embodiments, L2is optionally substituted C5 alkylene. In certain embodiments, L2is optionally substituted C6alkylene. In certain embodiments, L2is –(CH2)-. In certain embodiments, L2is –(CH2)2-. In certain embodiments, L2is –(CH2)3-. In certain embodiments, L2is –(CH2)4-. In certain embodiments, L2is –(CH2)5-. In certain embodiments, L2is –(CH2)6-.

[0094] In certain embodiments, L2is selected ,

[0095] As disclosed in Formula (TL), in certain embodiments, Y1isor, or a an ,,or. In certain embodiments, Y1isY1is. In certain embodiments, Y1. In certain embodiments, Y1. In certain embodiments, Y1is a bond.X1

[0096] As disclosed in Formula (TL), in certain embodiments, X1is a bond or optionally substituted C1-C6 aliphatic. In certain embodiments, X1is a bond. In certain embodiments, X1is a bond or unsubstituted C1-C6aliphatic. In certain embodiments, X1is unsubstituted C1-C6aliphatic. In certain embodiments, X1is a bond or optionally substituted C1-C6alkylene. In certain embodiments, X1is optionally substituted C1-C6alkylene. In certain embodiments, X1is a bond or unsubstituted C1-C6alkylene. In certain embodiments, X1is unsubstituted C1-C6 alkylene. In certain embodiments, X1is a bond or unsubstituted C1-C4 alkylene. In certain embodiments, X1is unsubstituted C1-C4 alkylene. In certain embodiments, X1is a bond or unsubstituted C1-C2 alkylene. In certain embodiments, X1is unsubstituted C1-C2 alkylene. In certain embodiments, X1is unsubstituted C2-C6 alkylene. In certain embodiments, X1is optionally substituted methylene. In certain embodiments, R2is optionally substituted C2alkylene. In certain embodiments, X1is optionally substituted C3alkylene. In certainembodiments, X1is optionally substituted C4alkylene. In certain embodiments, X1is optionally substituted C5alkylene. In certain embodiments, X1is optionally substituted C6alkylene. In certain embodiments, X1is –(CH2)-. In certain embodiments, X1is –(CH2)2-. In certain embodiments, X1is – (CH2)3-. In certain embodiments, X1is –(CH2)4-. In certain embodiments, X1is –(CH2)5-. In certain embodiments, X1is –(CH2)6-. XA

[0097] As disclosed in Formula (TL), in certain embodiments XAis a bond or optionally substituted C1-C6aliphatic. In certain embodiments, XAis a bond. In certain embodiments, XAis a bond or unsubstituted C1-C6 aliphatic. In certain embodiments, XAis unsubstituted C1-C6 aliphatic. In certain embodiments, XAis a bond or optionally substituted C1-C6 alkylene. In certain embodiments, XAis optionally substituted C1-C6 alkylene. In certain embodiments, XAis a bond or unsubstituted C1-C6 alkylene. In certain embodiments, XAis unsubstituted C1-C6 alkylene. In certain embodiments, XAis a bond or unsubstituted C1-C4alkylene. In certain embodiments, XAis unsubstituted C1-C4alkylene. In certain embodiments, XAis a bond or unsubstituted C1-C2alkylene. In certain embodiments, XAis unsubstituted C1-C2alkylene. In certain embodiments, XAis unsubstituted C2-C6alkylene. In certain embodiments, XAis optionally substituted methylene. In certain embodiments, R2is optionally substituted C2 alkylene. In certain embodiments, XAis optionally substituted C3 alkylene. In certain embodiments, XAis optionally substituted C4 alkylene. In certain embodiments, XAis optionally substituted C5alkylene. In certain embodiments, XAis optionally substituted C6alkylene. In certain embodiments, XAis –(CH2)-. In certain embodiments, XAis –(CH2)2-. In certain embodiments, XAis –(CH2)3-. In certain embodiments, XAis –(CH2)4-. In certain embodiments, XAis –(CH2)5-. In certain embodiments, XAis –(CH2)6-. R1

[0098] As disclosed in Formula (TL), in certain embodiments R1is selected from -OH, -OAc, -NR2, ,; wherein XZ is a bond or optionally substituted

[0099] In certain orIn certain embodiments, R1is -NR2. In certain- - embodiments, R1is -NEt2. In certain n certain embodiments, R1is . Incertain embodiments, R1is . In certain embodiments,certain embodiments, R1In certain . Incertaincertaincertaincertaincertain, ,.- embodiments, R is or C1-C6alkyl. In certain embodiments, R is C1-C4alkyl. In certain embodiments, R is C1-C2 alkyl. In certain embodiments, R is unsubstituted C1-C6 alkyl. In certain embodiments, R is unsubstituted C1-C4 alkyl. In certain embodiments, R is unsubstituted C1-C2 alkyl. In certain embodiments, R is methyl or ethyl. In certain embodiments, R is methyl. In certain embodiments, R is ethyl. X2, X3and X6

[0102] As disclosed in Formula (TL), in certain embodiments, X2, X3and X6are each independently a bond or optionally substituted C1-C12aliphatic. In certain embodiments, X2and X3are the same. In certain embodiments, X2and X3are different. In certain embodiments, X2, X3and X6are the same. In certain embodiments, X2, X3and X6are each different from each other. In certain embodiments, X2and X3are the same, and X6is different from the other two.

[0103] In certain embodiments, X2is a bond. In certain embodiments, X2is an optionally substituted C1-C12alkylene. In certain embodiments, X2is an optionally substituted C1-C12alkenylene. In certain embodiments, X2is an optionally substituted C1-C10aliphatic. In certain embodiments, X2is an optionally substituted C1-C10alkylene. In certain embodiments, X2is an optionally substituted C1-C10alkenylene. In certain embodiments, X2is an optionally substituted C1-C8 aliphatic. In certain embodiments, X2is an optionally substituted C1-C8 alkylene. In certain embodiments, X2is an optionally substituted C1-C8 alkenylene. In certain embodiments, X2is an optionally substituted C1-C6 aliphatic. In certain embodiments, X2is an optionally substituted C1-C6alkylene. In certain embodiments, X2is an optionally substituted C1-C6alkenylene. In certain embodiments, X2is an optionally substituted C2-C12aliphatic. In certain embodiments, X2is an optionally substituted C2-C12alkylene. In certain embodiments, X2is an optionally substituted C2-C12 alkenylene. In certain embodiments, X2is an optionally substituted C4-C12 aliphatic. In certain embodiments, X2is an optionally substituted C4-C12 alkylene. In certain embodiments, X2is an optionally substituted C4-C12 alkenylene. In certain embodiments, X2is an optionally substituted C4-C10 aliphatic. In certain embodiments, X2is an optionally substituted C4-C10alkylene. In certain embodiments, X2is an optionally substituted C4-C10alkenylene. In certain embodiments, X2is an optionally substituted C6- C8aliphatic. In certain embodiments, X2is an optionally substituted C6-C8alkylene. In certainembodiments, X2is an optionally substituted C6-C8alkenylene. In certain embodiments, X2is an optionally substituted C7-C9aliphatic. In certain embodiments, X2is an optionally substituted C7-C9alkylene. In certain embodiments, X2is an optionally substituted C7-C9 alkenylene. In certain embodiments, X2is a bond or an optionally substituted C1-C4 aliphatic. In certain embodiments, X2an optionally substituted C1-C4 alkylene. In certain embodiments, X2is an unsubstituted C1-C4 aliphatic. In certain embodiments, X2is an unsubstituted C1-C4alkylene. In certain embodiments, X2is an optionally substituted C1-C2aliphatic. In certain embodiments, X2is an optionally substituted C1-C2alkylene. In certain embodiments, X2is an unsubstituted C1-C2aliphatic. In certain embodiments, X2is an unsubstituted C1-C2 alkylene. In certain embodiments, X2is –(CH2)-. In certain embodiments, X2is –(CH2)2-. In certain embodiments, X2is –(CH2)3-. In certain embodiments, X2is –(CH2)4-. In certain embodiments, X2is –(CH2)5-. In certain embodiments, X2is –(CH2)6-. In certain embodiments, X2is –(CH2)7-. In certain embodiments, X2is –(CH2)8-. In certain embodiments, X2is –(CH2)9-. In certain embodiments, X2is –(CH2)10-. In certain embodiments, X3is –(CH2)11-. In certain embodiments, X3is –(CH2)12-.

[0104] In certain embodiments, X3is a bond. In certain embodiments, X3is an optionally substituted C1-C12 alkylene. In certain embodiments, X3is an optionally substituted C1-C12 alkenylene. In certain embodiments, X3is an optionally substituted C1-C10 aliphatic. In certain embodiments, X3is an optionally substituted C1-C10 alkylene. In certain embodiments, X3is an optionally substituted C1-C10 alkenylene. In certain embodiments, X3is an optionally substituted C1-C8aliphatic. In certain embodiments, X3is an optionally substituted C1-C8alkylene. In certain embodiments, X3is an optionally substituted C1-C8alkenylene. In certain embodiments, X3is an optionally substituted C1-C6aliphatic. In certain embodiments, X3is an optionally substituted C1-C6alkylene. In certain embodiments, X3is an optionally substituted C1-C6 alkenylene. In certain embodiments, X3is an optionally substituted C2-C12 aliphatic. In certain embodiments, X3is an optionally substituted C2-C12 alkylene. In certain embodiments, X3is an optionally substituted C2-C12 alkenylene. In certain embodiments, X3is an optionally substituted C4-C12aliphatic. In certain embodiments, X3is an optionally substituted C4-C12alkylene. In certain embodiments, X3is an optionally substituted C4-C12alkenylene. In certain embodiments, X3is an optionally substituted C4-C10aliphatic. In certain embodiments, X3is an optionally substituted C4-C10 alkylene. In certain embodiments, X3is an optionally substituted C4-C10 alkenylene. In certain embodiments, X3is an optionally substituted C6- C8 aliphatic. In certain embodiments, X3is an optionally substituted C6-C8 alkylene. In certain embodiments, X3is an optionally substituted C6-C8 alkenylene. In certain embodiments, X3is an optionally substituted C7-C9aliphatic. In certain embodiments, X3is an optionally substituted C7-C9alkylene. In certain embodiments, X3is an optionally substituted C7-C9alkenylene. In certain embodiments, X3is an optionally substituted C1-C4aliphatic. In certain embodiments, X3is an optionally substituted C1-C4 alkylene. In certain embodiments, X3is an unsubstituted C1-C4 aliphatic. In certain embodiments, X3is an unsubstituted C1-C4 alkylene. In certain embodiments, X3is anoptionally substituted C1-C2aliphatic. In certain embodiments, X3is an optionally substituted C1-C2alkylene. In certain embodiments, X3is an unsubstituted C1-C2aliphatic. In certain embodiments, X3is an unsubstituted C1-C2 alkylene. In certain embodiments, X3is –(CH2)-. In certain embodiments, X3is –(CH2)2-. In certain embodiments, X3is –(CH2)3-. In certain embodiments, X3is –(CH2)4-. In certain embodiments, X3is –(CH2)5-. In certain embodiments, X3is –(CH2)6-. In certain embodiments, X3is –(CH2)7-. In certain embodiments, X3is –(CH2)8-. In certain embodiments, X3is –(CH2)9-. In certain embodiments, X3is –(CH2)10-. In certain embodiments, X3is –(CH2)11-. In certain embodiments, X3is –(CH2)12-.

[0105] In certain embodiments, X6is a bond. In certain embodiments, X6is an optionally substituted C1-C12 alkylene. In certain embodiments, X6is an optionally substituted C1-C12 alkenylene. In certain embodiments, X6is an optionally substituted C1-C10 aliphatic. In certain embodiments, X6is an optionally substituted C1-C10 alkylene. In certain embodiments, X6is an optionally substituted C1-C10 alkenylene. In certain embodiments, X6is an optionally substituted C1-C8aliphatic. In certain embodiments, X6is an optionally substituted C1-C8alkylene. In certain embodiments, X6is an optionally substituted C1-C8alkenylene. In certain embodiments, X6is an optionally substituted C1-C6aliphatic. In certain embodiments, X6is an optionally substituted C1-C6 alkylene. In certain embodiments, X6is an optionally substituted C1-C6 alkenylene. In certain embodiments, X6is an optionally substituted C2-C12 aliphatic. In certain embodiments, X6is an optionally substituted C2-C12 alkylene. In certain embodiments, X6is an optionally substituted C2-C12alkenylene. In certain embodiments, X6is an optionally substituted C4-C12aliphatic. In certain embodiments, X6is an optionally substituted C4-C12alkylene. In certain embodiments, X6is an optionally substituted C4-C12alkenylene. In certain embodiments, X6is an optionally substituted C4-C10aliphatic. In certain embodiments, X6is an optionally substituted C4-C10 alkylene. In certain embodiments, X6is an optionally substituted C4-C10 alkenylene. In certain embodiments, X6is an optionally substituted C6- C8 aliphatic. In certain embodiments, X6is an optionally substituted C6-C8 alkylene. In certain embodiments, X6is an optionally substituted C6-C8alkenylene. In certain embodiments, X6is an optionally substituted C7-C9aliphatic. In certain embodiments, X6is an optionally substituted C7-C9alkylene. In certain embodiments, X6is an optionally substituted C7-C9alkenylene. In certain embodiments, X6is an optionally substituted C1-C4 aliphatic. In certain embodiments, X6is an optionally substituted C1-C4 alkylene. In certain embodiments, X6is an unsubstituted C1-C4 aliphatic. In certain embodiments, X6is an unsubstituted C1-C4 alkylene. In certain embodiments, X6is an optionally substituted C1-C2 aliphatic. In certain embodiments, X6is an optionally substituted C1-C2 alkylene. In certain embodiments, X6is an unsubstituted C1-C2aliphatic. In certain embodiments, X6is an unsubstituted C1-C2alkylene. In certain embodiments, X6is –(CH2)-. In certain embodiments, X6is –(CH2)2-. In certain embodiments, X6is –(CH2)3-. In certain embodiments, X6is –(CH2)4-. In certain embodiments, X6is –(CH2)5-. In certain embodiments, X6is –(CH2)6-. In certain embodiments, X6is –(CH2)7-. In certain embodiments, X6is –(CH2)8-. In certain embodiments, X6is –(CH2)9-. Incertain embodiments, X6is –(CH2)10-. In certain embodiments, X6is –(CH2)11-. In certain embodiments, X6is –(CH2)12-.

[0106] In certain embodiments, X2and X3are each independently a bond or an optionally substituted C6-C10 aliphatic. In certain embodiments, X2and X3are each independently a bond or an optionally substituted C6-C10 alkylene. In certain embodiments, X2and X3are each independently a bond or an unsubstituted C6-C10aliphatic. In certain embodiments, X2and X3are each independently a bond or an unsubstituted C6-C10alkylene. In certain embodiments, X2and X3are each independently a bond or an optionally substituted C7-C9aliphatic. In certain embodiments, X2and X3are each independently a bond or an optionally substituted C7-C9 alkylene. In certain embodiments, X2and X3are each independently a bond or an unsubstituted C7-C9 aliphatic. In certain embodiments, X2and X3are each independently a bond or an unsubstituted C7-C9 alkylene.

[0107] In certain embodiments, X2, X3and X6are each independently a bond or an optionally substituted C6-C10aliphatic. In certain embodiments, X2, X3and X6are each independently a bond or an optionally substituted C6-C10alkylene. In certain embodiments, X2, X3and X6are each independently a bond or an unsubstituted C6-C10aliphatic. In certain embodiments, X2, X3and X6are each independently a bond or an unsubstituted C6-C10 alkylene. In certain embodiments, X2, X3and X6are each independently a bond or an optionally substituted C7-C9 aliphatic. In certain embodiments, X2, X3and X6are each independently a bond or an optionally substituted C7-C9 alkylene. In certain embodiments, X2, X3and X6are each independently a bond or an unsubstituted C7-C9aliphatic. In certain embodiments, X2, X3and X6are each independently a bond or an unsubstituted C7-C9alkylene.

[0108] In certain embodiments, X2and X3are both a bond or an optionally substituted C6-C10aliphatic. In certain embodiments, X2and X3are both a bond or an optionally substituted C6-C10 alkylene. In certain embodiments, X2and X3are both a bond or an unsubstituted C6-C10 aliphatic. In certain embodiments, X2and X3are both a bond or an unsubstituted C6-C10 alkylene. In certain embodiments, X2and X3are both a bond or an optionally substituted C7-C9aliphatic. In certain embodiments, X2and X3are both a bond or an optionally substituted C7-C9alkylene. In certain embodiments, X2and X3are both a bond or an unsubstituted C7-C9aliphatic. In certain embodiments, X2and X3are both a bond or an unsubstituted C7-C9 alkylene.

[0109] In certain embodiments, X2, X3and X6are each a bond or an optionally substituted C6-C10 aliphatic. In certain embodiments, X2, X3and X6are each a bond or an optionally substituted C6-C10 alkylene. In certain embodiments, X2, X3and X6are each a bond or an unsubstituted C6-C10 aliphatic. In certain embodiments, X2, X3and X6are each a bond or an unsubstituted C6-C10alkylene. In certain embodiments, X2, X3and X6are each a bond or an optionally substituted C7-C9aliphatic. In certain embodiments, X2, X3and X6are each a bond or an optionally substituted C7-C9alkylene. In certain embodiments, X2, X3and X6are each a bond or an unsubstituted C7-C9 aliphatic. In certain embodiments, X2, X3and X6are each a bond or an unsubstituted C7-C9 alkylene.

