[go: up one dir, main page]

WO2025059607A1 - Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules - Google Patents

Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules Download PDF

Info

Publication number
WO2025059607A1
WO2025059607A1 PCT/US2024/046812 US2024046812W WO2025059607A1 WO 2025059607 A1 WO2025059607 A1 WO 2025059607A1 US 2024046812 W US2024046812 W US 2024046812W WO 2025059607 A1 WO2025059607 A1 WO 2025059607A1
Authority
WO
WIPO (PCT)
Prior art keywords
lnp
lipid
amino acid
acid sequence
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/046812
Other languages
English (en)
Other versions
WO2025059607A9 (fr
Inventor
Zhan Wang
Rui Zhang
Han GU
Apiwat WANGWEERAWONG
Lorenzo TOZZI
Rahul Palchaudhuri
Michael Monte
Giulia SCHIROLI
Lei Liu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tessera Therapeutics Inc
Original Assignee
Tessera Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tessera Therapeutics Inc filed Critical Tessera Therapeutics Inc
Publication of WO2025059607A1 publication Critical patent/WO2025059607A1/fr
Publication of WO2025059607A9 publication Critical patent/WO2025059607A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • HSCs Hematopoietic stem cells
  • LT-HSC rare long-term HSC
  • any therapeutic agent delivered systemically must evade or overwhelm myriad mechanisms that will prevent its delivery to the bone marrow microenvironment (for example, liver uptake, phagocytic cell uptake, complement activation, opsonization, phagocytic cell uptake, etc.).
  • the therapeutic agent must be delivered specifically to the self-renewing LT-HSCs population to provide a permanent cure.
  • compositions capable of effectively delivering a therapeutic agent to HSCs (e.g., LT-HSCs) in a patient.
  • the disclosure provides lipid nanoparticles (LNPs) or conjugates comprising a lipid nanoparticle (LNP) encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or reporter construct, for delivery to hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs).
  • a therapeutic agent e.g., a gene modifying system
  • HPCs hematopoietic progenitor cells
  • the LNPs and conjugates described herein can deliver the payload to an HSC and/or HPC more effectively than a baseline conjugate or baseline LNP.
  • the disclosure also provides conjugates comprising a lipid nanoparticle (LNP) encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or reporter construct, for delivery to HSCs and/or HPCs wherein the LNP has at least one targeting moiety on its surface.
  • a therapeutic agent e.g., a gene modifying system
  • reporter construct for delivery to HSCs and/or HPCs wherein the LNP has at least one targeting moiety on its surface.
  • the conjugates having at least one targeting moiety described herein will also be referred to as targeted LNPs or tLNPs.
  • the disclosure provides a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs and/or HPCs in vivo, wherein the LNP comprises an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or from the lipids in Table 1, Table 2 and Table 3, as set forth in the disclosure below, and a therapeutic agent encapsulated within the LNP.
  • LNP lipid nanoparticle
  • the disclosure provides conjugates comprising an LNP and a targeting moiety.
  • a targeting moiety of a targeted LNP enhances the ability of the conjugate or LNP to deliver the payload to HSCs and HPCs (collectively HSPCs).
  • the targeted LNPs can be formulated in a pharmaceutical composition and can be directly administered to a subject (e.g., patient) in need thereof (e.g., by in vivo administration). Once administered, the compositions can deliver significant quantities of a therapeutic agent to the HSCs (e.g., LT-HSCs) of the subject. Alternatively, the LNPs can be administered to isolated HSCs (e.g., LT-HSCs) ex vivo.
  • the payload can be a nucleic acid encoding a reporter gene, such as green fluorescent protein (GFP).
  • the payload can be a therapeutic agent, for example, a therapeutic peptide or protein, a nucleic acid comprising a therapeutic agent, or a nucleic acid encoding a therapeutic agent.
  • the therapeutic agent can be a genetic medicine, wherein the therapeutic agent is capable of modifying, altering or effecting a change in the genomic DNA of a cell (e.g., a cell in the subject).
  • the therapeutic agent is a gene therapy agent or gene editing agent.
  • the therapeutic agent is a gene modifying polypeptide.
  • the therapeutic agent is a gene modifying system.
  • the targeting moiety is an antibody, Fab fragment or single chain variable fragment (scFv), a DARPIN, a VHH domain antibody, a FN3 domain, a nanobody, a single domain antibody or a Centyrin.
  • the targeting moiety is a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin or a N-Acetylgalactosamine (GalNac).
  • the targeting moiety is a peptide or protein, such as a ligand or part of a ligand, that binds to a receptor (e.g., a receptor on the surface of an HSC or LT-HSC).
  • a receptor e.g., a receptor on the surface of an HSC or LT-HSC
  • the targeting moiety is a peptide or protein that binds to a receptor or ligand on the surface of an HSC, HPC or LT-HSC, wherein the affinity of the peptide or protein targeting moiety for the receptor or ligand on the surface of the HSC, HPC or LT-HSC is modulated via phage display.
  • the payload (e.g., therapeutic agent) delivered by the LNP or targeted LNP can be a small molecule, peptide or protein, non-coding RNA (ncRNA), gRNA, siRNA or miRNA, a nucleic acid (e.g., mRNA) encoding a peptide or protein (e.g., a protein for replacement gene therapy, or a protein for modifying or altering the genome or epigenome, e.g., a protein for gene editing), a nucleic acid encoding or comprising one or more components of a system for altering a genome (e.g., one or more components of a ribonucleoprotein (RNP) complex for editing or altering the genome or epigenome (e.g., for introducing insertion-deletion mutations (indels), base editing, epigenetic editing, or target-primed reverse transcription (TPRT), e.g., by a mechanism that requires a recombin
  • ncRNA non-
  • the therapeutic agent (i.e., payload) delivered by the LNP or targeted LNP can be a gene modifying protein, a nucleic acid encoding a gene modifying protein, or a gene modifying system, as described herein.
  • the disclosure provides a targeted LNP (conjugate) encapsulating one or more nucleic acids encoding components of a system for modifying or altering DNA (e.g., genomic DNA), wherein the targeted LNP comprises one or more targeting moieties, such as antibodies, Fab fragments, or scFvs, capable of binding to one or more proteins on the cell surface of a hematopoietic stem cell (HSC).
  • a targeted LNP conjugate
  • the targeted LNP comprises one or more targeting moieties, such as antibodies, Fab fragments, or scFvs, capable of binding to one or more proteins on the cell surface of a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • the targeted LNP is delivered in vivo to a subject (e.g., a human patient) in need thereof.
  • a subject e.g., a human patient
  • the nucleic acids encoding components of a system for modifying or altering a genome can be expressed, resulting in in vivo altering of the genome (e.g., gene editing) of the HSC.
  • LNPs comprising particular combinations of one or more targeting moieties (e.g., antibodies, Fab fragments or scFvs), ionizable lipids, and/or helper lipids are capable of enhancing delivery of a therapeutic payload, (e.g., a system capable of altering the genome, such as a gene modifying system) to HSCs following in vivo administration to a subject, thereby resulting in enhanced levels of therapeutic activity in the HSCs.
  • a therapeutic payload e.g., a system capable of altering the genome, such as a gene modifying system
  • an LNP or targeted LNP (conjugate) comprising an ionizable lipid as described herein is capable of delivering a payload (e.g., a therapeutic payload) to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% more HSCs, HSPCs or LT-HSCs than an LNP comprising a baseline lipid (such as V003).
  • a payload e.g., a therapeutic payload
  • an LNP comprising an ionizable lipid as described herein is capable of delivering a payload (e.g., a therapeutic payload) to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% more HSCs, HSPCs or LT-HSCs than an LNP comprising a baseline lipid (such as V003) in an animal model, as described herein.
  • the LNP is a targeted LNP.
  • a payload (e.g., a therapeutic payload) delivered to HSCs, HPCs or LT-HSCs by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as V003).
  • a baseline lipid such as V003
  • a payload (e.g., a therapeutic payload) delivered to HSCs, HSPCs or LT-HSCs by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as V003).
  • the LNP is a targeted LNP.
  • At least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% of HSCs (e.g., LT-HSCs) in a subject comprise a genome that have been modified or altered following administration of an LNP described herein, such as a targeted LNP encapsulating a therapeutic agent (e.g., a system for modifying, altering or editing a genome).
  • a targeted LNP encapsulating a therapeutic agent e.g., a system for modifying, altering or editing a genome.
  • the LNPs or targeted LNPs are delivered ex vivo to the isolated HSCs of a subject (e.g., human patient) in need thereof.
  • HSCs are first collected from the bone marrow of a subject.
  • the targeted LNPs of the disclosure can be mixed with the HSCs, thereby resulting in effective transduction of the payload (e.g., a gene modifying system) into the HSCs.
  • the modified HSCs can be administered to the patient for engraftment, as described in the art.
  • the LNPs of the present disclosure comprise one or more pegylated lipid molecules.
  • the targeting moiety e.g., antibody, Fab fragment or scFv
  • the targeted LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the targeting moiety.
  • LNPs comprising different pegylated lipids not covalently bound to a targeting moiety could show different toxicological properties when all other components of the LNPs (e.g., ionizable lipid) and relative proportions are the same.
  • LNPs comprising pegylated lipids with PEG lipid anchors having 16 carbon atoms (i.e., C16 PEG lipid anchors) or 18 carbon atoms (i.e., C18 PEG lipid anchors) could reduce or eliminate the toxicity associated with identical LNPs other than the pegylated lipid portion.
  • the pegylated lipid is 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000.). In some embodiments, the pegylated lipid is 1,2-Dipalmitoyl-rac- glycero-3-methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is C16 PEG ceramide. In some embodiments, the targeted LNPs comprising the C16 pegylated lipids show significantly reduced liver uptake than otherwise identical LNPS comprising C14 pegylated lipids.
  • the pegylated lipid has at least one C18 PEG lipid anchor. In some embodiments, the pegylated lipid has two C18 PEG lipid anchors (i.e., dialkyl chains of 18 carbons long). In some embodiments, the C18 pegylated lipid is 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the C18 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG- PEG2000).
  • the non-pegylated lipid is a sphingolipid.
  • the non- pegylated lipid is a sphingomyelin.
  • the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide.
  • the sphingomyelin is egg sphingomyelin.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 18% to about 32%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%.
  • mol% of the helper lipid refers to the mol% of the total lipid component of the LNP, which does not include the therapeutic agent (i.e., payload) or the targeting moiety.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is from about 22% to about 28%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%.
  • helper lipid e.g., DSPC or sphingomyelin
  • in vivo delivery of certain payloads following administration of the disclosed LNPs with these percentages of helper lipids provides enhanced transduction and/or expression of the payloads relative to LNPs with smaller or larger quantities of helper lipid.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, and the helper lipid is present at about 18 mol% to about 32 mol% of the lipid component; (ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting moieties are configured to target one or more receptors on the surface of HSCs selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or more nucleic acid molecules (e.g., RNA molecules) (e.g.,
  • the therapeutic agent comprises two or more nucleic acids, e.g., RNA molecules.
  • the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 1:1 to about 7:1. In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 4:1.
  • the molar ratio between the ionizable lipid and the non-pegylated helper lipid is from about 1:1 to about 3:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 1:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 2:1.
  • the molar ratio between the ionizable lipid and the non- pegylated helper lipid is from about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 2.5:1.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 092 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 092 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP)is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG- PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 093 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 093 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG- PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG-PEG2000.
  • the targeted LNP comprises Lipid 153 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 153 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 154 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 154 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG- PEG2000.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 155 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 155 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG- PEG2000.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 162 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 162 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG- PEG2000.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 163 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 163 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the pegylated lipid is DMG- PEG2000.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 169 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 169 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the pegylated lipid is DMG- PEG2000.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 176 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 176 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG- PEG2000.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 232 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 232 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the pegylated lipid is DMG- PEG2000.
  • the LNP or conjugate can comprise one or more cholesterol molecules.
  • the molar ratio between the cholesterol molecule and the non-pegylated helper lipid e.g., DSPC or sphingomyelin
  • the molar ratio between the cholesterol molecule and the non-pegylated helper lipid is from about 3:1 to about 0.5:1.
  • the molar ratio between the cholesterol molecule and the non- pegylated helper lipid is from about 2:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1.5:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 0.5:1.
  • the molar ratio between the cholesterol molecule and the non-pegylated helper lipid is from about 1:2 to about 0.8:1.
  • the LNP comprises a targeting moiety that targets a receptor on the surface of the HSC selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, and CD338.