[0110] In certain embodiments, X2and X3are both bonds. In certain embodiments, X2and X3are both –(CH2)6-. In certain embodiments, X2and X3are both –(CH2)7-. In certain embodiments, X2and X3are both –(CH2)8-. In certain embodiments, X2and X3are both –(CH2)9-. In certain embodiments, X2and X3are both –(CH2)10-. In certain embodiments, X2, X3and X6are each bonds. In certain embodiments, X2, X3and X6are each –(CH2)6-. In certain embodiments, X2, X3and X6are each – (CH2)7-. In certain embodiments, X2, X3and X6are each –(CH2)8-. In certain embodiments, X2, X3and X6are each –(CH2)9-. In certain embodiments, X2, X3and X6are each –(CH2)10-. Y2, Y3and Y4

[0111] As disclosed in Formula (TL), in certain embodiments, Y2, Y3and Y4are each independently or, wherein the bond marked with an "*" is attached to X2for Y2, or X3for Y3, or X6for embodiments, Y2and Y3are the same. In certain embodiments, Y2and Y3are different.In certain embodiments, Y2, Y3and Y4are all the same. In certain embodiments, Y2, Y3and Y4are all different. In certain embodiments, two of Y2, Y3and Y4are the same and the third is different. In certain embodiments, Y2and Y3are the same, and Y4is different from the other two.

[0112] In certain embodiments, Y2and Y3are each. In certain embodiments, Y2and Y3are each.In certain embodiments, Y2 andY3are each independently . In certain embodiments, Y2and Y3are each. In certain embodiments, Y2and Y3are each independentlyis. In certain embodiments, Y2is . In certain embodiments, Y2is. In certain embodiments, Y2is . In certain embodiments, Y2iscertain .In certain embodiments, Y3. In certainembodiments, Y3. In certain . In certainembodiments, Y3. In certain embodiments, Y3. In certain embodiments,certain embodiments, Y3. In certain embodiments, Y3and Y2. In certain embodiments, Y3and Y2are . In certainembodiments, Y3and Y2are . In certain embodiments, Y3and Y2are both.X4, X5and X7

[0113] As disclosed in Formula (TL), in certain embodiments, X4, X5and X7are each independently a bond or optionally substituted C1-C6 aliphatic. In certain embodiments, X4and X5are the same. In certain embodiments, X4and X5are different. In certain embodiments, X4, X5and X7are the same. In certain embodiments, X4, X5and X7are each different from each other. In certain embodiments, X4and X5are the same, and X7is different from the other two.

[0114] In certain embodiments, X4is a bond. In certain embodiments, X4is an optionally substituted C1-C6 aliphatic. In certain embodiments, X4is an optionally substituted C1-C6 alkylene. In certain embodiments, X4is an optionally substituted C1-C6 alkenylene. In certain embodiments, X4is an optionally substituted C2-C5 aliphatic. In certain embodiments, X4is an optionally substituted C2-C5alkylene. In certain embodiments, X4is an optionally substituted C2-C5alkenylene. In certain embodiments, X4is an optionally substituted C3-C4aliphatic. In certain embodiments, X4is an optionally substituted C3-C4 alkylene. In certain embodiments, X4is an optionally substituted C3-C4 alkenylene. In certain embodiments, X4is –(CH2)-. In certain embodiments, X4is –(CH2)2-. In certain embodiments, X4is –(CH2)3-. In certain embodiments, X4is –(CH2)4-. In certain embodiments, X4is – (CH2)5-. In certain embodiments, X4is –(CH2)6-.

[0115] In certain embodiments, X5is a bond. In certain embodiments, X5is an optionally substituted C1-C6aliphatic. In certain embodiments, X5is an optionally substituted C1-C6alkylene. In certain embodiments, X5is an optionally substituted C1-C6 alkenylene. In certain embodiments, X5is an optionally substituted C2-C5 aliphatic. In certain embodiments, X5is an optionally substituted C2-C5 alkylene. In certain embodiments, X5is an optionally substituted C2-C5 alkenylene. In certain embodiments, X5is an optionally substituted C3-C4 aliphatic. In certain embodiments, X5is an optionally substituted C3-C4alkylene. In certain embodiments, X5is an optionally substituted C3-C4alkenylene. In certain embodiments, X5is –(CH2)-. In certain embodiments, X5is –(CH2)2-. In certain embodiments, X5is –(CH2)3-. In certain embodiments, X5is –(CH2)4-. In certain embodiments, X5is – (CH2)5-. In certain embodiments, X5is –(CH2)6-.

[0116] In certain embodiments, X7is a bond. In certain embodiments, X7is an optionally substituted C1-C6 aliphatic. In certain embodiments, X7is an optionally substituted C1-C6 alkylene. In certain embodiments, X7is an optionally substituted C1-C6alkenylene. In certain embodiments, X7is an optionally substituted C2-C5aliphatic. In certain embodiments, X7is an optionally substituted C2-C5alkylene. In certain embodiments, X7is an optionally substituted C2-C5alkenylene. In certain embodiments, X7is an optionally substituted C3-C4aliphatic. In certain embodiments, X7is an optionally substituted C3-C4 alkylene. In certain embodiments, X7is an optionally substituted C3-C4 alkenylene. In certain embodiments, X7is –(CH2)-. In certain embodiments, X7is –(CH2)2-. In certain embodiments, X7is –(CH2)3-. In certain embodiments, X7is –(CH2)4-. In certain embodiments, X7is – (CH2)5-. In certain embodiments, X7is –(CH2)6-.

[0117] In certain embodiments, X4and X5are each independently a bond or an optionally substituted C2-C5aliphatic. In certain embodiments, X4and X5are each independently a bond or an optionally substituted C2-C5 alkylene. In certain embodiments, X4and X5are each independently a bond or an unsubstituted C2-C5 aliphatic. In certain embodiments, X4and X5are each independently a bond or an unsubstituted C2-C5 alkylene. In certain embodiments, X4and X5are each independently a bond or an optionally substituted C3-C4 aliphatic. In certain embodiments, X4and X5are each independently a bond or an optionally substituted C3-C4alkylene. In certain embodiments, X4and X5are each independently a bond or an unsubstituted C3-C4aliphatic. In certain embodiments, X4and X5are each independently a bond or an unsubstituted C3-C4alkylene.

[0118] In certain embodiments, X4, X5and X7are each independently a bond or an optionally substituted C2-C5 aliphatic. In certain embodiments, X4, X5and X7are each independently a bond oran optionally substituted C2-C5alkylene. In certain embodiments, X4, X5and X7are each independently a bond or an unsubstituted C2-C5aliphatic. In certain embodiments, X4, X5and X7are each independently a bond or an unsubstituted C2-C5 alkylene. In certain embodiments, X4, X5and X7are each independently a bond or an optionally substituted C3-C4 aliphatic. In certain embodiments, X4, X5and X7are each independently a bond or an optionally substituted C3-C4 alkylene. In certain embodiments, X4, X5and X7are each independently a bond or an unsubstituted C3-C4aliphatic. In certain embodiments, X4, X5and X7are each independently a bond or an unsubstituted C3-C4alkylene.

[0119] In certain embodiments, X4and X5are both a bond or an optionally substituted C2-C5 aliphatic. In certain embodiments, X4and X5are both a bond or an optionally substituted C2-C5 alkylene. In certain embodiments, X4and X5are both a bond or an unsubstituted C2-C5 aliphatic. In certain embodiments, X4and X5are both a bond or an unsubstituted C2-C5 alkylene. In certain embodiments, X4and X5are both a bond or an optionally substituted C3-C4aliphatic. In certain embodiments, X4and X5are both a bond or an optionally substituted C3-C4alkylene. In certain embodiments, X4and X5are both a bond or an unsubstituted C3-C4aliphatic. In certain embodiments, X4and X5are both a bond or an unsubstituted C3-C4 alkylene.

[0120] In certain embodiments, X4, X5and X7are each a bond or an optionally substituted C2-C5 aliphatic. In certain embodiments, X4, X5and X7are each a bond or an optionally substituted C2-C5 alkylene. In certain embodiments, X4, X5and X7are each a bond or an unsubstituted C2-C5aliphatic. In certain embodiments, X4, X5and X7are each a bond or an unsubstituted C2-C5alkylene. In certain embodiments, X4, X5and X7are each a bond or an optionally substituted C3-C4aliphatic. In certain embodiments, X4, X5and X7are each a bond or an optionally substituted C3-C4alkylene. In certain embodiments, X4, X5and X7are each a bond or an unsubstituted C3-C4 aliphatic. In certain embodiments, X4, X5and X7are each a bond or an unsubstituted C3-C4 alkylene.

[0121] In certain embodiments, X4and X5are both bonds. In certain embodiments, X4and X5are both –(CH2)6-. In certain embodiments, X4and X5are both –(CH2)5-. In certain embodiments, X4and X5are both –(CH2)4-. In certain embodiments, X4and X5are both –(CH2)3-. In certain embodiments, X4and X5are both –(CH2)2-. In certain embodiments, X4and X5are both –(CH2)-. In certain embodiments, X4, X5and X7are each bonds. In certain embodiments, X4, X5and X7are each –(CH2)6- . In certain embodiments, X4, X5and X7are each –(CH2)5-. In certain embodiments, X4, X5and X7are each –(CH2)4-. In certain embodiments, X4, X5and X7are each –(CH2)3-. In certain embodiments, X4, X5and X7are each –(CH2)2-. In certain embodiments, X4, X5and X7are each –(CH2)-. R2

[0122] As disclosed in Formula (TL), in certain embodiments, R2is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14aliphatic- R10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. In certain embodiments, R2is -CH(OR6)(OR7). In certain embodiments, R2is -CH(R6)(R7). In certain embodiments, R2is -CH(SR6)(SR7). In certain embodiments, R2is -R10. In certain embodiments, R2is optionally substituted C5-C18 aliphatic. In certain embodiments, R2is optionally substituted C1-C14 aliphatic-R10.

[0124] As disclosed in Formula (TL), in certain embodiments, R3is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14aliphatic- R11, wherein one or more methylene linkages of R3are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, - OC(O)-, -NHC(O)- or -C(O)O-. In certain embodiments, R3is -CH(OR8)(OR9). In certain embodiments, R3is -CH(R8)(R9). In certain embodiments, R3is -CH(SR8)(SR9). In certain embodiments, R3is -R11. In certain embodiments, R3is optionally substituted C5-C18aliphatic. In certain embodiments, R3is optionally substituted C1-C14aliphatic-R11.

[0125] In certain embodiments, R3is selected ,InorR12

[0127] As disclosed in Formula (TL), in certain embodiments, R12is -CH(OR13)(OR14), - CH(SR13)(SR14), -CH(R13)(R14), -R15, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14aliphatic-R15, wherein one or more methylene linkages of R12are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, -O-, -NH-, -S-, - SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. In certain embodiments, R12is - CH(OR13)(OR14). In certain embodiments, R12is -CH(R13)(R14). In certain embodiments, R12is - CH(SR13)(SR14). In certain embodiments, R12is -R15. In certain embodiments, R12is optionally substituted C5-C18 aliphatic. In certain embodiments, R12is optionally substituted C1-C14 aliphatic-R15. , ,orare are same, R6and R7

[0130] As disclosed in Formula (TL), in certain embodiments, R6and R7are each independently optionally substituted -C1-C14 aliphatic, -R10, or optionally substituted -C1-C14 aliphatic-R10; wherein one or more methylene linkages of R6and R7are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)- , -NHC(O)- or -C(O)O-.

[0131] In certain embodiments, R6and R7are the same. In certain embodiments, R6and R7are different.

[0132] In certain embodiments, R6is optionally substituted C1-C14 aliphatic. In certain embodiments, R6is optionally substituted C1-C14 alkyl. In certain embodiments, R6is optionally substituted C1-C14 branched alkyl. In certain embodiments, R6is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R6is optionally substituted C1-C14 alkenyl. In certain embodiments, R6is optionally substituted C1-C14branched alkenyl. In certain embodiments, R6is optionally substituted C1-C14straight chain alkenyl. In certain embodiments, R6is optionally substituted C4-C10alkyl. In certain embodiments, R6is optionally substituted straight chain C4-C10alkyl. In certain embodiments, R6is unsubstituted C6-C10 alkyl. In certain embodiments, R6is unsubstituted straight chain C4-C10 alkyl. In certain embodiments, R6is optionally substituted C6-C10 alkyl. In certain embodiments, R6is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R6is unsubstituted C4-C10 alkyl. In certain embodiments, R6is unsubstituted straight chain C6-C10alkyl. In certain embodiments, R6is optionally substituted –(CH2)5CH3. In certain embodiments, R6is optionally substituted – (CH2)6CH3. In certain embodiments, R6is optionally substituted –(CH2)7CH3. In certain embodiments, R6is optionally substituted –(CH2)8CH3. In certain embodiments, R6is optionally substituted – (CH2)9CH3.

[0133] In certain embodiments, R7is optionally substituted C1-C14aliphatic. In certain embodiments, R7is optionally substituted C1-C14alkyl. In certain embodiments, R7is optionally substituted C1-C14branched alkyl. In certain embodiments, R7is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R7is optionally substituted C1-C14 alkenyl. In certain embodiments, R7is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R7is optionally substituted C1-C14straight chain alkenyl. In certain embodiments, R7is optionally substituted C4-C10alkyl. In certain embodiments, R7is optionally substituted straight chain C4-C10alkyl. In certain embodiments, R7is unsubstituted C6-C10alkyl. In certain embodiments, R7is unsubstituted straight chain C4-C10alkyl. In certain embodiments, R7is optionally substituted C6-C10 alkyl. In certain embodiments, R7is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R7is unsubstituted C4-C10 alkyl. In certain embodiments, R7is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R7is optionally substituted –(CH2)5CH3. In certain embodiments, R7is optionally substituted – (CH2)6CH3. In certain embodiments, R7is optionally substituted –(CH2)7CH3. In certain embodiments, R7is optionally substituted –(CH2)8CH3. In certain embodiments, R7is optionally substituted – (CH2)9CH3.

[0134] In certain embodiments, each R6and R7are each independently selected from , , , , .

[0135] As disclosed in Formula (TL), in certain embodiments, R8and R9are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)- , -NHC(O)- or -C(O)O-.

[0136] In certain embodiments, R8and R9are the same. In certain embodiments, R8and R9are different.

[0137] In certain embodiments, R8is optionally substituted C1-C14 aliphatic. In certain embodiments, R8is optionally substituted C1-C14 alkyl. In certain embodiments, R8is optionally substituted C1-C14 branched alkyl. In certain embodiments, R8is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R8is optionally substituted C1-C14alkenyl. In certain embodiments, R8is optionally substituted C1-C14branched alkenyl. In certain embodiments, R8is optionally substituted C1-C14straight chain alkenyl. In certain embodiments, R8is optionally substituted C4-C10alkyl. In certain embodiments, R8is optionally substituted straight chain C4-C10 alkyl. In certain embodiments, R8is unsubstituted C6-C10 alkyl. In certain embodiments, R8is unsubstituted straight chain C4-C10alkyl. In certain embodiments, R8is optionally substituted C6-C10alkyl. In certain embodiments, R8is optionally substituted straight chain C6-C10alkyl. In certain embodiments, R8is unsubstituted C4-C10alkyl. In certain embodiments, R8is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R8is optionally substituted –(CH2)5CH3. In certain embodiments, R8is optionally substituted – (CH2)6CH3. In certain embodiments, R8is optionally substituted –(CH2)7CH3. In certain embodiments, R8is optionally substituted –(CH2)8CH3. In certain embodiments, R8is optionally substituted – (CH2)9CH3.

[0138] In certain embodiments, R9is optionally substituted C1-C14aliphatic. In certain embodiments, R9is optionally substituted C1-C14 alkyl. In certain embodiments, R9is optionally substituted C1-C14 branched alkyl. In certain embodiments, R9is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R9is optionally substituted C1-C14 alkenyl. In certain embodiments, R9is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R9is optionally substituted C1-C14straight chain alkenyl. In certain embodiments, R9is optionally substituted C4-C10alkyl. In certain embodiments, R9is optionally substituted straight chain C4-C10alkyl. In certain embodiments, R9is unsubstituted C6-C10alkyl. In certain embodiments, R9is unsubstituted straight chain C4-C10alkyl. In certain embodiments, R9is optionally substituted C6-C10 alkyl. In certain embodiments, R9is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R9is unsubstituted C4-C10 alkyl. In certain embodiments, R9is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R9is optionally substituted –(CH2)5CH3. In certain embodiments, R9is optionally substituted – (CH2)6CH3. In certain embodiments, R9is optionally substituted –(CH2)7CH3. In certain embodiments, R9is optionally substituted –(CH2)8CH3. In certain embodiments, R9is optionally substituted – (CH2)9CH3.

[0139] In certain embodiments, each R8and R9are each independently selected from , , , , .

[0140] As disclosed in Formula (TL), in certain embodiments, R13and R14are each independently optionally substituted -C1-C14 aliphatic, -R15, or optionally substituted -C1-C14 aliphatic-R15; wherein one or more methylene linkages of R13and R14are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, - OC(O)-, -NHC(O)- or -C(O)O-.

[0141] In certain embodiments, R13and R14are the same. In certain embodiments, R13and R14are different.

[0142] In certain embodiments, R13is optionally substituted C1-C14aliphatic. In certain embodiments, R13is optionally substituted C1-C14alkyl. In certain embodiments, R13is optionally substituted C1-C14branched alkyl. In certain embodiments, R13is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R13is optionally substituted C1-C14 alkenyl. In certain embodiments, R13is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R13is optionally substituted C1-C14straight chain alkenyl. In certain embodiments, R13is optionally substituted C4-C10alkyl. In certain embodiments, R13is optionally substituted straight chain C4-C10alkyl. In certain embodiments, R13is unsubstituted C6-C10alkyl. In certain embodiments, R13is unsubstituted straight chain C4-C10alkyl. In certain embodiments, R13is optionally substituted C6-C10 alkyl. In certain embodiments, R13is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R13is unsubstituted C4- C10 alkyl. In certain embodiments, R13is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R13is optionally substituted –(CH2)5CH3. In certain embodiments, R13is optionally substituted –(CH2)6CH3. In certain embodiments, R13is optionally substituted –(CH2)7CH3. In certain embodiments, R13is optionally substituted –(CH2)8CH3. In certain embodiments, R13is optionally substituted –(CH2)9CH3.