  • the LNP comprises a targeting moiety that targets CD117.
  • the LNP comprises a targeting moiety that targets CD33.
  • the LNP comprises a targeting moiety that targets CD34. In some embodiments, the LNP comprises a targeting moiety that targets CD38. In some embodiments, the LNP comprises a targeting moiety that targets CD43. In some embodiments, the LNP comprises a targeting moiety that targets CD46. In some embodiments, the LNP comprises a targeting moiety that targets CD49d. In some embodiments, the LNP comprises a targeting moiety that targets CD50. In some embodiments, the LNP comprises a targeting moiety that targets CD59. In some embodiments, the LNP comprises a targeting moiety that targets CD71. In some embodiments, the LNP comprises a targeting moiety that targets CD105. In some embodiments, the LNP comprises a targeting moiety that targets CD1117.
  • the LNP comprises a targeting moiety that targets CD123. In some embodiments, the LNP comprises a targeting moiety that targets CD164. In some embodiments, the LNP comprises a targeting moiety that targets CD184. In some embodiments, the LNP comprises a targeting moiety that targets CD338. [0043] In some embodiments, the LNP comprises a targeting moiety that comprises a sequence present in any of Tables S1 to S14. In some embodiments, the targeting moiety is encoded by a sequence present in any of Tables S1 to S14. In some embodiments, the LNP comprises two targeting moieties, wherein each targeting moiety comprises a sequence present in any of Tables S1 to S14.
  • the two targeting moieties are each encoded by a sequence present in any of Tables S1 to S14.
  • the CD34 targeting moiety comprises a Class II antibody or fragment thereof. In some embodiments, the CD34 targeting moiety comprises a Class III antibody or fragment thereof. In some embodiments the CD34 targeting moieity is insensitive or resistance to certain proteolytic enzymes. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by neuraminidase. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by chymopapain. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by glycoprotease.
  • the CD34 targeting moiety is insensitive or resistant to cleavage by neuraminidase, chymopapain, and glycoprotease. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by neuraminidase, but sensitive to cleavage by chymopapain and glycoprotease.
  • the conjugates comprising a Fab fragment are prepared by a method comprising: (i) contacting a Fab fragment (e.g., a Fab fragment that targets CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, or CD338) with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP (conjugate).
  • a Fab fragment e.g
  • the disclosure provides a conjugate comprising an LNP and a Fab fragment, wherein the LNP is covalently bonded to either or both of a first cysteine residue in the constant region of the heavy chain of the Fab fragment and/or a second cysteine residue in the constant region of light chain of the Fab fragment.
  • the Fab fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Fab fragment and the constant region of the light chain of the Fab fragment.
  • both the constant region of the heavy chain constant region of the heavy chain and the constant region of the light chain of the Fab fragment are covalently bonded to the LNP.
  • the Fab fragment is linked to the LNP through a thiosuccinimide moiety. In other of the foregoing embodiments, the Fab fragment is linked to the LNP through a dithiomalemide moiety.
  • the Fab fragment is linked to the LNP through a maleamic acid moiety.
  • the Fab fragment conjugated to the LNP is an IgG1 Fab fragment.
  • the cysteine at position 214 (Kabat numbering) of the light chain of the IgG1 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgG1 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgG1 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG1 Fab fragment are covalently bonded to the LNP.
  • the Fab fragment conjugated to the LNP is an IgG2 Fab fragment.
  • the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment are covalently bonded to the LNP.
  • the Fab fragment conjugated to the LNP is an IgG4 Fab fragment.
  • the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment are covalently bonded to the LNP.
  • the targeted LNPs are formed via a Click reaction between a first Click handle on the targeting moiety (e.g., antibody, Fab fragment, or scFv) and a second Click handle on the LNP, thereby generating a Click product.
  • the second Click handle can be on one or more of the lipids comprising the LNP.
  • the second Click handle is covalently bonded to a pegylated lipid comprising the LNP.
  • the Click product can be formed using a copper-catalyzed Click reaction.
  • the first or second Click handle comprises a cyclic derivative of the alkynyl group.
  • the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne. Cyclooctyne, and difluorinated cyclooctyne.
  • the click chemistry involves strain promoted cycloaddition of azides.
  • the click chemistry is based upon reaction of strained alkenes.
  • the Click product can be formed using copper-free Click chemistry.
  • the Click product can be formed between an azide and dibenzocyclooctene (DBCO).
  • DBCO dibenzocyclooctene
  • the Click product can be formed using a Staudinger reaction between an azide and a phosphine, hence producing an aza-ylide.
  • the Click product can be formed from an inverse electron demand Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the first or second Click handle.
  • TCO trans-cyclooctene
  • the first Click handle comprises a tetrazine (Tz) ring
  • the second Click handle comprises a TCO moiety.
  • the tetrazine ring is unsubstituted.
  • the tetrazine rung is methyltetrazine.
  • the tetrazine ring is a 6-methyl substituted tetrazine.
  • the Click product is formed by conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence.
  • an antibody, Fab fragment or single chain variable fragment (scFv) can be covalently linked to a first Click handle through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle on the LNP, thereby generating a Click product.
  • the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide.
  • the antibody Fab fragment or scFv is directly bonded to the enzyme recognition sequence.
  • the antibody Fab fragment or scFv is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C- terminus of the antibody, Fab fragment or scFv include, but are not limited to (GGGGS)v, (G)v, (EAAAK)v, (PAPAP)v, (AP)v and A(EAAAK)u ALEA(EAAAK)vA, wherein u is 1- 10 and v is 1-10.
  • a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs comprises: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any or the lipids in Table 1, Table 2 and Table 3; a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties binds to at least one of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4); and a therapeutic agent encapsulated within the LNP.
  • the plurality of targeting moieties can be added to the surface of the LNP by methods described in Section II of the Detailed Description.
  • a targeted LNP in some embodiments comprises two or more different targeting moieties, each of which may bind to the same or a different target.
  • a targeted LNP having two different targeting moieties which bind to the same or different targets may be referred to an a “dual binder” or “dual binding” targeted LNP.
  • each of the targeting moieties is conjugated to the lipid nanoparticle through a linker, wherein each linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the targeting moieties are antibodies or antigen binding fragments thereof. In other such embodiments, the targeting moieties are scFvs. In some embodiments, the targeting moieties are Fab fragments. [0055] In some embodiments, the plurality of targeting moieties binds to two or more of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4). In some embodiments, the plurality of targeting moieties binds to CD117 and CD34. In some embodiments, the plurality of targeting moieties binds to CD117 and CD71.
  • a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs comprises: a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties binds to CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4); and a therapeutic agent encapsulated within the LNP.
  • the disclosure provides a targeted lipid nanoparticle (LNP) for in vivo delivery of a therapeutic agent to HSCs (e.g., LT-HSCs), wherein the targeted LNP comprises: an ionizable lipid; a helper lipid; two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target on the surface of the HSCs (e.g., LT-HSCs); and one or more nucleic acids (e.g., two or more nucleic acids) encoding components of a system for modifying or altering genomic DNA (e.g., a gene modifying system), wherein the one or more nucleic acids (e.g., two or more nucleic acids) are RNA molecules.
  • the targeted LNP comprises: an ionizable lipid; a helper lipid; two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target
  • the dual binders can be added to the surface of the LNP by methods described in Section II of the Detailed Description.
  • each of the targeting moieties is conjugated to the lipid nanoparticle through a linker, wherein each linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the targeting moieties are antibodies or antigen binding fragments thereof.
  • the targeting moieties are scFvs.
  • the targeting moieties are Fab fragments.
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is an mRNA encoding a gene modifying polypeptide, as described herein.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9-RT fusion protein.
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is a guide RNA (gRNA).
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is a template RNA encoding a heterologous nucleic acid for use with a gene modifying polypeptide to insert the heterologous nucleic acid sequence into a DNA sequence, e.g., the genomic DNA of a cell.
  • the components of a system for modifying or altering genomic DNA have nuclease activity, e.g., nickase activity. In some embodiments, the components of a system for modifying or altering genomic DNA do not have nuclease activity. In some embodiments, the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) do not elicit a double- stranded break in the genomic DNA. In such embodiments, the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA.
  • a system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous sequence into the genomic DNA.
  • the targets on the surface of the HSC are selected from CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184 (CXCR4).
  • at least one of the targeting moieties binds to CD117.
  • at least one of the targeting moieties binds to CD34.
  • at least one of the targeting moieties binds to CD71.
  • the targeting moieties binds to CD164.
  • the targeted LNP is a dual binding LNP.
  • the two targeting moieties bind to CD117 and CD34.
  • the two targeting moieties bind to CD117 and CD71.
  • the ionizable lipid is V003 or a lipid from Table 1, Table 2, or Table 3.
  • the ionizable lipid is selected from Lipid 92, Lipid 93, Lipid 95, Lipid 96, Lipid 97, Lipid 97, Lipid 110, Lipid 132, Lipid 133, Lipid 134, Lipid 140, Lipid 141, Lipid 143, Lipid 144, Lipid 147, Lipid148, Lipid 149, Lipid 150, Lipid 151, Lipid 152, Lipid 153, Lipid 154, Lipid 155, Lipid 162, Lipid 163, Lipid 169, Lipid 170, Lipid 173, Lipid 174, Lipid 175, Lipid 176, Lipid 178, Lipid 179, Lipid 183, Lipid 184, and Lipid 232.
  • the ionizable lipid is Lipid 093. In some embodiments, the ionizable lipid is Lipid 092. In some embodiments, the ionizable lipid is Lipid 154. In some embodiments, the ionizable lipid is Lipid 232. [0063] In some embodiments, the helper lipid of the targeted LNP is DSPC or sphingomyelin. In some embodiments, the mo1% of the DSPC or sphingomyelin in the targeted LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the targeted LNP further comprises a pegylated lipid.
  • the pegylated lipid comprises at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated is DPPE-PEG2000 or DPG- PEG2000.
  • the targeted delivery of a system for modifying or altering genomic DNA results in synergistic enhancement of gene editing or modification of the target HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 5% of the genomes of the HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 10% of the genomes of the HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 15% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 20% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 25% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 20% to about 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 20% of the genomes of the HSCs or LT-HSCs.
  • a system for modifying or altering genomic DNA includes a gene modifying polypeptide and a template RNA for a nucleic acid sequence to be inserted at a specific location of the genomic DNA, hence resulting in modification of the genomic DNA.
  • the gene modifying system comprises a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • RT reverse transcriptase
  • a system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous nucleic acid sequence into a DNA sequence, e.g., genomic DNA.
  • TPRT target-primed reverse transcription
  • the disclosure provides a method of treating sickle cell disease in a subject (e.g., a human patient in need thereof), said method comprising administering to the subject a targeted lipid nanoparticle (LNP) comprising: an ionizable lipid; a helper lipid; two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target on the surface of the HSCs (e.g., LT-HSCs); and one or more nucleic acids (e.g., two or more nucleic acids) encoding components of a system for modifying or altering genomic DNA (e.g., a gene modifying system), wherein the two or more nucleic acids are RNA molecules.
  • LNP targeted lipid nanoparticle
  • the nucleic acid components do not introduce double stranded breaks into the genomic DNA.
  • greater than 5% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • greater than 10% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • greater than 15% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • greater than 20% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • greater than 30% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 5% to about 15% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 10% to about 30% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 10% to about 20% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • from about 15% to about 20% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 15% to about 25% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, and the helper lipid is present at about 18 mol% to about 32 mol% of the lipid component; (ii) means for binding a cell surface receptor selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises two or more RNA molecules (e.g., two or three RNA molecules).
  • FIG.1 shows an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).
  • FIG.2A shows assembly of an LNP encapsulating a therapeutic payload with a functional group (e.g., maleimide, 2,3-dibromomaleimide, sortase tag, etc.) to be reacted with an antibody or antigen-binding fragment thereof.
  • FIG.2B shows assembly of an LNP encapsulating a therapeutic payload using a post-insertion technique.
  • each Fab comprises two free cysteine residues with one free cysteine residue on the heavy chain and one free cysteine residue on the light chain.
  • the first Fab fragment and the second Fab fragment are contacted with an LNP comprising a plurality of thiol-reactive groups (e.g., maleimide or DBM) conjugated to the surface of the LNP, whereby a thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the first Fab Fragment and another thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the second Fab Fragment.