[0143] In certain embodiments, R14is optionally substituted C1-C14 aliphatic. In certain embodiments, R14is optionally substituted C1-C14 alkyl. In certain embodiments, R14is optionally substituted C1-C14 branched alkyl. In certain embodiments, R14is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R14is optionally substituted C1-C14alkenyl. In certain embodiments, R14is optionally substituted C1-C14branched alkenyl. In certain embodiments, R14is optionally substituted C1-C14straight chain alkenyl. In certain embodiments, R14is optionally substituted C4-C10alkyl. In certain embodiments, R14is optionally substituted straight chain C4-C10alkyl. In certain embodiments, R14is unsubstituted C6-C10 alkyl. In certain embodiments, R14is unsubstituted straight chain C4-C10 alkyl. In certain embodiments, R14is optionally substituted C6-C10 alkyl. In certain embodiments, R14is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R14is unsubstituted C4- C10alkyl. In certain embodiments, R14is unsubstituted straight chain C6-C10alkyl. In certain embodiments, R14is optionally substituted –(CH2)5CH3. In certain embodiments, R14is optionally substituted –(CH2)6CH3. In certain embodiments, R14is optionally substituted –(CH2)7CH3. In certain embodiments, R14is optionally substituted –(CH2)8CH3. In certain embodiments, R14is optionally substituted –(CH2)9CH3.

[0144] In certain embodiments, each R13and R14are each independently selected from , .R10, R11and R15

[0145] As disclosed in Formula (TL), in certain embodiments, each R10, R11and R15are independently an optionally substituted cyclic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.

[0146] In certain embodiments, each R10, R11and R15are independently an optionally substituted monocyclic C4-C14 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted monocyclic C6-C14 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted monocyclic C6-C8 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bicyclic C4-C14 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bicyclic C6-C14cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bicyclic C8-C14cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bicyclic C6-C10 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic C4-C14 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic C6-C14 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic C8-C14cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic C6-C10cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted multicyclic C4-C14cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted multicyclic C6-C14 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted multicyclic C8-C14 cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged multicyclic C4-C14cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged multicyclic C6-C14cycloalkyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged multicyclic C8-C14 cycloalkyl. In certain embodiments, two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl. In certain embodiments, two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C6-C14 cycloalkyl. In certain embodiments, two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C8-C14cycloalkyl.

[0147] In certain embodiments, each R10, R11and R15are independently an optionally substituted monocyclic 4-14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted monocyclic 6-14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted monocyclic C6-C8heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bicyclic 4-14 membered heterocyclyl. In certain embodiments, each R10and R11are independently an optionally substituted bicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bicyclic C8-C14 heterocyclyl. In certain embodiments, each R10and R11are independently an optionally substituted bicyclic 6-12 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic 4-14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic C8-C14 heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged bicyclic 6-12 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted multicyclic 4- 14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted multicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted multicyclic C8-C14heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged multicyclic 4- 14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged multicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10, R11and R15are independently an optionally substituted bridged multicyclic C8-C14heterocyclyl. In certain embodiments, two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl. In certain embodiments, two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic 6-14 membered heterocyclyl. In certain embodiments, two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic 8-14 membered heterocyclyl.

[0148] In certain embodiments, each R10, R11and R15is independently an optionally substituted monovalent cyclic group selected from , ,embodiments, each R10, R11and R15is independently a structure selected ,certain embodiments, each R10, R11and R15is independently a structure selected,,,,a , , ,aTable (I). Non-Limiting Examples of Ionizable Lipids of the Disclosure Cmpd Structure TL-1TL-5 O HO N O O N OOTL-11TL-17TL-23TL-29TL-35TL-41TL-46TL-51TL-56e (I) above, an enantiomer thereof, or any mixture of enantiomers thereof, or a pharmaceutically acceptable salt of any of the aforementioned. ii. Structural lipids

[0151] In some embodiments, an LNP comprises a structural lipid. In some embodiments, an LNP comprises two or more structural lipids. Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is a cholesterol analogue disclosed by Patel, et al., Nat Commun., 11, 983 (2020), which is incorporated herein by reference in its entirety. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combinations thereof. In some embodiments, a structural lipid is described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.

[0152] In some embodiments, a structural lipid is a cholesterol analog. Using a cholesterol analog may enhance endosomal escape as described in Patel et al., Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications (2020), which is incorporated herein by reference.

[0153] In some embodiments, a structural lipid is a phytosterol. Using a phytosterol may enhance endosomal escape as described in Herrera et al., Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery, Biomaterials Science (2020), which is incorporated herein by reference.

[0154] In some embodiments, a structural lipid contains plant sterol mimetics for enhanced endosomal release.

[0155] In some embodiments, the structural lipid is cholesteryl hemisuccinate (CHEMS). In some embodiments, the structural lipid is 3-(4-((2-(4-morpholinyl)ethyl)amino)-4-oxobutanoate) (Mochol).iii. PEGylated lipids

[0156] A PEGylated lipid is a lipid modified with polyethylene glycol. The term “PEGylated lipid” is used interchangeably herein with the shortened term “PEG lipid”.

[0157] In some embodiments, an LNP comprises one, two or more PEGylated lipid or PEG-modified lipid. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0158] In some embodiments, the PEGylated lipid is selected from (R)-2,3-bis(octadecyloxy)propyl- 1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG- PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG- DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C- DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k- DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG- PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE- PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, C18PEG750, C18PEG5000, C18PEG3000, C18PEG2000, C16PEG2000, C14PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X. In certain embodiments, the PEGylated lipid is PEG-DPPE. In certain embodiments, the PEGylated lipid is PEG2k-DPPE.

[0159] In some embodiments, the LNP comprises a PEGylated lipid disclosed in one of US 2019 / 0240354; US 2010 / 0130588; US 2021 / 0087135; WO 2021 / 204179; US 2021 / 0128488; US 2020 / 0121809; US 2017 / 0119904; US 2013 / 0108685; US 2013 / 0195920; US 2015 / 0005363; US 2014 / 0308304; US 2013 / 0053572; WO 2019 / 232095A1; WO 2021 / 077067; WO 2019 / 152557; US 2015 / 0203446; US 2017 / 0210697; US 2014 / 0200257; or WO 2019 / 089828A1, each of which isincorporated by reference herein in their entirety. In some embodiments, the LNP comprises a PEGylated lipid described or disclosed in PCT / US2023 / 072878 filed August 25, 2023, which is incorporated by reference herein in its entirety.

[0160] In some embodiments, the LNP comprises a PEGylated lipid substitute in place of the PEGylated lipid. All embodiments disclosed herein that contemplate a PEGylated lipid should be understood to also apply to PEGylated lipid substitutes. In some embodiments, the LNP comprises a polysarcosine-lipid conjugate, such as those disclosed in US 2022 / 0001025 A1, which is incorporated by reference herein in its entirety. In some embodiments the LNP comprises a polyoxazoline-lipid conjugate, such as those disclosed in US 2022 / 0249695 A1, which is incorporated by reference herein in its entirety.

[0161] In some embodiments, the LNP comprises a PEGylated lipid disclosed and described in PCT Application PCT / US2023 / 072878, which is incorporated by reference herein, in its entirety. In certain embodiments, the PEGylated lipid is a lipid of any one of formulas PL-I’, PL-I’’, PL-I, PL-Ia, PL-Ib, PL-Iaa, PL-Iab, PL-Iac, PL-Iad, PL-Iae, PL-Iaf, PL-Iag, PL-Iah, PL-Iba, PL-Ibb, PL-Ibc, PL- Ibd, PL-Ibe, PL-Ibf, PL-Ibg, PL-Ibh, PL-Ica, PL-Icb, PL-Icc, PL-Icd, PL-Id PL-Ie, PL-If, PL-Ig, PL- Ih, PL-Ii, PL-Iha, PL-Ihb, PL-Ihc, PL-Ihd, PL-Iia, PL-Iib, PL-Iic, PL-Iid, PL-Ij, PL-Ik, L-Il, PL-Im, PL-In, PL-Io, PL-Ip, PL-Iq, PL-Ioa, PL-Iob, PL-Ioc, PL-Iod, PL-Ioe, PL-Iof, PL-Iog, PL-Ioh, PL-Ipa, PL-Ipb, PL-Ipc, PL-Ipd, PL-Ipe, PL-Ipf, PL-Ipg, PL-Iph, PL-Iqa, PL-Iqb, PL-Iqc, PL-Iqd, PL-Ir, PL- Is, PL-It, PL-Iu, PL-Iv, PL-Iw, PL-Iva, PL-Ivb, PL-Ivc, PL-Ivd, PL-Iwa, PL-Iwb, PL-Iwc, PL-Iwd, PL-Ix, PL-Ixx, PL-Iy, PL-Iyy, PL-Iyyy, PL-Iz, PL-Izz, PL-Izzz, PL-II’, PL-II’’, PL-II, PL-IIc, PL-IId, PL-IIe, PL-IIf, PL-IIg, PL-IIh, PL-IIa, PL-IIb, PL-IIk, PL-IIm or PL-IIn.

[0162] In some embodiments, the PEGylated lipid is a compound of formula PL-I’:or a pharmaceutically acceptable salt thereof, wherein: A1is a saturated 5-6 membered carbocyclic ring or a saturated 5-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the carbocyclic ring and heterocyclic ring are substituted with t occurrences of R4; X1is -N(H)-, -N(C1-6alkyl)-, -C1-6aliphatic-N(H)-, -C1-6aliphatic-N(C1-6alkyl)-, -O- or -C1-6aliphatic-O-; L1is -C(O)(C1-6 aliphatic)C(O)-N(R)-, -C(O)(C1-6 aliphatic)-N(R)C(O)-, -C(O)(C1-6 aliphatic)C(O)O-, -C(O)(C1-6 aliphatic)C(O)-, -C(O)(C1-6 aliphatic)C(O)OCH2-, -C(O)(C1-6 aliphatic)-, -C(O)(C1-6 aliphatic)-N(R)-, or -C(O)-;L2and L3are independently a covalent bond or C1-6alkylene wherein one methylene unit of the C1-6alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -C(R5)=N-, or - C(R5)=N-O-; R1is H, C1-6 alkyl, -(C1-6 alkyl)-N3, -(C1-6 alkyl)-SH, or C3-8 alkynyl; R2and R3are independently a straight or branched C6-30alkyl, straight or branched C6-30alkenyl, or straight or branched C6-30alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of Rx; R4is C1-4 alkyl; R5is C1-6 alkyl or C2-14 alkenyl; each R is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each Rxis independently halogen, -CN, -OR, -SR, -C(O)R, -C(O)OR, or -OC(O)OR; n is an integer from 10-75, inclusive; m is 0, 1, 2, 3, or 4; and t is 0, 1, or 2.

[0163] In some embodiments, the PEGylated lipid is a compound of formula PL-II’:or a pharmaceutically acceptable salt thereof, wherein: X1is -N(H)-, -N(C1-6 alkyl)-, -C1-6 aliphatic-N(H)-, -C1-6 aliphatic-N(C1-6 alkyl)-, -O- or -C1-6 aliphatic-O-; L1is -C(O)(C1-6aliphatic)C(O)-, -C(O)(C1-6aliphatic)-, or -C(O)-; L2and L3are a covalent bond or C1-6alkylene wherein one methylene unit of the C1-6alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -C(R6)=N-, or -C(R6)=N- O-;R1is H, C1-6alkyl, -(C1-6alkyl)-N3, -(C1-6alkyl)-SH, or C3-8alkynyl; R2and R3are independently straight or branched C6-30alkyl, straight or branched C6-30alkenyl, or straight or branched C6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of Rx; R6is C1-6alkyl or C2-14alkenyl; each R is independently hydrogen or an optionally substituted group selected from C1-6aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each Rxis independently halogen, -CN, -OR, -SR, -C(O)R, -C(O)OR, or OC(O)OR; n is an integer from 10-75, inclusive; and m is 0, 1, 2, 3, or 4.

[0164] In some embodiments, the PEGylated lipid compound is one of those shown in Table (I-X), or a pharmaceutically acceptable salt thereof. Table (I-X). Exemplary PEGylated Compounds Compound StructureCompound Structure No.Compound Structure No.Compound Structure No.Compound Structure No.Compound Structure No.

[0165] In some embodiments, an LNP of the present disclosure comprises a phospholipid. In some embodiments, an LNP of the present disclosure comprises two or more phospholipids. AsPhospholipids useful in the compositions and methods may be selected from the non-limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn-phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1- stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3- phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin. In some embodiments, an LNP includes DSPC. In certain embodiments, an LNP includes DOPE. In some embodiments, an LNP includes both DSPC and DOPE.

[0166] In some embodiments, the LNP comprises a phospholipid selected from 1-pentadecanoyl-2- oleoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1- myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-glycero- 3-phosphocholine, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1- stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-palmitoyl-sn-glycero-3- phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero- 3-phosphocholine, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2- docosahexaenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo- inositol-3’,4’-bisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-3’,5’-bisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate), 1,2-dioleoyl-sn-glycero-3- phospho-(1'-myo-inositol-3',4',5'-trisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol- 3’-phosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’-phosphate), 1,2-dioleoyl-sn- glycero-3-phospho-(1'-myo-inositol-5'-phosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo- inositol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, and 1-(8Z-octadecenoyl)-2-palmitoyl-sn- glycero-3-phosphocholine.

[0167] In some embodiments, the LNP comprises a phospholipid selected from DSPS (Distearoylphosphatidylserine), DSPG (1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DSPA (1,2-Distearoyl-sn-glycero-3-phosphate), diPhyPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine), diPhy-diether-PC (1,2-di-O-phytanyl-sn-glycero-3-phosphocholine), diPhyPE (1,2-diphytanoyl-sn- glycero-3-phosphoethanolamine), diPhy-diether-PE (1,2-di-O-phytanyl-sn-glycero-3- phosphoethanolamine), diPhyPS (1,2-diphytanoyl-sn-glycero-3-phospho-L-serine), diPhyPG (1,2- diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), diPhyPA (1,2-diphytanoyl-sn-glycero-3- phosphate), Egg PA (L-α-phosphatidic acid), and Soy PA (L-α-phosphatidic acid).

[0168] In some embodiments, the LNP comprises a phospholipid selected from 18:1 (Δ9-Cis) PE (DOPE), 18:0-18:1 PE (SOPE), C16-18:1 PE, 16:0-18:1 PE (POPE), 18:1 BMP (S,R), 18:0-18:1 PC (SOPC), 16:0-18:1 PC (POPC), 4ME 16:0 Diether PE (4Me), 18:1 (Δ9-Trans) PE (DEPE), 16:1 PE(DPPE), and CL. In certain embodiments, the LNP comprises a phospholipid described or disclosed in Alvarez-Benedicto, et al. (Biomater. Sci., 2022, 10, 549) and Li, et al. (Asian Journal of Pharmaceutical Sciences, 2015, 10, 81-98).

[0169] In certain embodiments, the phospholipid is a sphingoid lipid or sphingolipid, such as, but not limited to sphingomyelin. As used herein, the terms “sphingoid lipid” and “sphingolipid” are meant to refer to a class of lipids containing a backbone comprising a sphingoid base. An exemplary sphingoid base is sphingosine. In certain embodiments, the LNP comprises a sphingolipid selected from Egg Sphingomyelin (Egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitamidooctadec-4-en-1-yl (2- (trimethylammonio)ethyl) phosphate), Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3- hydroxy-2-stearamidooctadec-4-en-1-yl (2-(trimethylammonio)ethyl) phosphate), Milk or Bovine Sphingomyelin (Milk SM / (2S,3R,E)-3-hydroxy-2-tricosanamidooctadec-4-en-1-yl (2- (trimethylammonio)ethyl) phosphate), 28:0 SM (N-octacosanoyl-D-erythro- sphingosylphosphorylcholine), 14:0 SM (N-myristoyl-D-erythro-sphingosylphosphorylcholine), 16:1 SM (N-palmitoleoyl-D-erythro-sphingosylphosphorylcholine), 12:0 Dihydro SM (N-lauroyl-D- erythro-sphinganylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (dihydro) (Sphinganine Phosphorylcholine), 24:1 SM (N- nervonoyl-D-erythro-sphingosylphosphorylcholine), 24:0 SM (N-lignoceroyl-D-erythro- sphingosylphosphorylcholine), 18:1 SM (N-oleoyl-D-erythro-sphingosylphosphorylcholine), 18:0 SM (N-stearoyl-D-erythro-sphingosylphosphorylcholine), 17:0 SM (N-heptadecanoyl-D-erythro- sphingosylphosphorylcholine), 16:0 SM (N-palmitoyl-D-erythro-sphingosylphosphorylcholine), 12:0 SM (N-lauroyl-D-erythro-sphingosylphosphorylcholine), 06:0 SM (N-hexanoyl-D-erythro- sphingosylphosphorylcholine), 02:0 SM (N-acetyl-D-erythro-sphingosylphosphorylcholine), 3-O- methyl Lyso SM (3-O-methyl-spingosylphosphorylcholine), 3-O-methyl-N-methyl Lyso SM (3-O- methyl-N-methyl-spingosylphosphorylcholine), and 3-N-methyl Lyso SM (3-N-methyl- spingosylphosphorylcholine).