  • thiol-reactive groups e.g., maleimide or DBM
  • FIG.8 shows some examples of a pegylated lipid bonded to a maleimide moiety.
  • FIG.9 shows some examples of non-pegylated lipid bonded to a maleimide moiety.
  • FIG.10 shows some examples of ionizable lipid bonded to a maleimide moiety.
  • FIG.11 shows some examples of sterols bonded to a maleimide moiety.
  • FIG.12 shows an exemplary schematic of conjugate formation.
  • a Fab fragment comprises a sortase tag.
  • the Fab fragment is contacted with an LNP in the presence of sortase, wherein the LNP comprises a plurality of polyglycine molecules conjugated to the surface of the LNP, whereby the Fab fragment is conjugated to the surface of the LNP through a sortase mediated ligation of the sortase tag and a polyglycine molecule of the plurality.
  • FIG.13 shows the formation of an LNP conjugated to the C-terminus of a Fab fragment through a sortase mediated ligation followed by a Click reaction.
  • FIG.14 shows the formation of an LNP conjugated to the C-terminus of a Fab fragment through a lipoic acid ligase mediated ligation followed by a Click reaction.
  • FIG.15 shows an enzymatic approach of site-specifically introducing a Tz ring onto a sugar moiety of an antibody.
  • FIG.16 shows an example of a light-induced crosslinking approach of site- specifically introducing a Tz ring onto an antibody.
  • FIG.17 shows an example of using a non-natural amino acid to site-specifically introduce a Tz ring onto an antibody or Fab fragment.
  • FIG.18 shows the results of transfection experiments in human CD34+ cells using LNPs conjugated to an anti-CD117 Fab fragment through sortase-mediated ligation. The transfection results are compared to unmodified (control) LNPs and LNPs modified with an anti-CD117 antibody produced using a random modification method.
  • FIG.18A shows mRNA transfection as quantified by flow cytometry in terms of % green fluorescent protein (%GFP+) cells.
  • FIG.18B shows transfection in terms of Median Fluorescence Intensity (MFI).
  • FIG.19A shows a schematic example for producing a site-specific conjugate through the reaction of an anti-CD117 Fab fragment with site-specific sortase recognition motif and an LNP.
  • FIG.19B shows a schematic of producing a conjugate through the reaction of a Fab fragment that has been modified using a random approach (NHS modification) and an LNP.
  • FIG.20 shows results of transfection into Kasumi-1 cells (CD117+ cell line) using LNPs conjugated to an anti-CD117 Fab fragment produced by site-specific (sortase- mediated) or by random modification.
  • FIG.20A shows mRNA transfection as quantified by flow cytometry in terms of % green fluorescent protein (%GFP+) cells.
  • FIG.20B shows transfection in terms of Median Fluorescence Intensity (MFI).
  • MFI Median Fluorescence Intensity
  • FIG.21 shows the results of in vivo experiments evaluating transduction of LNPs conjugated with one of three tested Fab fragments specific for anti-CD117 (Fab 1, Fab 5 or Fab 6) in HSCs and early progenitor cells (HSCs + early progenitors) (also referred to as HSPCs) compared to transduction using a base LNP lacking a conjugated anti-CD117 Fab fragment.
  • FIG.21A shows that up to 58% of HSCs + early progenitors receive the LNPs with the sortase-modified Fab fragments.
  • FIG.21B shows that up to 62% of long term (LT)- HSCs receive the LNPs with sortase-modified Fab fragments.
  • LT long term
  • FIG.22 shows the effect of increasing the mol% of helper lipid in the targeted LNPs conjugated with Fab fragments specific for CD117 on the transduction and expression of an mRNA encoding GFP in HSCs and early progenitor cells (HSCs + early progenitors) (also referred to as HSPCs).
  • HSCs + early progenitors also referred to as HSPCs.
  • FIG.22 shows the results of in vivo experiments comparing the transduction of targeted LNPs containing two different amounts of helper lipids (LNP A and LNP B).
  • LNPs A and B comprised 8% helper lipid (DSPC) and 22% helper lipid (DSPC), respectively. More than 80% of HSCs + early progenitors were transduced with LNP B.
  • FIG.23 compares the levels of transduction and MFI in a humanized mouse model administered LNPs comprising helper lipids at different percentages.
  • FIGs.23A and 23B demonstrate that targeted LNPs comprising ionizable lipid and 8% DSPC provide similar levels of GFP transduction and expression levels in HSPCs when compared to targeted LNPs comprising ionizable lipid and 8% (molar) sphingomyelin. Similar to experiments with DSPC as the helper lipid, increasing the sphingomyelin content to 22% (molar) results in substantially increased transduction and expression levels of the reporter protein.
  • FIGs.23C and 23D show results for transduction in LT-HSCs.
  • FIGs.31A-31F show the synthesis of Lipid 176.
  • FIG.32A shows enhanced transduction of GFP in LSK cells using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in an in vivo mouse model.
  • FIG.32B shows enhanced expression levels of GFP in LSK cells using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in an in vivo mouse model.
  • FIGs.32C and 32D shows enhanced transduction and/or expression levels of GFP in LSK cells using LNPs containing ionizable Lipid 154 or ionizable Lipid 155 relative to LNPs containing ionizable Lipid V003 in an in vivo mouse model.
  • FIGs.30E and 30F show enhanced transduction of GFP and/or enhanced GFP expression in in LT-HSCs using LNPs containing ionizable Lipid 154 or ionizable Lipid 155 relative to an LNPs containing ionizable Lipid V003.
  • FIG.33A shows enhanced transduction of GFP in LT-HSCs using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model.
  • FIG.33B shows enhanced expression of GFP in LT-HSCs using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model.
  • FIG.34A and FIG.34C show enhanced transduction of GFP in HSPCs or LT- HSPCs, respectively, using LNPs containing ionizable Lipid 153, Lipid 154, or Lipid 155 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model.
  • FIG. 34B and FIG.34D show enhanced expression of GFP in HSPCs or LT-HSCs, respectively, using LNPs containing ionizable Lipid 153 or ionizable Lipid 154 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model.
  • FIG.35 shows results of screening of targeted LNP (tLNP) in humanized NBSGW mice with increased quantities of helper lipid.
  • FIGs.35A and 35B show results for transduction and expression in HSPCs, respectively.
  • FIGs.35C and 35D show results for transduction and expression in LT-HSCs, respectively.
  • FIG.36 shows screening results of targeted LNP (tLNP) in nonhuman primates.
  • FIG.37A and 37B show in vitro screening results (% GFP+ cells and MFI, respectively) for targeting moieties capable of enhancing delivery of a targeted LNP for HSPCs, normalized against cells transfected with a base LNP lacking targeting moieties.
  • FIG.38 shows that a site-specific conjugation process, as described herein, can efficiently conjugate two targeting moieties (anti-CD117 Fab and anti-CD45 Fab, in this case) to the surface of LNPs at specific ratios.
  • FIG.39 show that tLNPs can effectively deliver a gene editing payload to human HSPCs in an animal model, leading to in vivo gene editing in the HSPCs.
  • FIG.39A and FIG.39B show that administering anti-CD34 tLNPs and anti-CD117 tLNPs resulted in detectable levels of B2M indels in HSPCs and LT-HSCs,but administering dual tLNPs conjugated to the anti-CD117 and anti-CD34 targeting moieties resulted in synergistically higher levels of gene editing in HSPCs.
  • FIG.39C shows that dual tLNPs conjugated to anti- CD117 and anti-CD71 targeting moieties, or antiCD34 and anti-CD71 targeting moieties, can substantially increase delivery of the gene editing payload to HSPCs and LT-HSCs, thereby resulting in an increase in B2M editing.
  • FIG.39D shows that doubling the amount of a single type of targeting moiety (anti-CD117) conjugated to the surface of tLNPs did not increase delivery of a payload to HSPCs or LT-HSCs.
  • FIGs.40A and 40B show that the administered anti-CD117 tLNPs can deliver payloads suitable for gene editing to human HSPCs in an animal model, leading to in vivo gene editing in HSPCs.
  • FIG.40A shows that tLNPs formulated with Lipid093 and the wtCas9-RT mRNA and B2M gRNA payload resulted in 8.1% indels in B2M in HSPCs.
  • FIG. 40B shows that the tLNPs formulated with Lipid154 and the nCas9-RT and B2M template RNA payload generated higher TPRT-mediated knockout of B2M in HPSCs relative to the tLNPs formulated with Lipid093 and the same payload (mean 5.2% with Lipid154 and mean 1.5% with Lipid93).
  • FIG.41B shows that the expression of GFP mRNA was higher in HSPCs and nearly four- fold higher in LT-HSCs in those NHPs dosed with the Lipid154 tLNPs compared to those dosed with the Lipid093 tLNPs.
  • FIG.42 shows that administration of the anti-CD117/anti-CD34 dual tLNPs formulated with Lipid093 and C14-PEG led to an average of 25% B2M indels in human HSPCs in vivo.
  • FIG.43 shows that anti-CD117/anti-CD34 dual-targeted tLNPs, formulated with Lipid154 and C16-PEG led to a mean 11.8% perfect gene modification in HSPCs and mean 14.3 perfect gene modification in LT-HSCs in vivo.
  • FIG.44B shows that HSPCs and LT-HSCs in the NHPs expressed GFP (MFI) following administration of each of the dual tLNPs.
  • MFI expressed GFP
  • FIG.45 shows that the dual targeted LNPS (tLNPs) formulated with the anti- CD117 and anti-CD34 targeting can effectively deliver a gene editing system to human HSCs and precursors in vivo, leading to B2M knockout in appreciable numbers of the cells.
  • FIGs.46A and 46B show that targeted LNPs (tLNPs) and dual tLNPs could deliver the B2M gene editing system to human HSPCs (FIG.46A) and LT-HSCs (FIG.46B) in vivo to generate B2M indels.
  • Antibody encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • Antibody fragment The term “antibody fragment” or an “antigen binding fragment” as used herein is a portion of a full-length antibody that binds the antigen to which the full length antibody binds. Examples of antibody fragments include Fv, Fab, Fab ⁇ , Fab ⁇ - SH, F(ab ⁇ )2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv, scFab); and multispecific antibodies formed from antibody fragments.
  • Antigen binding domain refers to that portion of antibody or a chimeric antigen receptor which binds an antigen.
  • an antigen binding domain binds to a cell surface antigen of a cell.
  • an antigen binding domain binds an antigen characteristic of a cancer, e.g., a tumor associated antigen in a neoplastic cell.
  • an antigen binding domain binds an antigen characteristic of an infectious disease, e.g. a virus associated antigen in a virus infected cell.
  • an antigen binding domain binds an antigen characteristic of a cell targeted by a subject’s immune system in an autoimmune disease, e.g., a 0-antigen.
  • an antigen binding domain is or comprises an antibody or antigen-binding portion thereof.
  • an antigen binding domain is or comprises an scFv or Fab.
  • Fab fragment refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain.
  • gRNA spacer A “gRNA spacer”, as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid.
  • gRNA scaffold refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid.
  • the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.
  • a “gene modifying polypeptide”, as used herein, refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell).
  • a nucleic acid sequence e.g., a sequence provided on a template nucleic acid
  • target DNA molecule e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell.
  • the gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, the gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate: 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) integration of the at least a portion of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence.
  • Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain.
  • heterologous constructs e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain.
  • Exemplary gene modifying polypeptides, and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in PCT/US2021/020948, which is
  • a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene.
  • a “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid. [0125] Gene modifying system: A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide, or a nucleic acid (e.g., an mRNA) encoding the gene modifying polypeptide, and a template nucleic acid.
  • domain refers to a structure of a biomolecule that contributes to a specified function of the biomolecule.
  • a domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non- contiguous sequences) of a biomolecule.
  • protein domains include, but are not limited to, an endonuclease domain, a DNA binding domain, a reverse transcriptase domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain.
  • Exogenous when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell, or organism by the hand of man.
  • a nucleic acid that is as added into an existing genome, cell, tissue, or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.
  • First Strand and Second Strand describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand the reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates.
  • the first strand refers to the strand of the target DNA upon which the reverse transcriptase domain initiates.
  • Heterologous The term “heterologous”, as used herein to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described.
  • a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to: (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions.
  • a heterologous regulatory sequence e.g., promoter, enhancer
  • a heterologous domain of a polypeptide or nucleic acid sequence e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide
  • a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both.
  • heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).
  • Mutation or Mutated when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence.