[0170] In some embodiments, the LNP comprises at least two phospholipids. In certain embodiments, at least a portion of the overall phospholipid content comprises a non- phosphatidylcholine phospholipid, wherein a “non-phosphatidylcholine phospholipid” is a phospholipid that does not comprise a phosphatidylcholine moiety. Exemplary non- phosphatidylcholine phospholipids include DOPE, DSPS, and DSPG. In certain embodiments, the LNP comprises at least 5 mol% of a non-phosphatidylcholine phospholipid. In certain embodiments, the LNP comprises at least 6 mol% of a non-phosphatidylcholine phospholipid. In certain embodiments, the LNP comprises at least 10 mol% of a non-phosphatidylcholine phospholipid.

[0171] In some embodiments, the LNP comprises a phospholipid comprising at least one constrained tail, such as those described by Gan, et al. (Bioeng Transl Med.2020 Sep; 5(3): e10161.). In certain embodiments, the phospholipid is one selected from:., 2007, 4584-4587.

[0173] In some embodiments, the LNP comprises a phospholipid disclosed in WO 2023 / 141470, which is incorporated by reference herein, in its entirety. In certain embodiments, the phospholipid is . 2022 / 040641,

[0175] In some embodiments, a phospholipid tail may be modified in order to promote endosomal escape as described in U.S.2021 / 0121411, which is incorporated herein by reference.

[0176] In some embodiments, the LNP comprises a phospholipid disclosed in one of US 2019 / 0240354; US 2010 / 0130588; US 2021 / 0087135; WO 2021 / 204179; US 2021 / 0128488; US 2020 / 0121809; US 2017 / 0119904; US 2013 / 0108685; US 2013 / 0195920; US 2015 / 0005363; US 2014 / 0308304; US 2013 / 0053572; WO 2019 / 232095A1; WO 2021 / 077067; WO 2019 / 152557; US 2017 / 0210697; or WO 2019 / 089828A1, each of which is incorporated by reference herein in their entirety.

[0177] In some embodiments, phospholipids disclosed in US 2020 / 0121809 have the following structure:wherein R1 and R2 are each independently a branched or straight, saturated or unsaturated carbon chain (e.g., alkyl, alkenyl, alkynyl). v. Targeting moieties

[0178] In some embodiments, the lipid nanoparticle further comprises a targeting moiety. The targeting moiety may be an antibody or a fragment thereof. The targeting moiety may be capable of binding to a target antigen. In certain embodiments, the lipid nanoparticle comprises more than one targeting moiety. In certain embodiments, the lipid nanoparticle comprises more than one targeting moiety, wherein the targeting moieties target at least two different receptors, and in some embodiments, the at least two different receptors are prevalent on different types of cells or tissues.

[0179] In some embodiments, the pharmaceutical composition comprises a targeting moiety that is operably connected to a lipid nanoparticle. In some embodiments, the targeting moiety is capable of binding to a target antigen. In some embodiments, the target antigen is expressed in a target organ. In some embodiments, the target antigen is expressed more in the target organ than it is in the liver.

[0180] In some embodiments, the targeting moiety is an antibody as described in WO2016189532A1, which is incorporated herein by reference. For example, in some embodiments, the targeted particles are conjugated to a specific anti-CD38 monoclonal antibody (mAb), which allows specific delivery of the siRNAs encapsulated within the particles at a greater percentage to B-cell lymphocytes malignancies (such as MCL) than to other subtypes of leukocytes.

[0181] In some embodiments, the targeting moiety targets a receptor selected from CD20, CCR7, CD3, CD4, CD5, CD8, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD35, CD40, CD45RA, CD45RO, CD52, CD62L, CD80, CD95, CD127, and CD137. In some embodiments, the targeting moiety targets a receptor selected from CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, 0X40, GITR, LAG3, ICOS, PD-1, leu- 12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7. In some embodiments, the targeting moiety targets a receptor selected from CD2, CD3, CD5 and CD7. In some embodiments, the targeting moiety targets a receptor selected from CD2, CD3, CD5, CD7, CD8, CD4, beta 7 integrin, beta 2 integrin, and C1q. In some embodiments, the targeting moiety targets CD117. In some embodiments, the targeting moiety targets CD90. In some embodiments, the targeting moiety targets a receptor selected from a mannose receptor, CD206 and C1q. In some embodiments, the targeting moiety is selected from T-cell receptor motif antibodies, T- cell α chain antibodies, T-cell β chain antibodies, T-cell γ chain antibodies, T-cell δ chain antibodies, CCR7 antibodies, CD3 antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8 antibodies, CD11b antibodies, CD11c antibodies, CD16 antibodies, CD19 antibodies, CD20 antibodies, CD21 antibodies, CD22 antibodies, CD25 antibodies, CD28 antibodies, CD34 antibodies, CD35 antibodies, CD40 antibodies, CD45RA antibodies, CD45RO antibodies, CD52 antibodies, CD56 antibodies,CD62L antibodies, CD68 antibodies, CD80 antibodies, CD90 antibodies, CD95 antibodies, CD117 antibodies, CD127 antibodies, CD133 antibodies, CD137 (4-1BB) antibodies, CD163 antibodies, F4 / 80 antibodies, IL-4Rα antibodies, Sca-1 antibodies, CTLA-4 antibodies, GITR antibodies GARP antibodies, LAP antibodies, granzyme B antibodies, LFA-1 antibodies, transferrin receptor antibodies, and fragments thereof. In certain embodiments, the targeting moiety is any one described or contemplated in US20230312713A1, US20230203538A1, US20230320995A1, US20160145348, and US20110038941, each of which is incorporated by reference herein in its entirety.

[0182] In some embodiments, the lipid nanoparticles may be targeted when conjugated / attached / associated with a targeting moiety such as an antibody, or a fragment thereof. vi. Zwitterionic amino lipids

[0183] In some embodiments, an LNP comprises a zwitterionic lipid. In some embodiments, an LNP comprising a zwitterionic lipid does not comprise a phospholipid. In some embodiments, an LNP comprising a zwitterionic lipid further comprises a phospholipid.

[0184] Zwitterionic amino lipids have been shown to be able to self-assemble into LNPs without phospholipids to load, stabilize, and release mRNAs intracellularly as described in U.S. Patent Application 20210121411, which is incorporated herein by reference in its entirety. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs as described in Liu et al., Membrane- destablizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing, Nat Mater. (2021), which is incorporated herein by reference in its entirety.

[0185] The zwitterionic lipids may have head groups containing a cationic amine and an anionic carboxylate as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013), which is incorporated herein by reference in its entirety. Ionizable lysine-based lipids containing a lysine head group linked to a long- chain dialkylamine through an amide linkage at the lysine α-amine may reduce immunogenicity as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013). vii. Additional lipid components

[0186] In some embodiments, the LNP compositions of the present disclosure further comprise one or more additional lipid components capable of influencing the tropism of the LNP. In some embodiments, the LNP further comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200 (see Cheng, et al. Nat Nanotechnol.2020 April; 15(4): 313– 320.; Dillard, et al. PNAS 2021 Vol.118 No.52.).

[0187] In some embodiments, an LNP of the present disclosure further comprises one or more additional ionizable lipids, such as, but not limited to those disclosed in one of US 2023 / 0053437; US 2019 / 0240354; US 2010 / 0130588; US 2021 / 0087135; WO 2021 / 204179; US 2021 / 0128488; US 2020 / 0121809; US 2017 / 0119904; US 2013 / 0108685; US 2013 / 0195920; US 2015 / 0005363; US2014 / 0308304; US 2013 / 0053572; WO 2019 / 232095A1; WO 2021 / 077067; WO 2019 / 152557; US 2017 / 0210697; or WO 2019 / 089828A1, each of which is incorporated by reference herein in their entirety. In certain embodiments, an LNP of the present disclosure further comprises one or more additional ionizable lipids selected from those disclosed in WO2023044343A1 or WO2023044333A1, both of which are incorporated by reference herein in their entirety.

[0188] In some embodiments, the LNP compositions of the present disclosure comprise, or further comprise one or more lipids selected from 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcarnosine (AC), 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl-sphingomyelin (SPM) (C18:l), N- lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa- 10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n- heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2-dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9- enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-dihydroxy-6-hydroxymethyl-1etrahydro- pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol-hemisuccinate-Nα-Histidinyl- Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12-pentacosadiynamide (h-Pegi- PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α-histidinyl-Nα- hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-myristoyl-2- hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N- dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB- phosphatidylethanolamine lipid (Rh-PE), purifiedsoy-derivedmixtureofphospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans-PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3- (bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine. B. Exemplary LNP Compositions

[0189] In some embodiments, provided herein are LNPs comprising (a): at least one Lipid of the Disclosure; (b) at least one PEG lipid; (c) at least one structural lipid; and (d) at least one non- ionizable lipid and / or a zwitterionic lipid. In some embodiments, the LNPs further comprise an additional ionizable lipid, besides a compound of Formula (AX).

[0190] In some embodiments, the PEG-lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

[0191] In some embodiments, the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.

[0192] In some embodiments, the non-ionizable lipid is a phospholipid selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn- glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L- serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

[0193] In some embodiments, the non-ionizable lipid is a phospholipid selected from the group consisting of Egg Sphingomyelin (Egg SM / ESM / (2S,3R,E)-3-hydroxy-2-palmitamidooctadec-4- en-1-yl (2-(trimethylammonio)ethyl) phosphate), Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3-hydroxy-2-stearamidooctadec-4-en-1-yl (2-(trimethylammonio)ethyl) phosphate), Milk or Bovine Sphingomyelin (Milk SM / (2S,3R,E)-3-hydroxy-2-tricosanamidooctadec-4-en-1-yl (2- (trimethylammonio)ethyl) phosphate), 28:0 SM (N-octacosanoyl-D-erythro- sphingosylphosphorylcholine), 14:0 SM (N-myristoyl-D-erythro-sphingosylphosphorylcholine), 16:1 SM (N-palmitoleoyl-D-erythro-sphingosylphosphorylcholine), 12:0 Dihydro SM (N-lauroyl-D- erythro-sphinganylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (dihydro) (Sphinganine Phosphorylcholine), 24:1 SM (N- nervonoyl-D-erythro-sphingosylphosphorylcholine), 24:0 SM (N-lignoceroyl-D-erythro- sphingosylphosphorylcholine), 18:1 SM (N-oleoyl-D-erythro-sphingosylphosphorylcholine), 18:0 SM (N-stearoyl-D-erythro-sphingosylphosphorylcholine), 17:0 SM (N-heptadecanoyl-D-erythro- sphingosylphosphorylcholine), 16:0 SM (N-palmitoyl-D-erythro-sphingosylphosphorylcholine), 12:0 SM (N-lauroyl-D-erythro-sphingosylphosphorylcholine), 06:0 SM (N-hexanoyl-D-erythro-sphingosylphosphorylcholine), 02:0 SM (N-acetyl-D-erythro-sphingosylphosphorylcholine), 3-O- methyl Lyso SM (3-O-methyl-spingosylphosphorylcholine), 3-O-methyl-N-methyl Lyso SM (3-O- methyl-N-methyl-spingosylphosphorylcholine), and 3-N-methyl Lyso SM (3-N-methyl- spingosylphosphorylcholine).

[0194] In some embodiments, (a) the PEG lipid is PEG2k-DMG or PEG2k-DSPE or a mixture thereof; (b) the structural lipid is cholesterol; and (c) the phospholipid, non-ionizable lipid or zwitterionic lipid is a sphingolipid or DSPC or a mixture thereof.

[0195] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0 mol% to about 10 mol% of PEG lipid; (b) about 0 mol% to about 30 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of a Lipid of the Disclosure.

[0196] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1 mol% to about 2 mol% of PEG lipid; (b) about 25 mol% to about 40 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of a Lipid of the Disclosure.

[0197] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 2 mol% of PEG lipid; (b) about 25 mol% structural lipid; (c) about 40 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 33 mol% of a Lipid of the Disclosure.

[0198] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0.5 mol% to about 10 mol% of PEG lipid; (b) about 0.5 mol% to about 50 mol% structural lipid; (c) about 5 mol% to about 45 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of a Lipid of the Disclosure.

[0199] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0.5 mol% to about 5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 35 mol% to about 45 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 40 mol% of a Lipid of the Disclosure.

[0200] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1.5 mol% to about 3.5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 35 mol% to about 45 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 40 mol% of a Lipid of the Disclosure.

[0201] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0.5 mol% to about 5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 5 mol% to about 15 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 45 mol% to about 55 mol% of a Lipid of the Disclosure.

[0202] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1.5 mol% to about 3.5 mol% of PEG lipid; (b) about 20 mol% to about 45 mol% structural lipid; (c) about 5mol% to about 15 mol% non-ionizable lipid (e.g., phospholipid), non-ionizable lipid or zwitterionic lipid; and (d) about 45 mol% to about 55 mol% of a Lipid of the Disclosure.

[0203] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 2.5 mol% of PEG lipid; (b) about 39 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of a Lipid of the Disclosure.

[0204] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1.5 mol% of PEG lipid; (b) about 40 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of a Lipid of the Disclosure.

[0205] In certain embodiments, the lipid component of the nanoparticle composition comprises about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 20 mol % to about 45 mol % ionizable lipid, about 30 mol % to about 60 mol % phospholipid, about 10 mol % to about 30 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 30 mol % to about 40 mol % ionizable lipid, about 35 mol % to about 45 mol % phospholipid, about 20 mol % to about 30 mol % structural lipid, and about 0.5 mol % to about 5 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 25 mol % to about 45 mol % ionizable lipid, about 35 mol % to about 50 mol % phospholipid, about 10 mol % to about 25 mol % structural lipid, and about 1 mol% to about 5 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In a particular embodiment, the lipid component comprises about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component comprises about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % structural lipid, and about 2 mol % of PEG lipid. In some embodiments, the phospholipid is DOPE or DSPC. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is a sphingolipid. In someembodiments, the phospholipid is sphingomyelin. In some embodiments, the phospholipid is egg sphingomyelin (ESM). In other embodiments, the PEG lipid is PEG-DMG (eg. PEG2K-DMG). In other embodiments, the PEG lipid is PEG-DSPE (eg. PEG2K-DSPE). In other embodiments, the PEG lipid is PEG-DMPE (eg. PEG2K-DMPE). In other embodiments, the structural lipid is cholesterol. In other embodiments, the PEG lipid is PEG-DMG and / or the structural lipid is cholesterol. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DSPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 33mol% ionizable lipid (eg. at least one ionizable lipid of a Formula described herein), about 40mol% of a sphingolipid, about 25mol% cholesterol and about 2mol% PEG2K-DMG. In some embodiments, the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is DSPC. In some embodiments, the PEG lipids is PEG2K- DSPE, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPE. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DLPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPS. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a sphingolipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and phosphatidylserine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a phosphoethanolamine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and phosphatidylserine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and a phosphoethanolamine lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 20mol% of a sphingolipid, about 20mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 10mol% of a sphingolipid, about 30mol% of a non- sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 30mol% of a sphingolipid, about 10mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizablelipid, about 20mol% sphingomyelin, about 20mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 10mol% sphingomyelin, about 30mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 30mol% sphingomyelin, about 10mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 30 mol% of the total lipid component of the LNP. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 25 mol% of the total lipid component of the LNP. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE.

[0206] In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % sphingomyelin, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPE, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DLPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPS, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 20 mol % sphingomyelin, about 20 mol% DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE.

[0207] In certain embodiments, the LNP comprises about 43mol% ionizable lipid, about 15mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 15mol% of a sphingolipid, about 25mol% of a non- sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K- DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE.

[0208] In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 3 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38 mol % structural lipid, and about 3.5 mol % of PEG lipid. In some embodiments, the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K- DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DPPE. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K- DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K- DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DMG. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K- DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K- DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DSPE.

[0209] In some embodiments, the LNP further comprises a targeting moiety. In some embodiments, the targeting moiety is an antibody or a fragment thereof.

[0210] In some embodiments, the LNP further comprises an active agent. In some embodiments, the active agent is a nucleic acid. In some embodiments, the nucleic acid is a ribonucleic acid. In some embodiments, the ribonucleic acid is at least one ribonucleic acid selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (lncRNA). In some embodiments, the nucleic acid is a messenger RNA (mRNA) or a circular RNA. In some embodiments, the mRNA comprises an open reading frame encoding a cancer antigen. In some embodiments, the mRNA comprises an open reading frame encoding an immune checkpoint modulator. In some embodiments, the mRNA comprises at least one motif selected from the group consisting of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and a 5' cap structure. In some embodiments, the nucleic acid is suitable for a genome editing technique. In some embodiments, the genome editing technique is clustered regularly interspaced short palindromic repeats (CRISPR) or transcriptionactivator-like effector nuclease (TALEN). In some embodiments, the nucleic acid is at least one nucleic acid suitable for a genome editing technique selected from the group consisting of a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a single guide RNA (sgRNA), and a DNA repair template. In some embodiments, the mRNA is at least 30 nucleotides in length. In some embodiments, the mRNA is at least 300 nucleotides in length. In some embodiments, the nucleic acid encodes a therapeutic protein. In some embodiments, the therapeutic protein is a CAR or TCR complex protein. In some embodiments, the CAR or TCR complex protein comprises an antigen binding domain specific for an antigen selected from the group: CD 19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule- 1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disaloganglioside GD3, TNF receptor family member, B cell maturation antigen (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), prostate- specific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD 117), Interleukin- 13 receptor subunit alpha-2, mesothelin, Interleukin 11 receptor alpha (IL-l lRa), prostate stem cell antigen (PSCA), Protease Serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y) antigen, CD24, Platelet-derived growth factor receptor beta (PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, Folate receptor alpha, HER2, HER3, Mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), Prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), Ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2), glycoprotein 100 (gplOO), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type- A receptor 2 (EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight-melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (0AcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 -related (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a. III. LNP payload

[0211] The instant specification describes compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of LNP-based RNA medicines (e.g., vaccines, gene therapies, or gene-editing therapeutics). In various embodiments, the LNP-based RNA medicines comprise an LNP delivery system (as described in detail herein) and an encapsulated cargo / payload (e.g., RNA in the case of RNA medicines).

[0212] In the case of RNA medicines, the payload can be one or more RNA molecules, including coding RNA (e.g., linear or circular mRNA) or non-coding RNA (e.g., guide RNA, pegRNA, or retron ncRNA).

[0213] In various other embodiments, the payloads can include any type of nucleic acid molecule, including coding RNA molecules (e.g., mRNA), guide RNAs for editing systems (e.g., Cas9 guides, Cas12a guides, base editor guides, and prime editor guides), other non-coding RNAs relating to editing systems (e.g., retron ncRNAs), small RNAs (sRNAs)—which refer to a wide variety of polymeric RNA molecules that are generally less than 200 nucleotides in length with various functionalities, such as RNA interference, and include small-interfering RNA (siRNA), microRNAs (miRNA), piwi-interacting RNA (piRNA), repeat associated small interfering RNA (rasiRNA), small nuclear RNA (snRNA or U-RNA), small nucleolar RNA (snoRNA), small rDNA-derived RNA (srRNA), rRNA fragment (tRF), and Y RNA-derived small RNA, tRNA, rRNA, and self-amplifying RNA (saRNA)—and DNA molecules, such as DNA vectors, DNA plasmids, HDR donors, oligonucleotides, primers, etc., and chimeric molecules comprising both DNA and RNA. The cargo nucleic acid molecules may be single-stranded or double-stranded. Such nucleic acid cargo may comprise exactly one molecule. Such nucleic acid cargo may comprise exactly two molecules. Such nucleic acid cargo may comprise exactly three molecules. Such nucleic acid cargo may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 distinct molecules. Such nucleic acid cargo may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 distinct molecules. Such nucleic acid cargo may comprise between 1-25, or 5-30, or 10-35, or 20-40, or up to 100, or more distinct molecules.

[0214] In various other aspects, the LNPs described herein may be used to deliver any payload of interest to a biological target, e.g., to a cell or a bodily tissue. The term “payload” refers to an active substance (i.e., not limited to RNA or DNA), such as a small molecule, polypeptide, peptide, carbohydrate, or nucleic acid molecule, and includes, without limitation, mRNA molecules (including linear and circular mRNA) or non-coding RNA molecules (e.g., guide RNAs, pegRNAs, retron ncRNAs) which are encapsulated within the LNPs described herein. In some embodiments, the LNP cargo may comprise an RNP or ribonucleoprotein, such as a gene editing nuclease protein complexed with a cognate guide RNA.

[0215] In various embodiments, the payload is an RNA molecule, which may be linear or circular and may comprise one or more functional nucleotide sequences of interest, which may include, but are not limited to coding and non-coding nucleotide sequences. In various embodiments, the non- coding nucleotide sequences may comprise regulatory elements that influence RNA post- transcriptional processing, nuclear translation control sequences, and sequences which encode one or more biological products of interest, e.g., a therapeutic protein or antigen, among other sequence elements that may impact the functioning of the RNA or its encoded products. As used herein, the term “coding region of interest” or “product coding region” or the like may be used to refer to theencoded one or more biological products of interest. Equivalently, a product coding region may be referred to as a “product expression sequence.”

[0216] In various embodiments herein, the specification refers to “originator constructs” (or “originator polynucleotide constructs”) and “benchmark constructs” (or “benchmark polynucleotide constructs”), which are embodiments of payloads comprising nucleic acid molecules, i.e., embodiments of linear and / or circular mRNA payloads, and which may comprise a product coding region that encodes a polypeptide, such as, but not limited to an antigen or a therapeutic protein or to components of a gene editing system (e.g., a programmable nuclease).

[0217] FIG.2 shows an example of an originator construct 100, which may be a linear or circular mRNA molecule. The originator construct 100 may include at least one product coding region 10 which is or encodes a polypeptide of interest, such as, but not limited to a vaccine antigen or a therapeutic protein. The originator construct 100 may contain 1 or 2 flanking regions 20. The flanking regions 20 may be located 5' to the product coding region 10 and / or 3' to the product coding region 10. In some instances the originator construct 100 does not contain a flanking region 20. The flanking region 20 of the originator construct 100 may include at least one regulatory region 30. At least one flanking region 20 of the originator polynucleotide construct 100 may include at least one identifier region 40. The identifier region 40 may be, but is not limited to, a barcode, label, signal and / or tag. Additionally, the identifier region 40 may be located within the product coding region 10 or may be located in the product coding region 10 and at least one flanking region 20.

[0218] In some embodiments, the originator construct comprises from about 5 to about 10,000 nucleotides in length. As a non-limiting examples, the length of the originator construct may be from 5 to 30, from 5 to 50, from 5 to 100, from 5 to 250, from 5 to 500, from 5 to 1,000, from 5 to 1,500, from 5 to 3,000 from 5 to 5,000, from 5 to 7,000, from 5 to 10,000 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 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 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 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 3,000 to 5,000, from 3,000 to 7,000, from 3,000 to 10,000, from 5,000 to 7,000, from 5,000 to 10,000, and from 7,000 to 10,000 nucleotides in length.

[0219] In some embodiments, the length of the product coding region is greater than about 5 nucleotides in length such as, but not limited to, at least or greater than about 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, 31, 32, 33, 34, 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, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 or more than 10,000 nucleotides in length.

[0220] In some embodiments, the flanking region may range independently from 0 to 10,000 nucleotides in length such as, but not limited to, at least 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, 30, 31, 32, 33, 34, 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, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length.

[0221] In some embodiments, the regulatory region may range independently from 0 to 3,000 nucleotides in length such as, but not limited to, at least 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, 30, 31, 32, 33, 34, 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 in length.

[0222] In some embodiments, the originator construct may be circularized. In other embodiments, the originator construct may be concatemerized.

[0223] Originator constructs which include at least one identifier 40 or “identifier region” 40 (e.g., barcodes, labels, signals and / or tags) may also be referred to as “benchmark constructs” or “benchmark polynucleotide constructs.” The benchmark construct may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more identifiers which may be the same or different throughout the benchmark polynucleotide construct.

[0224] In some embodiments, the identifier region may range independently from 1 to 3,000 nucleotides in length such as, but not limited to, at least 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, 30, 31, 32, 33, 34, 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. As a non-limiting example the identifier region may be 1-5 residues, 2-5 residues, 3-5 residues, 2-7 residues, 3-7 residues, 1-10 residues, 2-10 residues, 3-10 residues, 5-10 residues, 7-10 residues, 1-15 residues, 2-15 residues, 3-15 residues, 5-15 residues, 7-15 residues, 10-15 residues, 12-15 residues, 1-20 residues, 2- 20 residues, 3-20 residues, 5-20 residues, 7-20 residues, 10-20 residues, 12-20 residues, 15-20 residues, 17-20 residues, 1-25 residues, 2-25 residues, 3-25 residues, 5-25 residues, 7-25 residues, 10- 25 residues, 12-25 residues, 15-25 residues, 17-25 residues, 20-25 residues, 1-30 residues, 2-30 residues, 3-30 residues, 5-30 residues, 7-30 residues, 10-30 residues, 12-30 residues, 15-30 residues, 17-30 residues, 20-30 residues, 25-30 residues, 1-35 residues, 2-35 residues, 3-35 residues, 5-35 residues, 7-35 residues, 10-35 residues, 12-35 residues, 15-35 residues, 17-35 residues, 20-35 residues, 25-35 residues, 30-35 residues, 1-35 residues, 2-35 residues, 3-35 residues, 5-35 residues, 7- 35 residues, 10-35 residues, 12-35 residues, 15-35 residues, 17-35 residues, 20-35 residues, 25-35 residues, 30-35 residues, 1-40 residues, 2-40 residues, 3-40 residues, 5-40 residues, 7-40 residues, 10-40 residues, 12-40 residues, 15-40 residues, 17-40 residues, 20-40 residues, 25-40 residues, 30-40 residues, 35-40 residues, 1-45 residues, 2-45 residues, 3-45 residues, 5-45 residues, 7-45 residues, 10- 45 residues, 12-45 residues, 15-45 residues, 17-45 residues, 20-45 residues, 25-45 residues, 30-45 residues, 35-45 residues, 40-45 residues, 1-50 residues, 2-50 residues, 3-50 residues, 5-50 residues, 7- 50 residues, 10-50 residues, 12-50 residues, 15-50 residues, 17-50 residues, 20-50 residues, 25-50 residues, 30-50 residues, 35-50 residues, 40-50 residues, or 45-50 nucleotides in length.

[0225] In some embodiments, the identifier region in the benchmark construct overlaps with the product coding region. As used herein, "overlap" means that at least one nucleotide of the identifier region extends into the product coding region. In some aspects the identifier region overlaps with the product coding region by 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides or more than 50 nucleotides. In some aspects the identifier region overlaps with the product coding region by 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 2- 7 nucleotides, 3-7 nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides, 7-10 nucleotides, 1-15 nucleotides, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10-15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12-20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nucleotides, 3-25 nucleotides, 5-25 nucleotides, 7-25 nucleotides, 10-25 nucleotides, 12-25 nucleotides, 15-25 nucleotides, 17-25 nucleotides, 20-25 nucleotides, 1-30 nucleotides, 2-30 nucleotides, 3-30 nucleotides, 5-30 nucleotides, 7-30 nucleotides, 10-30 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, 20-30 nucleotides, 25-30 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-40 nucleotides, 2-40 nucleotides, 3-40 nucleotides, 5-40 nucleotides, 7-40 nucleotides, 10-40 nucleotides, 12-40 nucleotides, 15-40 nucleotides, 17-40 nucleotides, 20-40 nucleotides, 25-40 nucleotides, 30-40 nucleotides, 35-40 nucleotides, 1-45 nucleotides, 2-45 nucleotides, 3-45 nucleotides, 5-45 nucleotides, 7-45 nucleotides, 10-45 nucleotides, 12-45 nucleotides, 15-45 nucleotides, 17-45 nucleotides, 20-45 nucleotides, 25-45 nucleotides, 30-45 nucleotides, 35-45 nucleotides, 40-45 nucleotides, 1-50 nucleotides, 2-50nucleotides, 3-50 nucleotides, 5-50 nucleotides, 7-50 nucleotides, 10-50 nucleotides, 12-50 nucleotides, 15-50 nucleotides, 17-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides, 30-50 nucleotides, 35-50 nucleotides, 40-50 nucleotides, or 45-50 nucleotides.

[0226] In some embodiments, the benchmark polynucleotide construct comprises a product coding region and an identifier region. The identifier region may be located 5' to the product coding region, 3' to the product coding region, or the identifier region may overlap with the 5' end or the 3'end of the product coding region.

[0227] In some embodiments, the benchmark polynucleotide construct comprises a product coding region and two identifier regions. Each identifier region may independently be located 5' to the product coding region, 3' to the product coding region, or the identifier region may overlap with the 5' end or the 3'end of the product coding region.

[0228] As a non-limiting example, the first identifier region is located 5' to the product coding region and the second identifier region is located 3' to the product coding region. As a non-limiting example, the first and second identifier regions are located 5' to the product coding region. As a non-limiting example, the first and second identifier regions are located 3' to the product coding region.

[0229] As a non-limiting example, the first identifier region is inverted and is located 5' to the product coding region and the second identifier region is located 3' to the product coding region. As a non-limiting example, the first identifier region is inverted and is located 5' to the product coding region and the second identifier region is inverted and is located 3' to the product coding region. As a non-limiting example, the first identifier region is located 5' to the product coding region and the second identifier region is inverted and is located 3' to the product coding region. As a non-limiting example, the first and second identifier regions are both inverted and are located 5' to the product coding region. As a non-limiting example, the first and second identifier regions are located 5' to the product coding region and the first identifier region is inverted. As a non-limiting example, the first and second identifier regions are located 5' to the product coding region and the second identifier region is inverted. As a non-limiting example, the first and second identifier region are both inverted and located 3' to the product coding region. As a non-limiting example, the first and second identifier regions are located 3' to the product coding region and the first identifier region is inverted. As a non- limiting example, the first and second identifier regions are located 3' to the product coding region and the second identifier region is inverted.

[0230] As a non-limiting example, the first identifier region is located 5' to the product coding region and overlaps with the product coding region and the second identifier region is located 3' to the product coding region. As a non-limiting example, the first identifier region is located 5' to the product coding region and the second identifier region is located 3' to the product coding region and overlaps with the product coding region.

[0231] As a non-limiting example, the first and second identifier regions are located 5' to the product coding region and the second identifier region overlaps with the product coding region. As a non-limiting example, the first and second identifier regions are located 3' to the product coding region and the first identifier region overlaps with the product coding region.

[0232] As a non-limiting example, the first identifier region is inverted, is located 5' to the product coding region and overlaps with the product coding region, and the second identifier region is located 3' to the product coding region. As a non-limiting example, the first identifier region is inverted and is located 5' to the product coding region and the second identifier region is located 3' to the product coding region and overlaps with the product coding region. As a non-limiting example, the first identifier region is inverted, is located 5' to the product coding region, the second identifier region is located 3' to the product coding region, and both of the first and second identifier regions overlap with the product coding region.

[0233] As a non-limiting example, the first identifier region is inverted, is located 5' to the product coding region and overlaps with the product coding region, and the second identifier region is inverted and is located 3' to the product coding region. As a non-limiting example, the first identifier region is inverted and is located 5' to the product coding region and the second identifier region is inverted, is located 3' to the product coding region and overlaps with the product coding region. As a non-limiting example, the first identifier region is inverted and is located 5' to the product coding region, and the second identifier region is inverted and is located 3' to the product coding region, and both of the first and second identifier regions overlap with the product coding region.

[0234] As a non-limiting example, the first identifier region is located 5' to the product coding region and overlaps with the product coding region, and the second identifier region is inverted and is located 3' to the product coding region. As a non-limiting example, the first identifier region is located 5' to the product coding region and the second identifier region is inverted, is located 3' to the product coding region and overlaps with the product coding region. As a non-limiting example, the first identifier region is located 5' to the product coding region and the second identifier region is inverted and is located 3' to the product coding region, and both of the first and second identifier regions overlap with the product coding region.

[0235] As a non-limiting example, the first and second identifier regions are both inverted and are located 5' to the product coding region, and the second identifier region overlaps with the product coding region. As a non-limiting example, the first and second identifier regions are located 5' to the product coding region and the first identifier region is inverted, and the second identifier region overlaps with the product coding region. As a non-limiting example, the first and second identifier regions are located 5' to the product coding region and the second identifier region is inverted and overlaps with the product coding region. As a non-limiting example, the first and second identifier region are both inverted and located 3' to the product coding region, and the first identifier region overlap with the product coding region. As a non-limiting example, the first and second identifier regions are located 3' to the product coding region and the first identifier region is inverted and overlaps with the product coding region. As a non-limiting example, the first and second identifierregions are located 3' to the product coding region and the second identifier region is inverted, and the first product coding region overlap with the product coding region.

[0236] In some embodiments, at least one identifier moiety may be associated with the benchmark polynucleotide construct. The benchmark polynucleotide construct may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more identifier moieties associated with the benchmark polynucleotide construct which may be the same moiety or different moieties associated with the benchmark polynucleotide construct. Each identifier moiety may independently be located on the flanking region 5' to the product coding region, on the flanking region 3' to the product coding region, or the location of the identifier moiety may span the 5' end or the 3'end of the product coding region and a flanking region. In some aspects the location of the identifier moiety may include one or more nucleotides of the product coding region such as, but not limited to, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides or more than 50 nucleotides. In some aspects the location of the identifier moiety may include one or more nucleotides of the product coding region such as, but not limited to, 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 2-7 nucleotides, 3-7 nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides, 7-10 nucleotides, 1-15 nucleotides, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10-15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12-20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nucleotides, 3-25 nucleotides, 5-25 nucleotides, 7-25 nucleotides, 10-25 nucleotides, 12-25 nucleotides, 15-25 nucleotides, 17-25 nucleotides, 20-25 nucleotides, 1-30 nucleotides, 2-30 nucleotides, 3-30 nucleotides, 5-30 nucleotides, 7-30 nucleotides, 10-30 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, 20-30 nucleotides, 25-30 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-35 nucleotides, 2-35 nucleotides, 3-35 nucleotides, 5-35 nucleotides, 7-35 nucleotides, 10-35 nucleotides, 12-35 nucleotides, 15-35 nucleotides, 17-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, 30-35 nucleotides, 1-40 nucleotides, 2-40 nucleotides, 3-40 nucleotides, 5-40 nucleotides, 7-40 nucleotides, 10-40 nucleotides, 12-40 nucleotides, 15-40 nucleotides, 17-40 nucleotides, 20-40 nucleotides, 25-40 nucleotides, 30-40 nucleotides, 35-40 nucleotides, 1-45 nucleotides, 2-45 nucleotides, 3-45 nucleotides, 5-45 nucleotides, 7-45 nucleotides, 10-45 nucleotides, 12-45nucleotides, 15-45 nucleotides, 17-45 nucleotides, 20-45 nucleotides, 25-45 nucleotides, 30-45 nucleotides, 35-45 nucleotides, 40-45 nucleotides, 1-50 nucleotides, 2-50 nucleotides, 3-50 nucleotides, 5-50 nucleotides, 7-50 nucleotides, 10-50 nucleotides, 12-50 nucleotides, 15-50 nucleotides, 17-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides, 30-50 nucleotides, 35-50 nucleotides, 40-50 nucleotides, or 45-50 nucleotides.