  • a nucleic acid sequence may be mutated by any method known in the art. In some embodiments a mutation occurs naturally. In some embodiments a desired mutation can be produced by a system described herein.
  • Mutation Region refers to a region in a template RNA having one or more sequence difference relative to the corresponding sequence in a target nucleic acid.
  • the sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion.
  • Nucleic acid molecule “Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein.
  • the nucleic acid molecule can be double-stranded or single-stranded, circular, or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand.
  • SEQ ID NO: or “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complimentary to SEQ ID NO:1. The choice between the two is dictated by the context in which SEQ ID NO:1 is used.
  • nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.).
  • uncharged linkages for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages for example, phosphorothioates, phosphorodithioates, etc.
  • pendant moieties for example, polypeptides
  • intercalators for example, acridine
  • the CD33 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:88, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:89.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:89.
  • the CD164 targeting moiety comprises a class I antibody, as described in Watts et al., Regenerative Medicine (2021)6:33, which is incorporated by reference herein, or an antigen binding portion thereof.
  • the CD164 targeting moiety comprises a class II antibody, as described in Watts et al., Regenerative Medicine (2021)6:33, or an antigen binding portion thereof.
  • the target molecule is CD117, also referred to as c-kit, kit, or stem cell factor receptor (SCF-R).
  • the target cell is CD117+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD117.
  • Exemplary anti-CD117 antibodies or antigen-binding fragments thereof include Briquilimab (JSP-191, e.g., Jasper Therapeutics), LOP628 (e.g., Novartis Pharmaceuticals), NEG024 (e.g., Novartis Pharmaceuticals), NEG026 (e.g., Novartis Pharmaceuticals), NEG027 (e.g., Novartis Pharmaceuticals), NEG085 (e.g., Novartis Pharmaceuticals), NEG086 (e.g., Novartis Pharmaceuticals), NEG087 (e.g., Novartis Pharmaceuticals), GZQ167 (e.g., Novartis Pharmaceuticals), MGTA-117 (e.g., Magenta Therapeutics), Barzolvolimab (CDX ⁇ 0159, e.g., CellDex Therapeutics), D13A2 (e.g., Cell Signaling Technology), D3W6Y (e.g., Cell Signaling Technology), 11996-R018 (e.g., Sino
  • the CD117 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:427 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:428.
  • SEQ ID NOs:415-418 and 425-428 are shown in Table S2, with complementary determining regions (CDRs) marked in bold.
  • Table S2 Exemplary CD117 Targeting Moiety Sequences
  • the CD117 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:415, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:416.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:416.
  • the CD117 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:24, and/or a second Fab polypeptide (i.e., VL-CL) comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:25.
  • VH-CH1 first Fab polypeptide
  • VL-CL second Fab polypeptide
  • the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence FTFSDADMD (SEQ ID NO:32) or DADMD (SEQ ID NO:392), a CDR-H2 comprising the amino acid sequence RNKAGSYTTEYAASVKG (SEQ ID NO:33), a CDR-H3 comprising the amino acid sequence AREPKYWIDFDL (SEQ ID NO:34), a CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO:35), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:36), and a CDR-L3 comprising the amino acid sequence QQSYIAPYT (SEQ ID NO:37).
  • the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYYMA (SEQ ID NO:397), a CDR-H2 comprising the amino acid sequence NINYPGSSTYYLDSVKG (SEQ ID NO:398), a CDR-H3 comprising the amino acid sequence GDYYGTTYWY (SEQ ID NO:399), a CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO:35), a CDR-L2 comprising the amino acid sequence YTSRLQS (SEQ ID NO:400), and a CDR-L3 comprising the amino acid sequence QQGRRLWS (SEQ ID NO:401).
  • the CD117 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:425, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:426.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:426.
  • the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYWIG (SEQ ID NO:402), a CDR-H2 comprising the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO:403), a CDR-H3 comprising the amino acid sequence HGRGYNGYEGA (SEQ ID NO:404), a CDR-L1 comprising the amino acid sequence RASQGISSALA (SEQ ID NO:405), a CDR-L2 comprising the amino acid sequence DASSLES (SEQ ID NO:406), and a CDR-L3 comprising the amino acid sequence QQFNSYPLT (SEQ ID NO:407).
  • the CD117 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:427, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:428.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:428.
  • the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYNMH (SEQ ID NO:408), a CDR-H2 comprising the amino acid sequence VIYSGNGDTSYNQKFKG (SEQ ID NO:409), a CDR-H3 comprising the amino acid sequence RERDTR (SEQ ID NO:410), a CDR-L1 comprising the amino acid sequence RASESVDIYGNSFMH (SEQ ID NO:411), a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:412), and a CDR-L3 comprising the amino acid sequence QQNNEDPYT (SEQ ID NO:413).
  • the target molecule is CD34.
  • the target cell is CD34+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD34.
  • the anti-CD34 targeting moiety comprises a Fab fragment, an scFv, or a VHH domain.
  • anti- CD34 antibodies or antigen-binding fragments thereof include S20016E (e.g., BioLegend, Inc.), 581, RAM34, 1H6, 561, 563, QBEND/10, H4C8, L5F1, 2E10, 5B12, 4C8, 8G12, 2E9, 4H11, ICO-115, 3C8G12, BLR197J, CD34/4939, CD34/7719, CD34/7721, CS37, My10, OTI105C4, OTI10C8, OTI11A5, OTI11E1, OTI12F2, OTI39B1, OTI39B5, OTI51A1, OTI8F11, SPM123, SPM610, 15H1, 2C5, 4F11H3, 5A6, 5F3, 5L7E2, MEC14.7, PDM0-12, RM300, and SI16-01, as well as anti-CD34 antibodies or antigen-binding fragments thereof disclosed in any of: US 8,084,033; US 10,106,62,
  • the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:73, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:74. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:75, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:76.
  • the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:77, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:78. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:84, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:85. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:475, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:476.
  • the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:477, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:478. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:479, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:480. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:481, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:482. SEQ ID NOs:73-78, 84, 85, and 475-482 are shown in Table S3, with complementary determining regions (CDRs) marked in bold. Table S3: Exemplary CD34 Targeting Moiety Sequences
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:73, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:74.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:74.
  • the CD34 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:38, and/or a second Fab polypeptide (i.e., VL-CL) comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:39.
  • VH-CH1 first Fab polypeptide
  • VL-CL second Fab polypeptide
  • the CD34 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:40), a CDR-H2 comprising the amino acid sequence WINTNTGEPKYAEEFKG (SEQ ID NO:41), a CDR-H3 comprising the amino acid sequence GYGNYARGAWLAY (SEQ ID NO:42), a CDR-L1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO:43), a CDR-L2 comprising the amino acid sequence QVSNRFS (SEQ ID NO:44), and a CDR-L3 comprising the amino acid sequence FQGSHVPRT (SEQ ID NO:45).
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:75, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:76.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:76.
  • the CD34 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:40), a CDR-H2 comprising the amino acid sequence WINTNTGEPKYAEEFKG (SEQ ID NO:41), a CDR-H3 comprising the amino acid sequence GYGNYARGAWLAY (SEQ ID NO:42), a CDR-L1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO:43), a CDR-L2 comprising the amino acid sequence QVSNRFS (SEQ ID NO:44), and a CDR-L3 comprising the amino acid sequence FQGSHVPRT (SEQ ID NO:45).
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:77, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:78.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:78.
  • the CD34 targeting moiety comprises a CDR-H1 comprising the amino acid sequence RYGMH (SEQ ID NO:244), a CDR-H2 comprising the amino acid sequence VIWYDGRNKEYGDSVKG (SEQ ID NO:245), a CDR-H3 comprising the amino acid sequence PIVGGTNY (SEQ ID NO:246), a CDR-L1 comprising the amino acid sequence RASQSVSSDLA (SEQ ID NO:247), a CDR-L2 comprising the amino acid sequence GASTRATGIPA (SEQ ID NO:248), and a CDR-L3 comprising the amino acid sequence QQYNNWPIT (SEQ ID NO:249).
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:84, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:85.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:85.
  • the CD34 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPYNGYTFYNQKFKG (SEQ ID NO:251), a CDR-H3 comprising the amino acid sequence HFRYDGVFYYAMDY (SEQ ID NO:252), a CDR-L1 comprising the amino acid sequence TLSSQHSTFTIE (SEQ ID NO:253), a CDR-L2 comprising the amino acid sequence LKKDGSHSTGD (SEQ ID NO:254), and a CDR-L3 comprising the amino acid sequence GVGDTIKEQFVYV (SEQ ID NO:255).
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:475, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:476.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:476.
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:477, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:478.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:478.
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:479, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:480.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:480.
  • the CD34 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:481, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:482.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:482.
  • the target molecule is CD50, also referred to as ICAM-3.
  • the target cell is CD50+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD50.
  • anti-CD50 antibodies or antigen-binding fragments thereof include ICAM3-IB23 (e.g., Chugai Pharmaceuticals), clone 002 (e.g., Invitrogen), clone 003 (e.g., Invitrogen), 1019 (e.g., NeoBiotechnologies), MEM-171, MEM-04, EPR22890-230, EPR3994-123, EPR3995, 2873R, OTI1E7, OTI1C2, OIT1F4, OTI1F2, OTI1G4, OTI4F5, OTI4G8, OTI3A7, OTI4D4, OTI5D6, SPM505, 76203, 101, 101-1D2, 186-2G9, CG106, 2F8, MA4, BH10, JA4, ICO-60, ICAM3.1, BR-1, ICAM-3.3, CBR-IC3/1, CBR-IC3/2.1.2, as well as anti-CD50 antibodies or antigen-binding fragments thereof disclosed in any of
  • the CD50 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:328 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:329.
  • SEQ ID NOs:328 and 329 are shown in Table S4, with complementary determining regions (CDRs) marked in bold.
  • the CD50 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:46 or 54, and/or a second Fab polypeptide (i.e., VL-CL) comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:47.
  • VH-CH1 first Fab polypeptide
  • VL-CL second Fab polypeptide
  • the C-terminus of the first Fab polypeptide further comprises a sortase tag, such as is set forth in SEQ ID NO:83.
  • the CD50 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:328, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:329.
  • the CD50 targeting moiety comprises a CDR-H1 comprising the amino acid sequence TYWMH (SEQ ID NO:322), a CDR-H2 comprising the amino acid sequence YINPNTDYTEYNQKFK (SEQ ID NO:323), a CDR-H3 comprising the amino acid sequence SRDAYHGTY (SEQ ID NO:324), a CDR-L1 comprising the amino acid sequence RASQSISDYLH (SEQ ID NO:325), a CDR-L2 comprising the amino acid sequence YASQSIS (SEQ ID NO:326), and a CDR-L3 comprising the amino acid sequence QNGHNFPLT (SEQ ID NO:327).
  • the target molecule is CD71, also referred to as transferrin receptor.
  • the target cell is CD71+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD71.
  • Exemplary anti-CD71 antibodies or antigen-binding fragments thereof include CX- 2029 (e.g., CytomX Therapeutics), INA03 (e.g., Inatherys), D7G9X (e.g., Cell Signaling Technology), Ber-T9 (e.g., Santa Cruz Biotechnology), DF1513, OKT9, H68.4, 10F11, 3B82A1, MEM-189, T56/14, MEM-75, RM384, JF0956, MRQ-48, ICO-92, 3C11F11, 001, 040, 7H12, BGX.24, TFRC/2898R, TFRC/9106R, TFRC/3630, TFRC/1817, 66IG10, TFRC/1059, TFRC/1818, TFRC/1839, OTI6H9, TFRC/1396, 1E6, R17217.1.4, TFRC/1149, SAA1205, 7-1, 1H5, 3A12, 7C
  • the CD71 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:65, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:66. In some embodiments, the CD71 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:67, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:68. In some embodiments, the CD71 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:69, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:70.
  • the CD71 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:71, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:72.
  • SEQ ID NOs:65-72 are shown in Table S5, with complementary determining regions (CDRs) marked in bold.
  • the CD71 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:67, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:68.
  • the CD71 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWLG (SEQ ID NO:342), a CDR-H2 comprising the amino acid sequence DIYPGGDYPTYSEKFKV (SEQ ID NO:343), a CDR-H3 comprising the amino acid sequence SGNYDEVAY (SEQ ID NO:344), a CDR-L1 comprising the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:279), a CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO:345), and a CDR-L3 comprising the amino acid sequence SQSTHVPWT (SEQ ID NO:346).
  • the CD71 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:69, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:70.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:70.