[0237] In some embodiments, one identifier moiety may be associated with the benchmark polynucleotide construct. As a non-limiting example, the identifier moiety may be associated with the benchmark polynucleotide construct on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the identifier moiety may be associated with the benchmark polynucleotide construct on the 5' flanking region. As a non-limiting example, the identifier moiety may be associated with the benchmark polynucleotide construct on the 3' flanking region. As a non-limiting example, the identifier moiety may be associated with the benchmark polynucleotide construct on the 3' end of the benchmark polynucleotide construct. As a non-limiting example, the identifier moiety may be associated with the benchmark polynucleotide construct on the product coding region. As a non-limiting example, the benchmark polynucleotide construct comprises an identifier moiety and the location of the identifier moiety spans the 5' end of the product coding region and the 5' flanking region. As a non-limiting example, the benchmark polynucleotide construct comprises an identifier moiety and the location of the identifier moiety spans the 3' end of the product coding region and the 3' flanking region.

[0238] In some embodiments, two identifier moieties are associated with the benchmark polynucleotide construct. As a non-limiting example, the first identifier moiety and the second identifier moiety are located on the 5' flanking region. As a non-limiting example, the first identifier moiety and the second identifier moiety are located on the product coding region. As a non-limiting example, the first identifier moiety and the second identifier moiety are located on the 3' flanking region. As a non-limiting example, the first identifier moiety and the second identifier moiety are located on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the first identifier moiety and the second identifier moiety are located on the 3' end of the benchmark polynucleotide construct.

[0239] As a non-limiting example, the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 5' flanking region. As a non-limiting example, the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the product coding region. As a non-limiting example, the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 3' flanking region. As a non-limiting example, the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 5' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 3' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.

[0240] As a non-limiting example, the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the product coding region. As a non-limiting example, the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the 3' flanking region. As a non-limiting example, the first identifier moiety is located on the 5' flanking region and the location of the second identifier moiety spans the 5' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the 5' flanking region and the location of the second identifier moiety spans the 3' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.

[0241] As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 5' flanking region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the product coding region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the location of the second identifier moiety spans the 3' flanking region and the product coding region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' flanking region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.

[0242] As a non-limiting example, the first identifier moiety is located on the product coding region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the first identifier moiety is located on the product coding region and the second identifier moiety is located on the 5' flanking region. As a non-limiting example, the first identifier moiety is located on the product coding region and the location of the second identifier moiety spans the 5' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the product coding region and the location of the second identifier moiety spans the 3' flanking region and the product coding region. As a non-limiting example, thefirst identifier moiety is located on the product coding region and the second identifier moiety is located on the 3' flanking region. As a non-limiting example, the first identifier moiety is located on the product coding region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.

[0243] As a non-limiting example, the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 5' flanking region. As a non-limiting example, the location of the first identifier moiety spans the 3' flanking region and the product coding region and the location of the second identifier moiety spans the 5' flanking region and the product coding region. As a non-limiting example, the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the product coding region. As a non-limiting example, the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 3' flanking region. As a non-limiting example, the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 3'end of the benchmark polynucleotide construct.

[0244] As a non-limiting example, the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 5' flanking region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the product coding region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the location of the second identifier moiety spans the 3' flanking region and the product coding region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' flanking region. As a non-limiting example, the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.

[0245] As a non-limiting example, the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the 5' flanking region. As a non-limiting example, the first identifier moiety is located on the 3' flanking region and the location of the second identifier moiety spans the 5' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the product coding region. As a non-limiting example, the first identifier moiety is located on the 3' flanking region and the location of the second identifier moiety spans the 3' flanking region and the product coding region.As a non-limiting example, the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.

[0246] As a non-limiting example, the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct. As a non-limiting example, the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 5' flanking region. As a non-limiting example, the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 5' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the product coding region. As a non-limiting example, the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 3' flanking region and the product coding region. As a non-limiting example, the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 3' flanking region.

[0247] In some embodiments, three identifier moieties are associated with the benchmark polynucleotide construct. In some embodiments, four identifier moieties are associated with the benchmark polynucleotide construct. In some embodiments, five identifier moieties are associated with the benchmark polynucleotide construct. In some embodiments, six identifier moieties are associated with the benchmark polynucleotide construct. In some embodiments, seven identifier moieties are associated with the benchmark polynucleotide construct. In some embodiments, eight identifier moieties are associated with the benchmark polynucleotide construct. In some embodiments, nine identifier moieties are associated with the benchmark polynucleotide construct. In some embodiments, ten identifier moieties are associated with the benchmark polynucleotide construct.

[0248] In some embodiments, the product coding region encodes a biologically active molecule such as, but not limited to a therapeutic protein or an antigen. As used herein, the term "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. In some embodiments, the CROI encodes one or more prophylactically- or therapeutically-active proteins, polypeptides, or other factors. As a non- limiting example, the CROI may encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer. As another non-limiting example, the CROI may encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., CROI encodes Dystrophin), cardiovascular disease (e.g., CROI encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., CROI encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., CROI encodes GlyRal), an enkephalin, or a glutamate decarboxylase (e.g., CROI encodes GAD65, GAD67, or another isoform), lung disease (e.g., CROI encodes CFTR),hemophilia (e.g., CROI encodes Factor VIII or Factor IX), neoplasia (e.g., CROI encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notchl, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HI Fla, HIF3a, Met, HRG, Bcl2, PPARalpha, PPAR gamma, WT1 (Wilms Tumor), FGF Receptor Family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (Androgen Receptor), TSG101, IGF, IGF Receptor, Igfl (4 variants), Igf2 (3 variants), Igfl Receptor, Igf2 Receptor, Bax, Bcl2, caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), Kras, Ape), age-related macular degeneration (e.g., CROI encodes Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldlr), schizophrenia (e.g. Neuregulin (Nrgl), Erb4 (receptor for Neuregulin), Complexin-l (Cplxl), Tphl Tryptophan hydroxylase, Tph2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, 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-BP-global instability), VLDLR (Alzheimer's), Atxn7, Atxn10), fragile X syndrome (e.g., CROI encodes FMR2, FXRI, FXR2, mGLUR5), secretase related disorders (e.g., CROI encodes APH-1 (alpha and beta), Presenilin (Psenl), nicastrin (Ncstn), PEN-2), ALS (e.g., CROI encodes SOD1, ALS2, STEX, FUS, TARD BP, VEGF (VEGF-a, VEGF-b, VEGF-c)), autism (e.g., CROI encodes Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1), Alzheimer's disease (e.g., CROI encodes El, CHIP, UCH, UBB, Tau, LRP, PICALM, Clusterin, PS1, SORL1, CR1, Vldlr, Ubal, Uba3, CHIP28 (Aqpl, Aquaporin 1), Uchll, Uchl3, APP), inflammation (e.g., CROI encodes IL- 10, IL-1 (IL-Ia, IL-Ib), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cll), Parkinson's Disease (e.g., x-Synuclein, DJ-1, LRRK2, Parkin, PINK1), blood and coagulation disorders, such as, e.g., anemia, bare lymphocyte syndrome, bleeding disorders, hemophagocytic lymphohistiocytosis disorders, hemophilia A, hemophilia B, hemorrhagic disorders, leukocyte deficiencies and disorders, sickle cell anemia, and thalassemia (e.g., CROI encodes CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, FANCA, FAC A, FA1, FA, FA A, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BR1PI, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596, PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3, F8, FSC, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1), B-cell non-Hodgkin lymphoma or leukemia (e.g., CROI encodes BCL7A, BCL7, ALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP,CHIC2, BTL, FLT3, KIT, PBT, LPP, NPMI, NUP214, D9S46E, CAN, CAIN, RUNXI, CBFA2, AML1, WHSC1LI, NSD3, FLT3, AF1Q, NPMI, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABLI, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN), inflammation and immune related diseases and disorders (e.g., CROI encodes KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG, CXCL12, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation (e.g., CROI encodes IL-10, IL-1 (IL-IA, IL-IB), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL- 17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cII), JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4), metabolic, liver, kidney and protein diseases and disorders (e.g., CROI encodes TTR, PALB, APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, MODY3, SCOD1, SCOl, CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63), muscular / skeletal diseases and disorders (e.g., CROI encodes DMD, BMD, MYF6, LMNA, LMN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMDIA, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMNDl, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1), neurological and neuronal diseases and disorders (e.g., CROI encodes SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c), APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLOl, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP,SCA17, SNCA, NACP, PARK1, PARK4, DJI, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARKl, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16,MRX79, x-Synuclein, DJ- 1, Neuregulin-l (Nrgl), Erb4 (receptor for Neuregulin), Complexin-l (Cplxl), Tphl Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drdla), SLC6A , DAOA, DTNBP1, Dao (Daol), APH-l(alpha and beta), Presenilin (Psenl), Nicastrin, (Ncstn), PEN-2, Nosl, Parpl, Natl, Nat2, HTT, SBMA / SMAX1 / AR, FXN / X25, ATX3, TXN, ATXN2, DMPK, Atrophin-1, Atnl, CBP, VLDLR, Atxn7, and AtxnlO), and ocular diseases and disorders (e.g., Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin-D, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYAI, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYAI, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR, GLCIA, JO AG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NPG, CYP1BI, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, and VMD2).

[0249] In some embodiments, the product coding region of the RNA payloads described herein encodes a factor that can affect the differentiation of a cell. As a non-limiting example, 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.

[0250] In some embodiments, the product coding region of the RNA payloads described herein encodes a factor for transdifferentiating cells. Non-limiting examples of factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurrl, LmxlA, Bm2, Mytll, NeuroDl, FoxA2 for neural cells; and Hnf4a, Foxal, Foxa2 or Foxa3 for hepatic cells.

[0251] Additional product coding regions of the RNA payloads described herein are described elsewhere. A. Nucleic acid payloads

[0252] In various embodiments, the LNP compositions described herein can be used to deliver a nucleic acid or polynucleotide payload, e.g., a DNA HDR donor, a linear or circular mRNA, or a chimeric DNA / RNA guide.

[0253] In some embodiments, a LNP is capable of delivering a polynucleotide to a target cell, tissue, or organ. A polynucleotide, in its broadest sense of the term, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. RNAs useful in the compositions and methods described herein can be selected from the group consisting of but are not limited to, shortimers, antagomirs, antisense, ribozymes, short interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, a polynucleotide is mRNA. In some embodiments, a polynucleotide is circular RNA. In some embodiments, a polynucleotide encodes a protein, e.g., a vaccine antigen, a therapeutic protein, or a nucleobase editing enzyme. A polynucleotide may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[0254] In other embodiments, a polynucleotide is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0255] In some embodiments, a polynucleotide is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.

[0256] A polynucleotide may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'- UTR), at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, a polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, a polynucleotide (e.g., an mRNA) may include a 5'cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and / or a polyadenylation signal.

[0257] In various embodiments, the nucleic acid payloads may contain one or more modifications. Such modifications include various chemical and / or structural modifications. For example, in thecase of RNA, the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and / or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures). To date, hundreds of different RNA modifications have been characterized. Among them, several RNA modifications, including N6-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), 8-oxo-7,8- dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m5C), and N4-acetylcytidine (ac4C), have been shown to regulate mRNA stability, consequently affecting diverse cellular and biological processes. Any known modification to RNA or DNA is contemplated herein.

[0258] In some embodiments, a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3'-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and / or the coding region, 5'-UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5- substituted uridine (e.g., 5-methoxyu ridine), a 1-substituted pseudouridine (e.g., 1-methyl pseudouridine or 1-ethyl-pseudouridine), and / or a 5-substituted cytidine (e.g., 5-methyl-cytidine). In some embodiments, a polynucleotide contains only naturally occurring nucleosides. Nucleic acid modifications are well known in the art and are further discussed in the following references: (1) Crooke ST, Witztum JL, Bennett CF, Baker BF. RNA-Targeted Therapeutics. Cell Metab.2018 Apr 3;27(4):714-739. doi: 10.1016 / j.cmet.2018.03.004. Erratum in: Cell Metab.2019 Feb 5;29(2):501. PMID: 29617640; (2) JP, Wen W, Zhang F, Oberg KC, Zhang L, Cheng T, Zhang XB. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res.2021 Jan 25;49(2):969-985. doi: 10.1093 / nar / gkaa1251. PMID: 33398341; PMCID: PMC7826255; (3) Pradeep SP, Malik S, Slack FJ, Bahal R. Unlocking the potential of chemically modified peptide nucleic acids for RNA-based therapeutics. RNA.2023 Apr;29(4):434-445. doi: 10.1261 / rna.079498.122. Epub 2023 Jan 18. PMID: 36653113; PMCID: PMC10019372; (4) Haruehanroengra P, Zheng YY, Zhou Y, Huang Y, Sheng J. RNA modifications and cancer. RNA Biol.2020 Nov;17(11):1560-1575. doi: 10.1080 / 15476286.2020.1722449. Epub 2020 Feb 7. PMID: 31994439; PMCID: PMC7567502; (5) Heidenreich O, Pieken W, Eckstein F. Chemically modified RNA: approaches and applications. FASEB J.1993 Jan;7(1):90-6. doi: 10.1096 / fasebj.7.1.7678566. PMID: 7678566; (6) Zhang HY, Du Q, Wahlestedt C, Liang Z. RNA Interference with chemically modified siRNA. Curr Top Med Chem.2006;6(9):893-900. doi: 10.2174 / 156802606777303676. PMID: 16787282; (7) Jin G, Xu M, Zou M, Duan S. The Processing, Gene Regulation, Biological Functions, and Clinical Relevance of N4-Acetylcytidine on RNA: A Systematic Review. Mol Ther Nucleic Acids.2020 Jun 5;20:13-24. doi: 10.1016 / j.omtn.2020.01.037. Epub 2020 Feb 8. PMID: 32171170; PMCID: PMC7068197; (8) Gao M, Zhang Q, Feng XH, Liu J. Synthetic modified messenger RNA for therapeutic applications. Acta Biomater.2021 Sep 1;131:1-15. doi: 10.1016 / j.actbio.2021.06.020. Epub 2021 Jun 13. PMID: 34133982; PMCID: PMC8198544; (9) Filippova JA, Semenov DV, Juravlev ES, Komissarov AB, Richter VA, Stepanov GA. Modern Approaches for Identification of Modified Nucleotides in RNA. Biochemistry (Mosc).2017 Nov;82(11):1217-1233. doi: 10.1134 / S0006297917110013. PMID: 29223150; (10) Röthlisberger P, Berk C, Hall J. RNA Chemistry for RNA Biology. Chimia (Aarau).2019 May 29;73(6):368-373. doi: 10.2533 / chimia.2019.368. PMID: 31118118; and (11) Elkhalifa D, Rayan M, Negmeldin AT, Elhissi A, Khalil A. Chemically modified mRNA beyond COVID-19: Potential preventive and therapeutic applications for targeting chronic diseases. Biomed Pharmacother.2022 Jan;145:112385. doi: 10.1016 / j.biopha.2021.112385. Epub 2021 Oct 28. PMID: 34915673; PMCID: PMC8552589; (12) Boo SH, Kim YK. The emerging role of RNA modifications in the regulation of mRNA stability. Exp Mol Med.2020 Mar;52(3):400-408. doi: 10.1038 / s12276-020-0407-z. Epub 2020 Mar 24. PMID: 32210357; PMCID: PMC7156397; (13) Varshney D, Spiegel J, Zyner K, Tannahill D, Balasubramanian S. The regulation and functions of DNA and RNA G-quadruplexes. Nat Rev Mol Cell Biol.2020 Aug;21(8):459-474. doi: 10.1038 / s41580-020-0236-x. Epub 2020 Apr 20. PMID: 32313204; PMCID: PMC7115845; each of which are incorporated herein by reference in their entireties.

[0259] In some cases, a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the poly nucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.

[0260] In some embodiments, a polynucleotide molecule, formula, composition or method associated therewith comprises one or more polynucleotides comprising features as described in WO2002 / 098443, WO2003 / 051401, WO2008 / 052770, WO2009 / 127230, WO2006 / 122828, WO2008 / 083949, WO2010 / 088927, WO2010 / 037539, WO2004 / 004743, WO2005 / 016376, WO2006 / 024518, WO2007 / 095,976, WO2008 / 014979, WO2008 / 077592, WO2009 / 030481, WO2009 / 095226, WO2011 / 069586, WO2011 / 026641, WO2011 / 144358, WO2012 / 019780, WO2012 / 013326, WO2012 / 089338, WO2012 / 113513, WO2012 / 116811, WO2012 / 116810, WO2013 / 113502, WO2013 / 113501, WO2013 / 113736, WO2013 / 143698, WO2013 / 143699, WO2013 / 143700, WO2013 / 120626, WO2013 / 120627, WO2013 / 120628, WO2013 / 120629, WO2013 / 174409, WO2014 / 127917, WO2015 / 024669, WO2015 / 024668, WO2015 / 024667, WO2015 / 024665, WO2015 / 024666, WO2015 / 024664, WO2015 / 101415, WO2015 / 101414, WO2015 / 024667, WO2015 / 062738, WO2015 / 101416, all of which are incorporated by reference herein.

[0261] In some embodiments, a polynucleotide comprises one or more microRNA binding sites. In some embodiments, a microRNA binding site is recognized by a microRNA in a non-target organ. In some embodiments, a microRNA binding site is recognized by a microRNA in the liver. In some embodiments, a microRNA binding site is recognized by a microRNA in hepatic cells. B. Linear mRNA payloads

[0262] In various embodiments, the LNP-based RNA vaccines, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver an RNA payload that is a linear mRNA molecule.

[0263] In various embodiments, the LNP-based pharmaceutical compositions described herein, e.g., LNP-based gene editing systems, may include one or more linear mRNA molecules or linear mRNA payloads. In various embodiments, the mRNA payloads may encode one or more components of the herein described gene editing systems. For example, an mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., TALENS and zinc finger-binding domains) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB).

[0264] mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and / or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosinedeaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.

[0265] Ribonucleic acid (RNA) is a molecule that is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. The nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). Generally, 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. For example, the length of an RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., lncRNA), 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., lncRNA) sequence.

[0266] In various embodiments, the LNP-based RNA vaccines, RNA therapeutics, gene editing systems and pharmaceutical compositions thereof described herein can be used to deliver a mRNA payload that is a linear mRNA molecule. In embodiments, the mRNA payload may comprise one or more nucleotide sequences that encode a product of interest, such as, but not limited to a vaccine antigen, a component of a gene editing system (e.g., an endonuclease, a prime editor, etc.) and / or a therapeutic protein.

[0267] In some embodiments, the RNA payload may be a linear mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a protein of interest and which is capable of being translated to produce the encoded protein of interest in vitro, in vivo, in situ or ex vivo.

[0268] Generally, a mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. In some aspects, 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. As used herein, 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 affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G".

[0269] Generally, a coding region of interest in an mRNA used herein may encode a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the mRNA may 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 mRNA may encode a peptide of at least 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or a peptide that is no longer than 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0270] Generally, the length of the region of the mRNA encoding a product 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, 1000, 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).

[0271] In some embodiments, the mRNA has a total length that spans 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 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1 ,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1 ,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides).

[0272] In some embodiments, the region or regions flanking the region encoding the product 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).

[0273] In some embodiments, 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). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this embodiment, 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.

[0274] In some embodiments, 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. In some embodiments, the caping sequence is absent.

[0275] In some embodiments, 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.

[0276] In some embodiments, 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.

[0277] In some embodiments, 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. Untranslated Regions (UTRs)

[0278] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems and pharmaceutical compositions thereof described herein, may comprise at least one untranslated region (UTR) which flanks the region encoding the product of interest and / or is incorporated within the mRNA molecule. UTRs are transcribed by not translated. The mRNA payloads can include 5’ UTR sequences and 3’ UTR sequences, as well as internal UTRs.

[0279] The RNA payloads of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. Where nucleic acids are designed to encode at least one polypeptide of interest, the nucleic acid may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, 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. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the RNA payload molecules (e.g., linear and circular mRNA molecules) of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5′UTR and 3′UTR sequences are known and available in the art.

[0280] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise at least one UTR that may be selected from any UTR sequence listed in Tables 19 or 20 of U.S. Patent No.10,709,779, which is incorporated herein by reference. 5' UTR regions

[0281] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise at least one 5′ UTR.

[0282] In an embodiment, the 5’ UTR comprises a sequence provided in Table (II) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a 5’ UTR sequence provided in Table (II), or a variant or a fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, or six nucleotides of the 5’ UTR sequence provided in Table (II)). In an embodiment, the 5’ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29.

[0283] Table (II) – Exemplary nucleotide sequences of 5’ UTRs 5’ UTR Nucleotide Sequence Sequence Identifier ggaaaucgca aaauuugcuc uucgcguuag auuucuuuua SEQ ID NO: 1ggaaacuuua uuuaguguua cuuuauuuuc uguuuauuug SEQ ID NO: 11 uguuucuuca guggguuugu ucuaauuucc uuggccgccggaaauaaga gagaaaagaa gaguaagaag aaauauaaga SEQ ID NO: 28 gccaaaaaaa aaaaaccfirst codon of an mRNA transcript translated by a ribosome). A 5′ UTR does not encode a protein (is non-coding). Natural 5′UTRs have features that play roles in translation initiation. They harbor signatures like 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 (SEQ ID NO: 30), 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. 5’ UTR sequences are also known to be important for ribosome recruitment to the mRNA and have been reported to play a role in translation (Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6). In addition, 5’ UTR sequences may confer increased half-life, increased expression and / or increased activity of a polypeptide encoded by the RNA payload described herein.

[0285] In various embodiments, the RNA payload constructs contemplated herein may include 5’UTRs that are found in nature and those that are not. For example, the 5’UTRs can be synthetic and / or can be altered in sequence with respect to a naturally occurring 5’UTR. Such altered 5’UTRs can include one or more modifications relative to a naturally occurring 5’UTR, such as, for example, an insertion, deletion, or an altered sequence, or the substitution of one or more nucleotide analogs in place of a naturally occurring nucleotide.

[0286] 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.

[0287] 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.

[0288] In some embodiments of the disclosure, a 5′ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different mRNA. In another embodiment, a 5′ UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have beenmutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5′ UTRs include Xenopus or human derived alpha-globin or beta-globin (e.g., US8,278,063 and US9,012,219), human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus. CMV immediate-early 1 (IE1) gene (see US20140206753 and WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO: 31) (WO2014144196) may also be used. In another embodiment, 5′ UTR of a TOP gene is a 5′ UTR of a TOP gene lacking the 5′ TOP motif (the oligopyrimidine tract) (e.g., WO / 2015101414, WO2015101415, WO / 2015 / 062738, WO2015024667, WO2015024667; 5′ UTR element derived from ribosomal protein Large 32 (L32) gene (WO / 2015101414, WO2015101415, WO / 2015 / 062738)), 5′ UTR element derived from the 5′UTR of an hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5′ UTR element derived from the 5′ UTR of ATP5A1 (WO2015024667) can be used. In one embodiment, an internal ribosome entry site (IRES) is used as a substitute for a 5′ UTR.

[0289] In some embodiments, a 5′ UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 32 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC), SEQ ID NO: 33 (GGGAAATAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC), SEQ ID NO: 34 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC) and SEQ ID NO: 35 (GGGAAATAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC). 3' UTR regions

[0290] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise at least one 3′ UTR.3′ UTRs may be heterologous or synthetic.

[0291] A 3′ UTR is region of an mRNA that is directly downstream (3′) from the stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3′ UTR does not encode a protein (is non-coding). Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) 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) (SEQ ID NO: 36) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-α. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well- studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented toincrease 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 will lead to HuR binding and thus, stabilization of the message in vivo.

[0292] 3' UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) 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. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. 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 will lead to HuR binding and thus, stabilization of the message in vivo.

[0293] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of the mRNA payloads described herein. For example, 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. Alternatively, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0294] In some embodiments, 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. As a non-limiting example, the feature can be a UTR. As another example, the feature can be introns or portions of introns sequences.

[0295] Those of ordinary skill in the art will understand that 5′ UTRs that are heterologous or synthetic may be used with any desired 3′ UTR sequence. For example, a heterologous 5′ UTR may be used with a synthetic 3′ UTR with a heterologous 3′ UTR.

[0296] Non-UTR sequences may also be used as regions or subregions within an RNA payload construct. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.

[0297] Combinations of features may be included in flanking regions and may be contained within other features. For example, the polypeptide coding region of interest in an mRNA payload may be flanked by a 5′ UTR which may contain a strong Kozak translational initiation signal and / or a 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / ordifferent genes such as the 5′ UTRs described in US Patent Application Publication No.20100293625 and PCT / US2014 / 069155, herein incorporated by reference in its entirety

[0298] It should be understood that any UTR from any gene may be incorporated into the regions of an RNA payload molecule (e.g., a linear mRNA). Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ UTR 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. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.

[0299] In some embodiments, a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.

[0300] It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

[0301] In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[0302] The untranslated region may also include translation enhancer elements (TEE). As a non- limiting example, the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art. 5' Capping

[0303] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise a 5’ cap structure.

[0304] 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. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.

[0305] 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.

[0306] 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.

[0307] Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0308] 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.

[0309] 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.

[0310] For example, the Anti-Reverse Cap Analog (ARCA) 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).

[0311] Another exemplary cap is 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).

[0312] While 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.

[0313] mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, 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). For example, 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. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro- inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. 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).

[0314] In some embodiments, the 5' terminal caps may include endogenous caps or cap analogs.

[0315] In some embodiments, 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. IRES Sequences

[0316] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise one or more IRES sequences.

[0317] In some embodiments, the mRNA may contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, 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 onefunctional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes. Non-limiting examples of IRES sequences that can be used include without limitation, those from picornaviruses (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).

[0318] In some embodiments, the IRES is from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV- Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BNS, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVBS, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G. Poly-A tails and 3’ stabilizing region

[0319] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise a poly-A tail.

[0320] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecules in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the free 3' hydroxyl end. The process, called polyadenylation, adds a poly-A tail of a certain length.

[0321] In some embodiments, 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) and no more than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 nucleotides in length. In some embodiments, 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 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0322] In some embodiments, 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.

[0323] In this context 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. In this context, 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. Further, engineered binding sites and conjugation of mRNA for poly-A binding protein may enhance expression.

[0324] Additionally, 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.

[0325] In some embodiments, 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 sequencesin both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. Stop Codons

[0326] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise one or more translation stop codons. Translational stop codons, UAA, UAG, and UGA, are an important component of the genetic code and signal the termination of translation of an mRNA. During protein synthesis, stop codons interact with protein release factors and this interaction can modulate ribosomal activity thus having an impact translation (Tate WP, et al., (2018) Biochem Soc Trans, 46(6):1615-162).

[0327] A stop element as used herein, refers to a nucleic acid sequence comprising a stop codon. The stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA. In an embodiment, a stop element comprises two consecutive stop codons. In an embodiment, a stop element comprises three consecutive stop codons. In an embodiment, a stop element comprises four consecutive stop codons. In an embodiment, a stop element comprises five consecutive stop codons.

[0328] In some embodiments, 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.

[0329] In other embodiments, the stop codon may be selected from one or more of the following stop elements of Table (III): Table (III): Additional stop elements Nucleotide sequence (5’ to 3’) Sequence IdentifierUAAAGCUCCSEQ ID NO: 47UAGGGUUAASEQ ID NO: 48codon. In a further embodiment the addition stop codon may be TAA. MicroRNA binding sites and other regulatory elements

[0331] In various embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein, may comprise one or more regulatory elements, including, but not limited to microRNA (miRNA) binding sites, structured mRNA sequences and / or motifs, artificial binding sites to bind to endogenous nucleic acid binding molecules, and combinations thereof. Chemically unmodified nucleotides

[0332] In some embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein are not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT). Chemically modified nucleotides

[0333] In some embodiments, the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein comprise, in some embodiments, comprises at least one chemical modification.

[0334] The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties. With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids. Polypeptides, as provided herein, are also considered “modified” of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.

[0335] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise various (more than one) different modifications. In some embodiments, a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside ornucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).

[0336] Modifications of polynucleotides include, without limitation, those described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications. Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).

[0337] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post- synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.

[0338] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[0339] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non- standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker may be incorporated into polynucleotides of the present disclosure.

[0340] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0341] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of pseudouridine (ψ), N1- methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0342] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine and α-thio-adenosine. In some embodiments, polynucleotides includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0343] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl- pseudouridine (m1ψ). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA such as mRNA polynucleotides)comprise 2-thiouridine (s2U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine (mo5U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyl uridine. In some embodiments polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A) and 5-methyl-cytidine (mC).

[0344] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modifiedwith 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0345] Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl- cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl- cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0346] In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and In some embodiments, a modified nucleobase is a modified cytosine. nucleosides having a modified uridine include 5-cyano uridine, and 4′-thio uridine.

[0347] The polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0348] The polynucleotide may contain 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 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0349] The polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracilcan be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). C. Circular mRNA payloads

[0350] In various embodiments, the LNP-based RNA vaccines, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver an RNA payload that is a circular mRNA molecule or “oRNA.” The circular mRNA molecule may encode a CROI, such as a vaccine antigen, cancer antigen, or therapeutic protein as described in this specification.

[0351] In various embodiments, the LNP-based pharmaceutical compositions described herein, e.g., LNP-based gene editing systems, may include one or more circular mRNA molecules or “oRNAs.” In various embodiments, the circular mRNA payloads may encode one or more components of the herein described gene editing systems or other therapeutic protein of interest. For example, a circular mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., TALENS and zinc finger-binding domains) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB).

[0352] The circular mRNA payloads may also encode, depending upon the nature of the gen...

Claims

CLAIMS 1. A compound of Formula (TL’’)or a A1is selected from CH and N; A2is selected from CH and N; L1is optionally substituted C1-C8 aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-;, wherein the bond marked with an "*" is attached to L1; of -O-, -NR-, and -S-;group of a bond, -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -NRC(O)-, - C(O)NR-,-NRC(O)O-, or -OC(O)NR-; each Z4is independently selected from =CR- and =N-; L2is optionally substituted C1-C8aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R1is selected from the group consisting of -OH, -OAc, -NR2, ,,XZis a bond or optionally substituted C1-C6aliphatic; RZis NR2or OH; X1and XAare each independently a bond or optionally substituted C1-C6aliphatic; Y1is selected from the group consisting of or, and bond; wherein the bond marked with an "*" is attached to X1; and X6is independently a bond or optionally substituted C1-C12 aliphatic;each of Y2, Y3, and Y4is independently selected from the group consisting of or; wherein the bond marked with an "*" is attached to X2, X3, of X6, as appropriate;and X7is independently optionally substituted C1-C6 aliphatic; R2is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, - NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R3is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R11, wherein one or more methylene linkages of R3are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, - NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R12is -CH(OR13)(OR14), -CH(SR13)(SR14), -CH(R13)(R14), -R15, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14aliphatic-R15, wherein one or more methylene linkages of R12are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, -O-, - NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R6and R7are each independently optionally substituted -C1-C14aliphatic, -R10, or optionally substituted - C1-C14aliphatic-R10; wherein one or more methylene linkages of R6and R7are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-;R8and R9are each independently optionally substituted -C1-C14aliphatic, -R11, or optionally substituted - C1-C14aliphatic-R11; wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R13and R14are each independently optionally substituted -C1-C14aliphatic, -R15, or optionally substituted -C1-C14aliphatic-R15; wherein one or more methylene linkages of R13and R14are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each of R10, R11, and R15is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.

2. The compound of claim A, wherein the compound is of Formula (TL):or a pharmaceutically acceptable salt thereof, wherein: A1is selected from CH and N; A2is selected from CH and N; L1is optionally substituted C1-C8 aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; Z1is selected from the group consisting of,,, wherein the bond marked with an "*" is attached to L1;of -O-, -NR-, and -S-; Z3is selected from the group consisting of a bond, -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -NRC(O)-, - C(O)NR-,-NRC(O)O-, or -OC(O)NR-; each Z4is independently selected from =CR- and =N-; L2is optionally substituted C1-C8 aliphatic, wherein one or more methylene linkages of L1are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; R1is selected from the group consisting of -OH, -OAc, -NR2, ,,a or RZis NR2or OH; X1and XAare each independently a bond or optionally substituted C1-C6aliphatic; Y1is selected from the group consisting of or, and bond; wherein the bond marked with an "*" is attached to X1;and X6is independently a bond or optionally substituted C1-C12aliphatic; each of Y2, Y3, and Y4is independently selected from the group consisting of or; wherein the bond marked with an "*" is attached to X2, X3, of X6, as appropriate;and X7is independently optionally substituted C1-C6 aliphatic; R2is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R10, wherein one or more methylene linkages of R2are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R3is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R11, wherein one or more methylene linkages of R3are eachoptionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R12is -CH(OR13)(OR14), -CH(SR13)(SR14), -CH(R13)(R14), -R15, optionally substituted C5-C18aliphatic, or optionally substituted C1-C14aliphatic-R15, wherein one or more methylene linkages of R12are each optionally and independently replaced with an optionally substituted C3-C8cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R6and R7are each independently optionally substituted -C1-C14 aliphatic, -R10, or optionally substituted -C1-C14 aliphatic-R10; wherein one or more methylene linkages of R6and R7are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R8and R9are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8and R9are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R13and R14are each independently optionally substituted -C1-C14 aliphatic, -R15, or optionally substituted -C1-C14 aliphatic-R15; wherein one or more methylene linkages of R13and R14are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each of R10, R11, and R15is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10, R11or R15are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.

3. The compound of claim 2, wherein the compound is of Formula (TL-A), or a pharmaceutically acceptable salt thereof.

4. The compound of claim 2, wherein the compound is of Formula (TL-B):or a pharmaceutically acceptable salt thereof.

5. The compound of claim 2, wherein the compound is of Formula (TL-B’):or a pharmaceutically acceptable salt thereof.

6. The compound of claim 2, wherein the compound is of Formula (TL-C):or a pharmaceutically acceptable salt thereof.

7. The compound of claim 2, wherein the compound is of Formula (TL-D):or a pharmaceutically acceptable salt thereof.

8. The compound of claim 2, wherein the compound is of Formula (TL-E):or a pharmaceutically acceptable salt thereof.

9. The compound of claim 2, wherein the compound is of Formula (TL-E’):or a pharmaceutically acceptable salt thereof.

10. The compound of claim 2, wherein the compound is of Formula (TL-E’’):or a pharmaceutically acceptable salt thereof.

11. The compound of claim 2, wherein the compound is of Formula (TL-F):or a pharmaceutically acceptable salt thereof.

12. The compound of claim 2, wherein the compound is of Formula (TL-F’):or a pharmaceutically acceptable salt thereof.

13. The compound of claim 2, wherein the compound is of Formula (TL-F’’):or a pharmaceutically acceptable salt thereof.

14. The compound of any one of claims 1-4, wherein R1is -OH.

15. The compound of any one of claims 1-4, .

16. The compound of any one of claims 1-4, wherein R1is .

17. The compound of any of claims 2-16, wherein X1is18. The compound of any of claims 2-16, wherein X1is optionally substituted C2-C4alkylene.

19. The compound of claim 17, wherein X1is selected from the group consisting of -(CH2)2-, -(CH2)3- and -(CH2)4-.

20. The compound of claim 19, wherein X1is -(CH2)2-.

21. The compound of claim 19, wherein X1is -(CH2)3-.

22. The compound of claim 19, wherein X1is -(CH2)4-.

23. The compound of any one of claims 1-22, wherein L1is unsubstituted C1-C8aliphatic, wherein one methylene linkage of L1is optionally replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-. ,25. The compound of claim 23, wherein The compound of claim 22, wherein , or26. The compound of any one of claims 1-25, wherein L2is unsubstituted C1-C8aliphatic, wherein one methylene linkage of L1is optionally replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-. ,.