  • the CD71 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYYMN (SEQ ID NO:256), a CDR-H2 comprising the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO:257), a CDR-H3 comprising the amino acid sequence DLPLVYTGFAY (SEQ ID NO:258), a CDR-L1 comprising the amino acid sequence SGDNLRHYYVY (SEQ ID NO:259), a CDR-L2 comprising the amino acid sequence GDSKRPS (SEQ ID NO:347) or GDSKRPSGIPE (SEQ ID NO:260), and a CDR- L3 comprising the amino acid sequence QTYTGGASLV (SEQ ID NO:261).
  • the CD71 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:71, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:72.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:72.
  • the CD71 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWLG (SEQ ID NO:342), a CDR-H2 comprising the amino acid sequence DIYPGGDYPTYSEKFKV (SEQ ID NO:343), a CDR-H3 comprising the amino acid sequence SGNYDEVAY (SEQ ID NO:344), a CDR-L1 comprising the amino acid sequence RSSQSLVH (SEQ ID NO:348) or RSSQSLVHSNGNTYLH (SEQ ID NO:279), a CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO:345), and a CDR-L3 comprising the amino acid sequence SQSTHVPWT (SEQ ID NO:346).
  • the CD71 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:65, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:66.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:66.
  • the CD71 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:48, and/or a second Fab polypeptide (i.e., VL-CL) comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:49.
  • VH-CH1 first Fab polypeptide
  • VL-CL second Fab polypeptide
  • the CD71 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO:349), a CDR-H2 comprising the amino acid sequence YIWSGGSTDYASWA (SEQ ID NO:350), a CDR-H3 comprising the amino acid sequence RYGTSYPDYGDASGFDP (SEQ ID NO:351), a CDR-L1 comprising the amino acid sequence RASQSISSYLA (SEQ ID NO:352), a CDR-L2 comprising the amino acid sequence RASTLAS (SEQ ID NO:353), and a CDR-L3 comprising the amino acid sequence QQNYASSNVDNT (SEQ ID NO:354).
  • the target molecule is CD133, also referred to as PROM1, PROML1, prominin-1, AC133, CORD12, MCDR2, MSTP061, RP41, or STGD4.
  • the target cell is CD133+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD133.
  • anti-CD133 antibodies or antigen-binding fragments thereof include A3G6K (e.g., Cell Signaling Technology), D2V8Q (e.g., Cell Signaling Technology), W6B3C1, W6B3, TMP4, 2F8C5, 5E3, EMK08, and BLR093G, as well as anti-CD133 antibodies or antigen-binding fragments thereof disclosed in any of: US 11,098,109; US 11,220,551; Glumac et al. Prostate.2018 Sep; 78(13): 981–991; etc., each hereby incorporated by reference in its entirety.
  • CD38 [0184]
  • the target molecule is CD38.
  • the target cell is CD38+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD38.
  • anti-CD38 antibodies or antigen-binding fragments thereof include daratumumab (e.g., Janssen Biotech, Inc.), isatuximab (e.g., Sanofi), felzartamab, (MOR202, e.g., MorphoSys AG), TAK-079 (e.g., Takeda), SAR442085 (e.g., Sanofi), CM313, 6E12D, AT1, AT2, AT13/5, HB7, 5C5C3, HIT2, 3C6G4, PD01-49, RM388, 1G3, 4G3, OKT10, BLR123H, 6448R, 7017R, 4247R, rCD38/8334, CD38/8075R, CD38/81114R, CD38/8335R, CD38/8994R, ZR351, 4328, rCD38/6447
  • the CD38 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:92, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:93.
  • SEQ ID NOs:92 and 93 are shown in Table S6, with complementary determining regions (CDRs) marked in bold.
  • the CD38 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:92, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:93.
  • the CD38 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYYMN (SEQ ID NO:256), a CDR-H2 comprising the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO:257), a CDR-H3 comprising the amino acid sequence DLPLVYTGFAY (SEQ ID NO:258), a CDR-L1 comprising the amino acid sequence SGDNLRHYYVY (SEQ ID NO:259), a CDR-L2 comprising the amino acid sequence GDSKRPSGIPE (SEQ ID NO:260), and a CDR-L3 comprising the amino acid sequence QTYTGGASLV (SEQ ID NO:261).
  • the target molecule is CD43, also referred to as leukosialin or sialophorin.
  • the target cell is CD43+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD43.
  • anti-CD43 antibodies or antigen-binding fragments thereof include AT1413, UMG-1, MT-1, S11, CD43-10G7, MEM-59, 1G10, 3G8, 84-3C1, SP55, 9D10L2, PD01-28, 4-29-5-10-21, DFT-1, 1094, 839, 1766R, 2049R, rSPN/6563, SPN/6562R, 2A11D6, SPN/839, SPN/3388, DF-T1, SPN/1094, SPM503, OTI2D4, OTI2C7, OTI3D11, OTI6A4, OTI4G8, OTI3D11, OTI4F6, Bra7G, A2F9, A2F7, and A2F8, as well as anti-CD43 antibodies or antigen-binding fragments thereof disclosed in any of: US 7,674,605; US 9,746,474; US 10,676,531; US 11,174,318; US 11,524,989; KR2012008
  • the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:118, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:119. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:120, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:121.
  • the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:122, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:123. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:124, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:125. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:126, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:127.
  • the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:128, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:129. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:130, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:131. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:132, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:133. SEQ ID NOs:118-133 are shown in Table S7, with complementary determining regions (CDRs) marked in bold. Table S7: Exemplary CD43 Targeting Moiety Sequences
  • the CD43 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:118, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:119.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:118.
  • the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFKG (SEQ ID NO:262), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).
  • the CD43 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:120, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:121.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:121.
  • the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYYMN (SEQ ID NO:267), a CDR-H2 comprising the amino acid sequence RINPNSGDSFYNQKFKG (SEQ ID NO:268), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).
  • the CD43 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:122, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:123.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:123.
  • the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFQG (SEQ ID NO:269), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).
  • the CD43 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:124, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:125.
  • the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFKG (SEQ ID NO:262), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).
  • the CD43 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:126, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:127.
  • the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFQG (SEQ ID NO:269), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).
  • the CD43 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:128, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:129.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:129.
  • the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO:270), a CDR-H2 comprising the amino acid sequence YISSGSGNFYYVDTVKG (SEQ ID NO:271), a CDR-H3 comprising the amino acid sequence STYYHGSRGAMDY (SEQ ID NO:272), a CDR-L1 comprising the amino acid sequence SASSSVSSMY (SEQ ID NO:273), a CDR-L2 comprising the amino acid sequence DTSKMASGVPI (SEQ ID NO:274), and a CDR-L3 comprising the amino acid sequence QQWSSYPPIT (SEQ ID NO:275).
  • the CD43 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:132, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:133.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:133.
  • the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SSPNWWT (SEQ ID NO:282), a CDR-H2 comprising the amino acid sequence EIYYGGRVSYNSALRS (SEQ ID NO:283), a CDR-H3 comprising the amino acid sequence QKNIGCGYSSCFIS (SEQ ID NO:284), a CDR-L1 comprising the amino acid sequence KSSQTILQRSNHLNYLA (SEQ ID NO:285), a CDR-L2 comprising the amino acid sequence WASTRESGVPD (SEQ ID NO:286), and a CDR-L3 comprising the amino acid sequence HQYYTTPQT (SEQ ID NO:287).
  • the target molecule is CD45.
  • the target cell is CD45+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD45.
  • Exemplary anti-CD45 antibodies or antigen-binding fragments thereof include YAML501.4, YAML568, HI30, YTH-24, YTH- 54, JE03-05, 2D1, CD45-2B11, MEM-28, 4E9B2, UCHL1, BL-178-12C7, RM291, 4R0B0, 4A8A4C7A2, F10-89-4, MEM-55, 158-D3, 6O19, 1975R, 1460, 1461, 3881R, OTI4C11, UCH-L1 or UCHL-1, OTI1B7, OTI2F1, OTI3F2, PD7/26, OTI2E7, OTI2H1, OTI1F6, OTI3C8, OTI2
  • the CD45 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:134, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:135.
  • the CD45 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:136, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:137.
  • the CD45 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:138, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:139.
  • SEQ ID NOs:134-139 are shown in Table S8, with complementary determining regions (CDRs) marked in bold.
  • the CD45 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:134, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:135.
  • the CD45 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWMT (SEQ ID NO:288), a CDR-H2 comprising the amino acid sequence SISSSGGSIYYPDSVKG (SEQ ID NO:289), a CDR-H3 comprising the amino acid sequence DERWAGAMDA (SEQ ID NO:290), a CDR-L1 comprising the amino acid sequence KASQNINKNLD (SEQ ID NO:291), a CDR-L2 comprising the amino acid sequence ETNNLQTGIPS (SEQ ID NO:292), and a CDR-L3 comprising the amino acid sequence YQHNSRFT (SEQ ID NO:293).
  • the CD45 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:136, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:137.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:137.
  • the CD45 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NFWMT (SEQ ID NO:294), a CDR-H2 comprising the amino acid sequence SISSSGGSIYYPDSVKD (SEQ ID NO:295), a CDR-H3 comprising the amino acid sequence LHYYSGGGDA (SEQ ID NO:296), a CDR-L1 comprising the amino acid sequence KASQNINKYLD (SEQ ID NO:297), a CDR-L2 comprising the amino acid sequence YTNNLHTGIPS (SEQ ID NO:298), and a CDR-L3 comprising the amino acid sequence LQHSSRWT (SEQ ID NO:299).
  • the CD45 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:138, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:139.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:139.
  • the CD45 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWMT (SEQ ID NO:288), a CDR-H2 comprising the amino acid sequence SISSSGGSIYYPDSVKD (SEQ ID NO:295), a CDR-H3 comprising the amino acid sequence LYYYSGGGDA (SEQ ID NO:300), a CDR-L1 comprising the amino acid sequence KASQDINKYLD (SEQ ID NO:301), a CDR-L2 comprising the amino acid sequence NTNNLHTGIPS (SEQ ID NO:302), and a CDR-L3 comprising the amino acid sequence LQHISRWT (SEQ ID NO:303).
  • the target molecule is CD46.
  • the target cell is CD46+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD46.
  • Exemplary anti-CD46 antibodies or antigen-binding fragments thereof include SB1HGNY, YS5, YS12, 8E2, MEM-258, JB25- 49, 7D12, 23GB5720, ARC0428, 001, 5A8C11, M037, 05, 1E3D1, M177, 122.2, 169-1- E4.3, 3F1, 2E10, 1B6, and MCP/931, as well as anti-CD46 antibodies or antigen-binding fragments thereof disclosed in any of: US 10,533,056; etc., each hereby incorporated by reference in its entirety.
  • the CD46 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:419, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:420.
  • the CD46 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:421, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:422.
  • the CD46 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:423, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:424.
  • SEQ ID NOs:419-424 are shown in Table S9, with complementary determining regions (CDRs) marked in bold. Table S9: Exemplary CD46 Targeting Moiety Sequences
  • the CD46 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:419, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:420.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:420.
  • the CD46 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAMH (SEQ ID NO:304), a CDR-H2 comprising the amino acid sequence FIRSDGSKKYYADSVKG (SEQ ID NO:305), a CDR-H3 comprising the amino acid sequence HGNYFDS (SEQ ID NO:306), a CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO:307), a CDR-L2 comprising the amino acid sequence AASTLQS (SEQ ID NO:308), and a CDR-L3 comprising the amino acid sequence QQLASYPLT (SEQ ID NO:309).
  • the CD46 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:421, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:422.
  • the CD46 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYAMH (SEQ ID NO:310), a CDR-H2 comprising the amino acid sequence VISYDGNNKYYADSVKG (SEQ ID NO:311), a CDR-H3 comprising the amino acid sequence GGGYFDL (SEQ ID NO:312), a CDR-L1 comprising the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO:313), a CDR-L2 comprising the amino acid sequence GNNNRPS (SEQ ID NO:314), and a CDR-L3 comprising the amino acid sequence SSYTSGTWL (SEQ ID NO:315).
  • the CD46 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:423, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:424.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:424.
  • the CD46 targeting moiety comprises a CDR-H1 comprising the amino acid sequence TYGMH (SEQ ID NO:316), a CDR-H2 comprising the amino acid sequence FISYDGDEKYYADSVKG (SEQ ID NO:317), a CDR-H3 comprising the amino acid sequence ASGYGMGILDY (SEQ ID NO:318), a CDR-L1 comprising the amino acid sequence QGDSLRSYYVS (SEQ ID NO:319), a CDR-L2 comprising the amino acid sequence GQNNRPS (SEQ ID NO:320), and a CDR-L3 comprising the amino acid sequence HSRDSSGTHLRV (SEQ ID NO:321).
  • the target molecule is CD49d.
  • the target cell is CD49d+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD49d.
  • Exemplary anti-CD49d antibodies or antigen-binding fragments thereof include Natalizumab (e.g., Biogen), Vedolizumab (e.g., Takeda), and 9F10.
  • the CD49d targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:94, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:95.
  • the CD49d targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:96, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:97.
  • SEQ ID NOs:94-97 are shown in Table S10, with complementary determining regions (CDRs) marked in bold.
  • the CD49d targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:94, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:95.
  • the CD49d targeting moiety comprises a CDR-H1 comprising the amino acid sequence DTYIH (SEQ ID NO:330), a CDR-H2 comprising the amino acid sequence RIDPANGYTKYDPKFQG (SEQ ID NO:331), a CDR-H3 comprising the amino acid sequence EGYYGNYGVYAMDY (SEQ ID NO:332), a CDR-L1 comprising the amino acid sequence KTSQDINKYMA (SEQ ID NO:333), a CDR-L2 comprising the amino acid sequence YTSALQP (SEQ ID NO:334), and a CDR-L3 comprising the amino acid sequence LQYDNLWT (SEQ ID NO:335).
  • the CD49d targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:96, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:97.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:97.
  • the CD49d targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYWMH (SEQ ID NO:336), a CDR-H2 comprising the amino acid sequence EIDPSESNTNYNQKFKG (SEQ ID NO:337), a CDR-H3 comprising the amino acid sequence GGYDGWDYAIDY (SEQ ID NO:338), a CDR-L1 comprising the amino acid sequence RSSQSLAKSYGNTYLS (SEQ ID NO:339), a CDR-L2 comprising the amino acid sequence GISNRFS (SEQ ID NO:340), and a CDR-L3 comprising the amino acid sequence LQGTHQPYT (SEQ ID NO:341).
  • the target molecule is CD49f. In some embodiments, the target cell is CD49f+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD49f. Exemplary anti-CD49f antibodies or antigen-binding fragments thereof include MP-4F10, GoH3, NKI-GoH3, 450-30A, 5HCLC, SR45-00, 5H14L18, 4G10, CL6957, and MAB-5A. CD59 [0211] In some embodiments, the target molecule is CD59. In some embodiments, the target cell is CD59+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD59.
  • Exemplary anti-CD59 antibodies or antigen-binding fragments thereof include MEM-43, OV9A2, JM10-71, p282(H19), QA19A13, 2491C, 2E11B6, 193-27, 029, and 108.
  • CD90 [0212]
  • the target molecule is CD90.
  • the target cell is CD90+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD90.
  • Exemplary anti-CD90 antibodies or antigen-binding fragments thereof include 5E10, HIS51, 53-2.1, F15-42-1, G7, and YKIX337.217.
  • the CD90 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:148, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:149.
  • the CD90 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:150, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:151.
  • SEQ ID NOs:148-151 are shown in Table S11, with complementary determining regions (CDRs) marked in bold.
  • the CD90 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:148, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:149.
  • the CD90 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYYVH (SEQ ID NO:373), a CDR-H2 comprising the amino acid sequence WVNPNSGDTNYAQKFQG (SEQ ID NO:374), a CDR-H3 comprising the amino acid sequence DGDEDWY (SEQ ID NO:375), a CDR-L1 comprising the amino acid sequence RASQGISRSLV (SEQ ID NO:376), a CDR-L2 comprising the amino acid sequence AASTLQS (SEQ ID NO:308), and a CDR-L3 comprising the amino acid sequence LQHNTYPFT (SEQ ID NO:377).
  • the CD90 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:150, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:151.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:151.
  • the CD90 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO:349), a CDR-H2 comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO:378), a CDR-H3 comprising the amino acid sequence GARMDV (SEQ ID NO:379), a CDR-L1 comprising the amino acid sequence KSSQSVLSSSKNKNYLA (SEQ ID NO:380), a CDR-L2 comprising the amino acid sequence WASTRQS (SEQ ID NO:381), and a CDR-L3 comprising the amino acid sequence QQHYSIPVT (SEQ ID NO:382).
  • the target molecule is CD105. In some embodiments, the target cell is CD105+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD105. Exemplary anti-CD105 antibodies or antigen-binding fragments thereof include QA19A14, O99E5, 43A3, SN6, MJ7/18, 3A9, MEM-226, ARC0446, 209701, 2D5E8 JE60-59, 103, and 23GB3745. CD110 [0217] In some embodiments, the target molecule is CD110. In some embodiments, the target cell is CD110+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD110.
  • Exemplary anti-CD110 antibodies or antigen-binding fragments thereof include 1D6B7, S16017A, 1H2, and ARC2257.
  • CD123 [0218]
  • the target molecule is CD123.
  • the target cell is CD123+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD123.
  • Exemplary anti-CD123 antibodies or antigen-binding fragments thereof include pivekimab (IMGN632, e.g., ImmunoGen), talacotuzumab (e.g., Johnson & Johnson), S18016C, QA18A25, 6H6, 7G3, 5B11, 017, and 32703.
  • the CD123 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:148, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:149.
  • the CD123 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:98, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:99.
  • the CD123 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SSIMH (SEQ ID NO:355), a CDR-H2 comprising the amino acid sequence YIKPYNDGTKYNEKFKG (SEQ ID NO:356), a CDR-H3 comprising the amino acid sequence EGGNDYYDTMDY (SEQ ID NO:357), a CDR-L1 comprising the amino acid sequence RASQDINSYLS (SEQ ID NO:358), a CDR-L2 comprising the amino acid sequence RVNRLVD (SEQ ID NO:359), and a CDR-L3 comprising the amino acid sequence LQYDAFPYT (SEQ ID NO:360).
  • the CD123 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:100, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:101.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:101.
  • the CD123 targeting moiety comprises a CDR-H1 comprising the amino acid sequence DYYMK (SEQ ID NO:361), a CDR-H2 comprising the amino acid sequence DIIPSNGATFYNQKFKG (SEQ ID NO:362), a CDR-H3 comprising the amino acid sequence SHLLRASWFAY (SEQ ID NO:363), a CDR-L1 comprising the amino acid sequence ESSQSLLNSGNQKNYLT (SEQ ID NO:364), a CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO:365), and a CDR-L3 comprising the amino acid sequence QNDYSYPYT (SEQ ID NO:366).
  • the target molecule is CD135. In some embodiments, the target cell is CD135+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD135. Exemplary anti-CD135 antibodies or antigen-binding fragments thereof include BV10A4H2, 4G8, A2F10, 7B7C3, BV10A4, 66903, OTI7D6, and FLT3/2458.
  • CD184 [0223] In some embodiments, the target molecule is CD184. In some embodiments, the target cell is CD184+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD184.
  • Exemplary anti-CD184 antibodies or antigen-binding fragments thereof include ulocuplumab (e.g., Bristol-Myers Squibb), 12G5, 1D9, QA18A64, 2B11, 21H17L11, HL2424, 2F10, 2D4, ARC0381, 9D9, 4B5E4, 2G9, and 2A9.
  • the CD184 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:146, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:147.
  • SEQ ID NOs:146 and 147 are shown in Table S13, with complementary determining regions (CDRs) marked in bold.
  • Table S13 Exemplary CD184 Targeting Moiety Sequences variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:146, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:147.
  • the target molecule is CD201.
  • the target cell is CD201+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD201.
  • Exemplary anti-CD201 antibodies or antigen-binding fragments thereof include clones 1560, 041, RCR-227, mRCR-16, 1F5C4, 011, and 016.
  • the CD201 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:152, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:153.
  • the CD201 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:152, and/or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:153.
  • the CD201 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO:383), a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO:384), a CDR-H3 comprising the amino acid sequence GYSDAFDI (SEQ ID NO:385), a CDR-L1 comprising the amino acid sequence RASQGIRNDLG (SEQ ID NO:386), a CDR-L2 comprising the amino acid sequence AASRLQS (SEQ ID NO:387), and a CDR-L3 comprising the amino acid sequence HQAYSYPLT (SEQ ID NO:388).
  • the CD201 targeting moiety is an sdAb comprising a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:102.
  • the CD201 targeting moiety is an sdAb comprising a CDR-H1 comprising the amino acid sequence YYAIG (SEQ ID NO:389), a CDR-H2 comprising the amino acid sequence CISSSDGSTYYADSVKG (SEQ ID NO:390), and a CDR-H3 comprising the amino acid sequence SLGGAWVVAGNCPALDFNS (SEQ ID NO:391).
  • CD338 [0230]
  • the target molecule is CD338.
  • the target cell is CD338+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD338.
  • Exemplary anti-CD338 antibodies or antigen-binding fragments thereof include 5D3, 3G8, BXP-21, and EPR20080.
  • GPR56 [0231]
  • the target molecule is GPR56.
  • the target cell is GPR56+.
  • the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to GPR56.
  • Exemplary anti-GPR56 antibodies or antigen-binding fragments thereof include CG4, 4C3, 3A1D7, and 1443CT642.85.27, as well as anti-GPR56 antibodies or antigen-binding fragments thereof disclosed in Chatterjee et al. (2021) J Biol Chem.296: 100261, hereby incorporated by reference in its entirety.
  • the LNP generally includes a lipid that has polyethylene glycol (PEG) spacer functionalized with a reactive moiety such as a thiol, amine, maleimide or carboxylic acid group.
  • PEG polyethylene glycol
  • the functionalized lipid of the LNP reacts with a complementary group that is covalently bonded to a targeting moiety, hence generating a conjugate of the LNP and the targeting moiety.
  • the targeting moiety is site-specifically conjugated to the surface of the LNP.
  • Conjugates prepared by site-specific methods have a high density of the targeting moiety on the surface of the LNP.
  • the conjugate can comprise more than one targeting moiety (e.g., antibody, Fab fragment, or scFv) per LNP.
  • the conjugate can comprise more than 10 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP.
  • the conjugate can comprise more than 20 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP.
  • the conjugate can comprise more than 30 targeting moieties (e.g., antibodies, Fab fragments, or scFvs). In some embodiments, the conjugate can comprise more than 50 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 75 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 100 targeting moieties (e.g., antibodies, Fab fragments, or scFvs).
  • targeting moieties e.g., antibodies, Fab fragments, or scFvs.
  • the conjugate can comprise from about 50 to about 200 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP.
  • targeting moieties e.g., antibodies, Fab fragments, or scFvs
  • the conjugate can comprise from about 10 to about 30 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP.
  • the weight ratio between a targeting moiety on the surface of the LNP and the payload (e.g., RNA) encapsulated in the LNP can be 1:20, 1:15, 1:10, 1:9, 1:8, 1:7.1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1.
  • the conjugates prepared by site-specific methods can have two or more different targeting moieties on the surface of the LNP, wherein one of the targeting moieties binds to CD164.
  • the conjugate can have two different targeting moieties.
  • the conjugate can have three different targeting moieties on the surface of the LNP, wherein one of the targeting moieties binds to CD164.
  • the conjugate can have two different targeting moieties, wherein the mass ratio of the two targeting moieties on the surface of the LNP is 20:1, 15:1, 10:1, 9:1, 8:1, 7:1.6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, or 1:20.
  • the weight ratio between the one or more targeting moieties on the surface of the LNP and the payload (e.g., RNA) encapsulated in the LNP can be 1:20, 1:15, 1:10, 1:9, 1:8, 1:7.1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1.
  • IgG antibodies consist of four polypeptide chains linked by disulfide bonds. The two polypeptide chains of low molecular weight are call light chains (L).
  • the light chains consist of a variable light chain domain (VL) and a constant light chain domain (CL).
  • the heavy chains consist of a variable heavy light domain (V H ) and three constant heavy chain domains (CH1, CH2, and CH3).
  • the Fab region of the antibody includes the VL, CL, VH, and C H 1 domains.
  • the Fc region includes the constant heavy chain domains C H 2, and C H 3.
  • a hinge region of the IgG antibody covalently links the CH1 domain to the CH2 domain.
  • the two heavy chains of IgG antibodies are connected in the hinge region by a variable number of disulfide bonds depending on the IgG subclass. Different subclasses of IgG antibodies have varying numbers of interchain disulfide bonds.