29. The compound of any one of theZ1is selected from * , with an30. The compound of any one of the preceding claims, wherein Z1is selected from * .

31. any one is selected from .

32. The compound of any one of the preceding claims wherein Y1is a bond.

33. The compound of any one of the preceding claims, wherein Y1 .

34. The compound of any one of the preceding claims, wherein Y1is a bond.

35. The compound of any one of the preceding claims, wherein XAis a bond.

36. The compound of any one of the preceding claims, wherein XAis optionally substituted C2-C4alkylene.

37. The compound any one of the preceding claims, wherein XAis selected from the group consisting of - (CH2)2-, -(CH2)3- and -(CH2)4-. ,39. The compound of any one of the preceding claims, wherein -X1-Y1-XA-R1 .

40. The compound of any one of the preceding claims, wherein X2is an optionally substituted C4-C10alkylene.

41. The compound of any one of the preceding claims, wherein X2is an unsubstituted C4-C10alkylene.

42. The compound of any one of the preceding claims, wherein X2is an unsubstituted C8alkylene.

43. The compound of any one of the preceding claims, wherein X3is an optionally substituted C4-C10alkylene.

44. The compound of any one of the preceding claims, wherein X3is an unsubstituted C4-C10alkylene.The compound of any one of the preceding claims, wherein X3is an unsubstituted C8 alkylene.

45. The compound of any one of the preceding claims, wherein X6is an optionally substituted C4-C10 alkylene.

46. The compound of any one of the preceding claims, wherein X6is an unsubstituted C4-C10 alkylene.

47. The compound of any one of the preceding claims, wherein X6is an unsubstituted C8 alkylene.

48. The compound of any one of the preceding claims, wherein Y2.

49. The compound of any one of the preceding claims, wherein Y3.

50. The compound of any one of the preceding claims, wherein Y4.

51. The compound of any one of the preceding claims, wherein X4is an optionally substituted C2-C5alkylene.

52. The compound of any one of the preceding claims, wherein X4is an unsubstituted C2-C5alkylene.

53. The compound of any one of the preceding claims, wherein X5is an optionally substituted C2-C5alkylene.

54. The compound of any one of the preceding claims, wherein X5is an unsubstituted C2-C5alkylene.

55. The compound of any one of the preceding claims, wherein X5is an unsubstituted C2alkylene.

56. The compound of any one of the preceding claims, wherein X7is an optionally substituted C2-C5alkylene.

57. The compound of any one of the preceding claims, wherein X7is an unsubstituted C2-C5alkylene.The compound of any one of the preceding claims, wherein X7is an unsubstituted C2alkylene.

58. The compound of any one of the preceding claims, wherein R2is -CH(R6)(R7).

59. The compound of any one of the preceding claims, wherein R2is -CH(OR6)(OR7).

60. The compound of any one of the preceding claims, wherein R2is -CH(SR6)(SR7).

61. The compound of any one of the preceding claims, wherein R2is -R10.

62. The compound of any one of the preceding claims, wherein R2is optionally substituted C1-C14 aliphatic-R10.

63. The compound of any one of the preceding claims, wherein R2is selected from ,.<img src='' class="img-anchor img-center" img-id="IMGF000517_0001" / >67. The compound of any one of the preceding claims, wherein R3is -CH(SR8)(SR9< / sup>).

68. The compound of any one of the preceding claims, wherein R3is -R11< / sup>.

69. The compound of any one of the preceding claims, wherein R3is optionally substituted C1-C14 aliphatic-R11< / sup>.

70. The compound of any one of the preceding claims, wherein R3is selected from ,.<img src='' class="img-anchor img-center" img-id="IMGF000518_0001" / >74. The compound of any one of the preceding claims, wherein R12is -CH(SR13)(SR14< / sup>).

75. The compound of any one of the preceding claims, wherein R12is -R15< / sup>.

76. The compound of any one of the preceding claims, wherein R12is optionally substituted C1-C14 aliphatic-R15< / sup>.

77. The compound of any one of the preceding claims, wherein R12is selected from ,.acceptable salt therefore.

80. A pharmaceutical composition comprising: a) at least one lipid nanoparticle comprising at least one compound of any one of claims 1-79; and b) at least one nucleobase editing system.

81. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a CRISPR-Cas gene editing system.

82. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a prime editing system or components thereof.

83. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a retron editing system.

84. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a TnpB editing system.

85. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises an integrase editing system.

86. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises an integrase editing system.

87. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises an epigenetic editing system.

88. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a gene writing system.

89. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a gene inactivating system.

90. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises zinc finger nuclease.

91. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a TALE Nuclease, a TALE nickase, Zinc Finger (ZF) Nuclease, ZF Nickase, meganuclease, or a combination thereof.

92. The pharmaceutical composition of claim 80, wherein the nucleobase editing system comprises a meganuclease.

93. The pharmaceutical composition of any one of claims 80-92, wherein the at least one lipid nanoparticle further comprises: i) at least one structural lipid; ii) at least one phospholipid; and iii) at least one PEGylated lipid.

94. The pharmaceutical composition of any one of claims 80-93, wherein the at least one structural lipid is selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta- sitosterol-acetate and any combinations thereof.

95. The pharmaceutical composition of any one of claims 80-94, wherein the at least one phospholipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn-phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3- phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3- phospho-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0- 18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy- sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

96. T The pharmaceutical composition of any one of claims 80-95, wherein the at least one PEGylated lipid is selected from those depicted in Table (I-X).

97. The pharmaceutical composition of any one of claims 80-95, wherein the at least one PEGylated lipid is selected from (R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG- PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG- DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEGDMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG- PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE- PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

98. The pharmaceutical composition of any one of claims 80-96, wherein the LNP further comprises at least one additional lipid component selected from 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcarnosine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl- sphingomyelin (SPM) (C18:l), N-lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl- phosphoethanolamine (DHPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl- sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2-dioleoylglyceryl-3- hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}- ethoxy)-ethyl]-3-(3,4,5-1rihydroxy-6-hydroxymethyl-1etrahydro-pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE),dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol- hemisuccinate-Nα-Histidinyl-Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12- pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α-histidinyl-Nα- hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane- carboxamide] (MCC-PE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1- myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB- PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3- [phosphoethanolamine-N-dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn- glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB- phosphatidylethanolamine lipid (Rh-PE), purifiedsoy-derivedmixtureofphospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans-PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3- (bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine.

99. A method of delivering a nucleobase editing system to a subject in need thereof, the methodcomprising administering to the subject the pharmaceutical composition of any one of claims 80-98.

100. The pharmaceutical composition of any one of claims 80-99 for use as a medicament.

101. Use of a pharmaceutical composition of any one of claims 80-99 for the manufacture of a medicament for delivery of a nucleobase editing system.

102. A lipid nanoparticle (LNP) comprising a compound of any one of claims 1-79, or a pharmaceutically acceptable salt thereof.

103. The LNP of claim 102, further comprising: (a) a PEG-lipid (b) a structural lipid; and (c) a non-ionizable lipid and / or a zwitterionic lipid.

104. The LNP of claim 103, wherein the lipid nanoparticle further comprises an additional ionizable lipid, besides a compound of Formula (TL).

105. The LNP of claim 103 or 104, wherein the PEG-lipid is selected from those depicted in Table (I-X).

106. The LNP of claim 103 or 104, wherein the PEG-lipid is selected from the group consisting of PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

107. The LNP of any one of claims 103-106, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, an alpha-tocopherol.

108. The LNP of any one of claims 103-107, wherein the non-ionizable lipid is a phospholipid selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn- glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L- serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

109. The LNP of any one of claims 103-108, further comprising a targeting moiety.

110. The LNP of claim 109, wherein the targeting moiety is an antibody or a fragment thereof.

111. The LNP of any one of claims 103-110, further comprising an active agent.

112. The LNP of claim 111, wherein the active agent is a nucleic acid.

113. The LNP of claim 112, wherein the nucleic acid is a ribonucleic acid.

114. The LNP of claim 113, wherein the ribonucleic acid is at least one ribonucleic acid selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (lncRNA).

115. The LNP of claim 114, wherein the nucleic acid is a messenger RNA (mRNA) or a circular RNA.

116. The LNP of claim 115, wherein the mRNA includes an open reading frame encoding a cancer antigen.

117. The LNP of claim 115, wherein the mRNA includes an open reading frame encoding an immune checkpoint modulator.

118. The LNP of any one of claims 115-117, wherein the mRNA includes at least one motif selected from the group consisting of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and a 5' cap structure.

119. The LNP of claim 112, wherein the nucleic acid is suitable for a genome editing technique.

120. The LNP of claim 119, wherein the genome editing technique is clustered regularly interspaced short palindromic repeats (CRISPR) or transcription activator-like effector nuclease (TALEN).

121. The LNP of claim 112, wherein the nucleic acid is at least one nucleic acid suitable for a genome editing technique selected from the group consisting of a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a single guide RNA (sgRNA), and a DNA repair template.

122. The LNP of claim 115, wherein the mRNA is at least 30 nucleotides in length.

123. The LNP of claim 115, wherein the mRNA is at least 300 nucleotides in length.

124. A pharmaceutical composition comprising a LNP of any one of claims 103-123, and a pharmaceutically acceptable carrier.

125. The pharmaceutical composition of claim 124, formulated for intravenous or intramuscular administration.

126. The pharmaceutical composition of claim 124, which is formulated for intravenous administration.

127. A method for delivering a nucleic acid to a cell comprising contacting the cell with a LNP of any one of claims 103-123 or a pharmaceutical composition of any one of claims 99-101.

128. A method for treating a disease characterized by a deficiency of a functional protein, the method comprising administering to a subject having the disease, a LNP formulation comprising a LNP of any one of claims 103-123, wherein the mRNA encodes the functional protein or a protein having thesame biological activity as the functional protein.

129. A method for treating a disease characterized by overexpression of a polypeptide, comprising administering to a subject having the disease a LNP formulation comprising a LNP of any one of claims 103-123 and a siRNA, wherein the siRNA targets expression of the overexpressed polypeptide.

130. A pharmaceutical composition comprising: a) at least one lipid nanoparticle (LNP) comprising at least one ionizable lipid selected from those in any one of claims 1-79; and b) at least one gene editing system capable of editing, modifying or altering a polynucleotide sequence that results in treatment of a hemoglobinopathy.

131. The pharmaceutical composition of claim 129, wherein the at least one gene editing system comprises a gene editing system that corrects a sickle cell mutation by repairing the β-globin gene.

132. The pharmaceutical composition of any one of claims 129-130, wherein the at least one gene editing system comprises a gene editing system that corrects a sickle cell mutation through homology directed repair.

133. The pharmaceutical composition of any one of claims 129-130, wherein the at least one gene editing system comprises a gene editing system that corrects a sickle cell mutation through non-homologous end-joining (NHEJ), targeting aberrant splicing sites in introns I and II, resulting in restoration of functional β-globin transcripts.

134. The pharmaceutical composition of any one of claims 129-130, wherein the at least one gene editing system comprises a gene editing system that corrects a sickle cell mutation through a single base pair gene correction.

135. The pharmaceutical composition of any one of claims 129-130, wherein the at least one gene editing system comprises a gene editing system that corrects a sickle cell mutation by executing a single base pair correction, converting the aberrant thymine to adenine or cytosine. 136..The pharmaceutical composition of claim 129-130, wherein the at least one gene editing system comprises a gene editing system that reactivates γ-globin production.

137. The pharmaceutical composition of any one of claims 129 and 135, wherein the at least one geneediting system comprises a gene editing system that eliminates an HbF inhibitory sequence, or a portion thereof.

138. The pharmaceutical composition of any one of claims 129 and 135, wherein the at least one gene editing system comprises a gene editing system that targets HBG1 and / or HBG2 promoters, resulting in disruption of binding sites for γ-globin transcriptional repressors.

139. The pharmaceutical composition of any one of claims 129 and 135, wherein the at least one gene editing system comprises a gene editing system that disrupts BCL11A binding sites.

140. The pharmaceutical composition of any one of claims 129 and 135, wherein the at least one gene editing system comprises a gene editing system that disrupts the GATA1 motif in the erythroid- specific intronic enhancer of BCL11A.

141. The pharmaceutical composition of any one of the previous claims, wherein the at least one gene editing system comprises a gene editing system, or components thereof, disclosed in any one of: US Patents 0 7<img src='' class="img-anchor img-center" img-id="IMGF000528_0001" / >142. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a CRISPR-Cas gene editing system or components thereof, or a polynucleotide encodingthe same.

143. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a Cas9 nuclease, or a polynucleotide encoding the same.

144. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a Cas12a nuclease, or a polynucleotide encoding the same.

145. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a prime editing system or components thereof, or a polynucleotide encoding the same.

146. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a retron editing system or components thereof, or a polynucleotide encoding the same.

147. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a TnpB editing system or components thereof, or a polynucleotide encoding the same.

148. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises an integrase editing system or components thereof, or a polynucleotide encoding the same.

149. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a base editing system or components thereof, or a polynucleotide encoding the same.

150. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises an epigenetic editing system or components thereof, or a polynucleotide encoding the same.

151. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a gene writing system or components thereof, or a polynucleotide encoding the same.

152. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a gene inactivating system or components thereof, or a polynucleotide encoding the same.

153. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises zinc finger nuclease or components thereof, or a polynucleotide encoding the same.

154. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a TALE Nuclease, a TALE nickase, Zinc Finger (ZF) Nuclease, ZF Nickase,meganuclease, or a combination thereof, or components thereof, or a polynucleotide encoding any of the same.

155. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises a meganuclease, or a polynucleotide encoding the same.

156. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises at least one guide RNA (gRNA).

157. The pharmaceutical composition of any one of the preceding claims, wherein the gene editing system comprises at least one guide RNA, or a portion thereof, disclosed in any one of: US Patents 11242525 11572543 11268077 11466271 9957501 10808020 7. , nanoparticle further comprises: i) at least one structural lipid; ii) at least one phospholipid; and iii) at least one PEGylated lipid.

159. The pharmaceutical composition of any one of the preceding claims, wherein the at least one structural lipid is selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol,ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and any combinations thereof.

160. The pharmaceutical composition of any one of the preceding claims, wherein the at least one phospholipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn-phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3- phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3- phospho-L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0- 18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

161. The pharmaceutical composition of any one of the preceding claims, wherein the at least one PEGylated lipid is selected from (R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG- PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG- DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG- PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE- PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

162. The pharmaceutical composition of any one of the preceding claims, wherein the LNP further comprises at least one additional lipid component selected from 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcarnosine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl- sphingomyelin (SPM) (C18:l), N-lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl- sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2-dioleoylglyceryl-3- hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}- ethoxy)-ethyl]-3-(3,4,5-1rihydroxy-6-hydroxymethyl-1etrahydro-pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), a cell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol- hemisuccinate-Nα-Histidinyl-Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12- pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α-histidinyl-Nα- hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane- carboxamide] (MCC-PE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1- myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB- PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3- [phosphoethanolamine-N-dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn- glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB- phosphatidylethanolamine lipid (Rh-PE), purifiedsoy-derivedmixtureofphospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans-PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine.

163. The pharmaceutical composition of any one of the preceding claims, wherein the LNP further comprises one or more targeting moieties.

164. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises: (a) a PEG lipid selected from PEG2k-DMG or PEG2k-DSPE or a mixture thereof; (b) cholesterol; (c) a phospholipid selected from sphingolipid or DSPC or a mixture thereof.

165. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises: (a) about 0 mol% to about 10 mol% of PEG lipid; (b) about 0 mol% to about 30 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid; and (d) about 30 mol% to about 60 mol% of at least one ionizable lipid of any one of claims 1-79.

166. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises: (a) about 1 mol% to about 2 mol% of PEG lipid; (b) about 25 mol% to about 40 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid; and (d) about 30 mol% to about 60 mol% of at least one ionizable lipid of any one of claims 1-79.

167. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises: (a) about 2 mol% of PEG lipid; (b) about 25 mol% structural lipid;(c) about 40 mol% phospholipid; and (d) about 33 mol% of at least one ionizable lipid of any one of claims 1-79.

168. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises: (a) about 2.5 mol% of PEG lipid; (b) about 39 mol% structural lipid; (c) about 10 mol% phospholipid; and (d) about 48.5 mol% of at least one ionizable lipid of any one of claims 1-79.

169. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises: (a) about 1.5 mol% of PEG lipid; (b) about 40 mol% structural lipid; (c) about 10 mol% phospholipid; and (d) about 48.5 mol% of at least one ionizable lipid of any one of claims 1-79.

170. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises an mRNA or a circRNA encoding a CRISPR nuclease and a guide RNA capable of enacting a genetic edit that disrupts the GATA1 motif in the erythroid-specific intronic enhancer of BCL11A.

171. The pharmaceutical composition of any one of the preceding claims, wherein the lipid nanoparticle comprises an mRNA or a circRNA encoding a CRISPR nuclease and a guide RNA capable of enacting a genetic edit that disrupts BCL11A binding sites.

172. A method of treating a hemoglobinopathy in a patient in need thereof, the method comprising administering to the subject a pharmaceutical composition of any one of the preceding claims.

173. The pharmaceutical composition of any of the preceding claims for use as a medicament in the treatment of a hemoglobinopathy.

174. Use of a pharmaceutical composition of any of the preceding claims for the manufacture of a medicament for delivery of a gene editing system capable of treating a hemoglobinopathy.

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