  • the light chain is covalently linked to the heavy chain via a disulfide bond between the light chain and the heavy chain.
  • this natural interchain disulfide bond is also referred to as the CL-CH1 disulfide bond to distinguish it from disulfide bonds present in the hinge region.
  • Therapeutic antibodies of type IgG1 possess an intermolecular disulfide bond between Cys233 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.
  • Therapeutic antibodies of type IgG4 possess an intermolecular disulfide bond between Cys127 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.
  • Therapeutic antibodies of type IgG2 possess an intermolecular disulfide bond between Cys135 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.
  • Proteolytic cleavage of an IgG antibody results in the formation of a Fab fragment known as a F(ab’)2 fragment.
  • the F(ab’)2 fragment does not include the CH2 domain or the CH3 domain. However, the hinge region of the antibody is retained in a F(ab’)2 fragment.
  • the F(ab’)2 fragment includes disulfide bonds that covalently link two Fab fragments.
  • Reduction of the disulfide bond in the F(ab’) 2 generates two F(ab’) fragments.
  • the sulfhydryl (thiol) groups of the F(ab’) could potentially react with a thiol-reactive group on the surface of an LNP, hence generating a conjugate.
  • site-specific conjugation is challenging.
  • the reduction of the F(ab’) 2 to the F(ab’) fragments could also disrupt the natural interchain disulfide bonds between the CL and CH1 regions of the Fab fragments, hence further compromising site-specific conjugation.
  • Fab fragment e.g., a Fab fragment that binds to CD164
  • LNP precursor lipid nanoparticle
  • the term “precursor LNP” or “base LNP” refers to an LNP that has been functionalized with a reactive moiety (e.g., thiol-reactive group or polyglycine) prior to reacting with the Fab fragment.
  • the process for conjugating a targeting moiety involves reducing the natural interchain disulfide bond between the CL and CH1 domains of a Fab fragment, and reacting the reduced Fab fragment with a thiol-reactive group (e.g., a maleimide or DBM group) covalently bonded to the surface of a precursor LNP, thus forming a conjugate.
  • a thiol-reactive group e.g., a maleimide or DBM group
  • the reduced Fab fragment can be reacted with a lipid that has been chemically modified (functionalized) with a thiol-reactive group (e.g., maleimide or DBM group).
  • the resultant lipid can then be inserted into a preexisting LNP, thus generating a conjugate.
  • Fab fragments used for conjugation may be used by recombinant methods.
  • the Fab fragments generated recombinant are designed not to include a hinge region at the C-terminus.
  • the recombinantly generated Fab fragments include only one disulfide bond between the C L -C H 1 and domains.
  • the CL-CH1 can then be reduced and the resultant free thiol groups can be used as anchors to conjugate the Fab fragment to the surface of an LNP.
  • the Fab fragment is of the IgG class, the IgM class, or the IgA class.
  • the Fab fragment is of the IgG class and has an IgGl, IgG2, IgG3, or IgG4 isotype.
  • the Fab fragment is a native protein.
  • the Fab fragment is an engineered protein.
  • the disclosure provides methods of making a targeted LNP, said method comprising: (i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (C L ) and the constant heavy chain domain 1 (CH1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.
  • the disclosure provides a conjugate produced by a method comprising: (i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chain domain 1 (C H 1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.
  • the thiol-reactive group e.g., maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl
  • a lipid molecule to create a modified lipid wherein the thiol-reactive group is covalently attached to the lipid where it is capable of reacting with at least one free cysteine residue of the reduced Fab fragment (either on the heavy or light chain of the Fab fragment).
  • the reaction between the thiol-reactive group and the at least one free cysteine residue can be completed prior to or after formation of the LNP with the modified lipid.
  • the various components comprising the LNP and a therapeutic payload can be mixed with lipid molecules, including one or more lipids that comprise a thiol-reactive group, thus generating an LNP that comprises a plurality of thiol-reactive groups (each thiol-reactive group shown schematically as a “functional group” in FIG.2A).
  • the thiol-reactive group can then be reacted with at least one free cysteine residue of the Fab fragment, hence generating a conjugate.
  • a lipid that has been modified with the thiol-reactive group can be directly reacted with at least one free cysteine residue of a Fab fragment.
  • the resultant modified lipid attached to the Fab fragment can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.
  • Any suitable reducing reagent can be used to reduce the interchain disulfide bond of the Fab fragment. Examples of reducing reagents include, but are not limited to, 2- mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP), and combinations thereof.
  • the reducing reagent is a mild reducing reagent.
  • mild reducing reagents include, e.g., DTT, TCEP, and DTE.
  • the reducing reagent is TCEP.
  • Any suitable reaction conditions can be used for the reduction of the interchain disulfide bond in step (i).
  • the reduction reaction can occur in water, aqueous buffer, or cell culture media.
  • the reduction reaction is performed at physiological pH (e.g., about 7.4).
  • the reduction reaction is performed at physiological temperature (e.g., about 37°C).
  • the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reduction reaction is performed at ambient temperature (e.g., about 23 ⁇ to about 25°C). In some embodiments, the reduction reaction is performed at about 0°C to about 4°C. [0246] In some embodiments, excess reducing agent is removed following step (i), prior to conjugation to the precursor LNP. In some embodiments, excess reducing agent is not removed following step (i), prior to conjugation to the precursor LNP.
  • the thiol-reactive groups on the LNP comprises any suitable reactive group, including but not limited to, maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl.
  • the thiol-reactive group is maleimide.
  • maleimide reacts with one of the two free cysteine residues of the Fab fragment (either on the heavy or light chain) to form a thiosuccinimide moiety.
  • maleimide reacts with a free cysteine residue on the heavy chain of the Fab fragment.
  • maleimide reacts with a free cysteine residue on the light chain of the Fab fragment.
  • two maleimide groups each react with the Fab fragment, wherein one maleimide reacts with a free cysteine residue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.
  • Any suitable conditions can be used for the reaction between the thiol-reactive group and at least one of the two free cysteine residues of the Fab fragment in step (ii).
  • the reaction can occur in water, aqueous buffer, or cell culture media.
  • the reaction is performed at physiological pH (e.g., about 7.4).
  • the reaction is performed at physiological temperature (e.g., about 37°C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reaction is performed at ambient temperature (e.g., about 23 ⁇ to about 25°C). In some embodiments, the reaction is performed at about 0°C to about 4°C. [0250] A schematic showing the reduction of an interchain disulfide bond in a Fab fragment is depicted in FIG.3.
  • the Fab fragment depicted in FIG.3 can be produced recombinantly without a hinge region at the C-terminus. Accordingly, in such embodiments, the Fab fragment only comprises a single interchain disulfide bond, which is located in the C L -C H 1 interface of the Fab fragment. As shown in FIG.3, the interchain disulfide bond is located between the heavy chain and the light chain of the Fab fragment. The reduction reaction breaks the covalent linkage forming the disulfide bond, thereby generating two free cysteine residues that can react in a subsequent step with a thiol- reactive group.
  • FIG.4 shows an exemplary schematic of conjugate formation, as described herein.
  • increasing the number of thiol-reactive groups on the LNP increases the number of Fab fragments conjugated to two thiol-reactive groups.
  • decreasing the number of thiol-reactive groups on the LNP decreases the number of Fab fragments conjugated to two thiol-reactive groups.
  • the heavy chain and the light chain remain intact on the surface of the LNP, thus forming a functional Fab fragment that is capable of engaging with a receptor on a targeted cell.
  • the thiol-reactive group is maleimide, as shown in FIG.5.
  • step (ii) i.e., the lipoic acid ligase catalyzed reaction
  • step (ii) is carried out in the presence of a mutated lipoic acid ligase, wherein the tryptophan reside (W) at position of the lipoic acid ligase is mutated.
  • W37 mutants are described in Cohen et al., 2012, ChemBioChem, 13, 888-894.
  • the mutant lipoic acid ligase is selected from W37V, W37I, W37T, W37L, W37C, W37A, and W37G.
  • the average LNP diameter of the targeted LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the targeted LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation ranges from about l mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.
  • An LNP of the disclosure may optionally comprise one or more coatings.
  • an LNP may be formulated in a capsule, film, or tablet having a coating.
  • a capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.
  • Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.
  • in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio).
  • R 2 is C3alkyl. In some embodiments, R 2 is C4alkyl. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y is . In some embodiments, Y is . , . In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl.
  • compounds of formula (I) are compounds of formula (I-C): formula (I-C), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R 1 is C4-10alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C6-8alkyl.
  • R 1 is C 6 alkyl.
  • R 2 is C 3 alkyl.
  • R 2 is C 4 alkyl.
  • X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine. [0343] In some embodiments, compounds of formula (I) are compounds of formula (I-D):
  • R 1 is C6-8alkyl. In some embodiments, R 1 is C 6 alkyl. In some embodiments, R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl.
  • X is O. In some embodiments, X is methylene. In some embodiments, m is 2.
  • compounds of formula (I) are compounds of formula (I-E): formula (I-E), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R 1 is C4-10alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C6-8alkyl.
  • R 1 is C 6 alkyl.
  • R 2 is C 3 alkyl.
  • R 2 is C 4 alkyl.
  • R 1 is C 6-8 alkyl. In some embodiments, R 1 is C6alkyl. In some embodiments, R 2 is C3alkyl. In some embodiments, R 2 is C4alkyl.
  • X is O. In some embodiments, X is methylene. In some embodiments, m is 2.
  • m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine. [0349] In some embodiments, compounds of formula (I) are compounds of formula (I-G):
  • R 1 is C6-8alkyl. In some embodiments, R 1 is C 6 alkyl. In some embodiments, R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl.
  • X is O. In some embodiments, X is methylene. In some embodiments, m is 2.
  • m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine. [0351] In some embodiments, compounds of formula (I) are compounds of formula (I-H):
  • R 1 is C6-8alkyl. In some embodiments, R 1 is C 6 alkyl. In some embodiments, R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl.
  • X is O. In some embodiments, X is methylene. In some embodiments, m is 2.
  • m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (II): formula (II), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; and Y is a lipophilic tail, branched or unbranched.
  • Y is .
  • Y is .
  • Y is .
  • Y is .
  • X is O.
  • X is methylene.
  • m is 2.
  • m is 3.
  • m is 4.
  • compounds of formula (II) are compounds of formula (II-
  • Y is a lipophilic tail, branched or unbranched.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid formula (III), or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle; and Y is a lipophilic tail, branched or unbranched.
  • Y is .
  • Y is .
  • compounds of formula (III) are compounds of formula (III- A): formula (III-A), or a pharmaceutically acceptable salt thereof, wherein: Y is a lipophilic tail, branched or unbranched. [0360] In some embodiments of the formula (III-A), Y is , . [0361] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 2.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (IV):
  • compounds of formula (IV) are compounds of formula (IV-A): or a pharmaceutically acceptable salt thereof, wherein: p is 2, 3, 4, 5, or 6; R 5 is C 4-10 alkyl; and R 6 is C 4-10 alkyl.
  • the ionizable lipid has one of the structures depicted below: Lipid 153
  • the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)- heptatriaconta- 6,9,28,3 l-tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3- nonyldocosa-l3, l6-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US9,867,888 (incorporated by reference herein in its entirety).
  • the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of WO2015/095340 (incorporated by reference herein in its entirety).
  • the ionizable lipid is Di((Z)-non-2-en-1-yl) 9-((4- dimethylamino)butanoyl)oxy)heptadecanedioate (L319), , e.g. as synthesized in Example 7, 8, or 9 of US2012/0027803 (incorporated by reference herein in its entirety).
  • the ionizable lipid is 1,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2- hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010/053572 (incorporated by reference herein in its entirety).
  • the ionizable lipid is; Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl 3- (1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946 (incorporated by reference herein in its entirety).
  • ICE Imidazole cholesterol ester
  • the mol% of the ionizable lipid in the LNP is from about 25% to about 65%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 35% to about 60%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 40% to about 50%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 45% to about 50%.
  • the compounds disclosed herein may include an asymmetric center and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms.
  • the present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another and “diastereomers,” which refers to stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.
  • the present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms.
  • Optically active (+) and (-), or (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization.
  • Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate using, for example, chiral high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • the LNPs e.g., targeted LNPs, of the disclosure comprise one or more ionizable lipids.
  • exemplary helper lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl.
  • Additional exemplary lipids include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.
  • non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl
  • non-cationic lipids are described in WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety.
  • the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018/0028664, incorporated herein by reference in its entirety.
  • the helper lipid is a sphingolipid.
  • the non-pegylated lipid is a sphingomyelin.
  • the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide.
  • the sphingomyelin is egg sphingomyelin.
  • the helper lipid comprises 5-40% (mol), 8%-30%, 10%- 28%, 20%-36%, 22%-32%, or 10-15% (mol) of the total lipid present in the lipid nanoparticle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 32%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 21% to about 23%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%.
  • helper lipid e.g., DSPC or sphingomyelin
  • in vivo delivery of certain payloads following administration of the disclosed LNPs (e.g., targeted LNPs) with these percentages of helper lipids provides enhanced transduction and expression of the payloads relative to targeted LNPs with smaller or larger quantities of helper lipid.
  • the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 1:1 to about 7:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 4:1.
  • the molar ratio between the ionizable lipid and the non-pegylated helper lipid is from about 1:1 to about 3:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 1:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 2:1.
  • the molar ratio between the ionizable lipid and the non-pegylated helper lipid is from about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 2.5:1.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid in Table 1, Table 2 or Table 3 and DSPC.
  • the LNP e.g., targeted LNP
  • the LNP comprises an ionizable lipid in Table 1, Table 2 or Table 3 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 25%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 22% to about 28%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP e.g., targeted LNP
  • the LNP comprises an ionizable lipid of Formula I and DSPC.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid of Formula I and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 22% to about 28%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 093 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 093 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 20%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 092 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 092 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%.
  • the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 153 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 153 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 25%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP e.g., targeted LNP comprises Lipid 154 and DSPC.
  • the LNP e.g., targeted LNP, comprises Lipid 154 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 25%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. [0383] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 155 and DSPC.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 155 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 162 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 163 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 169 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 169 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 176 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 176 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 178 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 178 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid 183 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 183 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP, e.g., targeted LNP comprises Lipid V003 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid V003 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53- coprostanol, choiesteryl-(2 , -hydroxy)-ethyl ether, choiesteryl-(4'-hydroxy)-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue, e.g., choiesteryl-(4'-hydroxy)-buty1 ether.
  • the component providing membrane integrity such as a sterol
  • the component providing membrane integrity can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle.
  • such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
  • the molar ratio between the cholesterol molecule and the non-pegylated helper lipid is from about 6:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 3:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 0.5:1.
  • the ratio between the cholesterol molecule and the non-pegylated helper lipid is from about 1.5:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:2 to about 0.8:1.
  • the LNPs e.g., targeted LNPs
  • the LNPs can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule.
  • PEG polyethylene glycol
  • conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof.
  • the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
  • PEG-lipid conjugates include, but are not limited to, PEG- diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), 1,2-dimyristoyl-sn-glycerol, methoxypoly ethylene glycol (DMG-PEG-2K), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0- (2',3'-di(tetradecanoyloxy)propyl-l-0-(DAG)
  • exemplary PEG-lipid conjugates are described, for example, in US5,885,6l3, US6,287,59l, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety.
  • a PEG- lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety.
  • a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (l-[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-
  • the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].
  • the PEG-lipid comprises a structure selected from: .
  • lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.
  • the pegylated lipid has at least one C16 (palmitoyl) PEG lipid anchor. In some embodiments, the pegylated lipid has two C16 PEG lipid anchors (i.e., dialkyl chains of 16 carbons long).
  • the pegylated lipid is 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000.). In some embodiments, the pegylated lipid is 1,2-Dipalmitoyl-rac- glycero-3-methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is C16 PEG ceramide. In some embodiments, the targeted LNPs comprising the C16 pegylated lipids show reduced liver uptake than otherwise identical LNPs comprising C14 pegylated lipids.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG-PEG2000.
  • the pegylated lipid has at least one C18 PEG lipid anchor.
  • the pegylated lipid has two C18 PEG lipid anchors (i.e., dialkyl chains of 18 carbons long).
  • the C18 pegylated lipid is 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the C18 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG- PEG2000). [0400] In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle.
  • one or more additional compounds can also be included in the LNPs, e.g., targeted LNPs, of the disclosure. Those compounds can be administered separately or the additional compounds can be included in the lipid nanoparticles of the invention.
  • the lipid nanoparticles can contain other compounds, such as a payload, e.g., a therapeutic agent, as described herein.
  • the lipid nanoparticles can contain one or more nucleic acids.
  • the lipid nanoparticles can contain at least a first nucleic acid and a second nucleic acid, wherein the second nucleic acid is different than the first nucleic acid.
  • lipid nanoparticle or a formulation comprising lipid nanoparticles
  • reactive impurities e.g., aldehydes or ketones
  • comprises less than a preselected level of reactive impurities e.g., aldehydes or ketones
  • a lipid reagent is used to make a lipid nanoparticle formulation, and the lipid reagent may comprise a contaminating reactive impurity (e.g., an aldehyde or ketone).
  • a lipid regent may be selected for manufacturing based on having less than a preselected level of reactive impurities (e.g., aldehydes or ketones).
  • aldehydes can cause modification and damage of RNA, e.g., cross-linking between bases and/or covalently conjugating lipid to RNA (e.g., forming lipid-RNA adducts).
  • a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.
  • a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • a lipid nanoparticle formulation is produced using a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • the lipid nanoparticle formulation is produced using a plurality of lipid reagents, and each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph. [0404] In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.
  • each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph.
  • the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%
  • the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • the lipid nanoparticle formulation comprises: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.
  • one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • total aldehyde content and/or quantity of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS/MS), e.g., according to the method described in Example 34.
  • LC liquid chromatography
  • MS/MS tandem mass spectrometry
  • reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleic acid molecule (e.g., an RNA molecule, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents.
  • a nucleic acid molecule e.g., an RNA molecule, e.g., as described herein
  • reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleotide or nucleoside (e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents.
  • a nucleotide or nucleoside e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein
  • reactive impurities e.g., aldehydes
  • LNPs e.g., targeted LNPs
  • the LNPs can be used to deliver payloads to cells, e.g., HSCs or HSC progenitors.
  • the payload may be a therapeutic agent.
  • the LNPs can be used to deliver a payload into long-term HSC (LT-HSC) subpopulations.
  • LT-HSC long-term HSC
  • a targeted LNP as described herein can be used to deliver a payload to cells expressing cell-surface receptors targeted by the targeting moiety, e.g., antibody, Fab fragment, scFv or nanobody component, of the targeted LNP (conjugate).
  • the payload is one or more nucleic acids.
  • the payload is one or more RNA molecules.
  • the payload is an mRNA (e.g., an mRNA encoding an enzyme).
  • the RNA molecule is a non-coding RNA (ncRNA).
  • the payload is an RNA template (for example, an RNA template for reverse transcription, e.g., for Target Primed Reverse Transcription (TPRT)).
  • the payload is a siRNA or a microRNA (miRNA).
  • the payload is a guide RNA.
  • the payload is a tRNA.
  • the payload is an antisense oligonucleotide (ASO).
  • the payload is a DNA molecule, for example, a DNA plasmid, closed-ended DNA (ceDNA), or a small circular DNA (e.g., a minicircle or nanoplasmid).
  • Nucleic acid payloads can be linear, circular, covalently closed, single- stranded, double-stranded, or hybrid RNA/DNA molecules.
  • the payload is a small molecule.
  • the payload is a peptide or protein.
  • the conjugates disclosed herein can include two or more payloads, for example, selected from mRNA, ncRNA, guide RNA, siRNA, miRNA, ASO, DNA vector, small molecule, peptide, or protein.
  • the payload can be a reporter protein (e.g., GFP) or a nucleic acid encoding the reporter protein.
  • the LNPs described herein can be used to deliver a therapeutic of interest to a cell, e.g., an HSC or HSC progenitor.
  • the LNP e.g., targeted LNP
  • the therapeutic agent can be a therapeutic peptide or protein, a nucleic acid comprising a therapeutic agent, or a nucleic acid encoding a therapeutic agent.
  • the therapeutic agent can be a genetic medicine (e.g., for gene therapy or gene editing), wherein the therapeutic agent is capable of modifying, altering or effecting a change in the genomic DNA of a cell (e.g., an HSC (e.g., LT-HSC) or HSC progenitor in the subject).
  • the therapeutic agent is a gene therapy agent or gene editing agent.
  • the therapeutic agent is a gene modifying polypeptide, as described herein.
  • the therapeutic agent is a gene modifying system, as described herein.
  • the therapeutic agent can be a peptide or protein, such as an enzyme, or a nucleic acid (e.g., mRNA or DNA) encoding the peptide or protein (e.g., an enzyme).
  • the enzyme can be a nuclease, recombinase, integrase, transposase, retrotransposase, helicase, transcriptase, polymerase, reverse transcriptase, deaminase, methylase, demethylase, or ligase, or can have a combination of enzymatic activities thereof.
  • the therapeutic agent can be a peptide or protein, or a nucleic acid encoding the peptide or protein, for use as a replacement gene therapy.
  • the therapeutic agent can be a peptide or protein, or a nucleic acid encoding the peptide or protein, for use in modifying or altering the genome or epigenome of a cell, e.g., an HSC or HSC progenitor of a subject.
  • the therapeutic agent can comprise one or more components of a system for modifying or altering the genome or epigenome of a cell, e.g., an HSC or HSC progenitor of a subject.
  • the system for modifying or altering the genome or epigenome of a cell of a subject comprises one or more proteins, one or more nucleic acids (e.g., RNA and/or DNA), or combinations thereof.
  • the therapeutic agent can be one or more components of a ribonucleoprotein (RNP) complex for modifying or altering the genome or epigenome of a cell, e.g., an HSC or HSC progenitor.
  • RNP ribonucleoprotein
  • the therapeutic agent can be a protein, or a nucleic acid (e.g., mRNA) encoding the protein, and/or an RNA molecule (e.g., a gRNA) for guiding the protein to a particular location in the genome or epigenome, wherein the protein is capable of modifying or altering the genome or epigenome as a nuclease, recombinase, integrase, transposase, helicase, reverse transcriptase, deaminase, methylase, demethylase, or ligase, or combinations thereof.
  • the therapeutic agent is a nuclease.
  • the nuclease cleaves DNA (e.g., both strands of the DNA), thereby introducing insertion and/or deletion (indel) mutations in DNA, e.g., genomic DNA.
  • the nuclease is a nickase (i.e., it cleaves a single stand of DNA).
  • the nuclease is mutated such that it is inactive or comprises reduced nuclease activity.
  • the therapeutic agent can be used to introduce a substitution in DNA, e.g., genomic DNA, via base editing.
  • the therapeutic agent can be used for epigenome editing.
  • the therapeutic agent can be used to introduce an indel or a substitution in DNA, e.g., genomic DNA, by inducing target-primed reverse transcription (TPRT).
  • the therapeutic agent (i.e., payload) delivered by the LNP e.g., targeted LNP
  • the therapeutic agent can be a gene modifying protein, a nucleic acid encoding a gene modifying protein, or a gene modifying system, as described herein.
  • the therapeutic agent can be a small molecule.
  • the therapeutic agent can be an siRNA or miRNA.
  • the LNPs, e.g., targeted LNPs, of the disclosure can be used to deliver gene editing components into HSCs or HSC progenitors.
  • the LNPs, e.g., targeted LNPs can be used to deliver a CRISPR-Cas system into HSCs.
  • the LNPs, e.g., targeted LNPs can be used to deliver a Class 1 (type I, type III, or type IV) CRISPR system into HSCs.
  • the LNPs, e.g., targeted LNPs can be used to deliver a Class 2 (type II, type V, or type VI) CRISPR system into HSCs.
  • the LNPs can be used to deliver a CRISPR-Cas9 system, or a nucleic acid encoding one or more components of the CRISPR- Cas9 system, into HSCs (e.g., LT-HSCs).
  • the LNPs e.g., targeted LNPs
  • the LNPs can comprise two RNA molecules, such as an RNA comprising a guide RNA (gRNA) and an mRNA encoding the Cas protein.
  • the Cas is Cas9 or Cas12a.
  • the Cas is Cas9.
  • the Cas is Cas12a.
  • the gRNA is a single guide RNA (sgRNA).
  • the LNPs, e.g., targeted LNPs comprise a payload consisting of or comprising a Cas9 or an mRNA encoding a Cas9.
  • the LNPs e.g., targeted LNPs
  • the LNPs comprise a payload consisting of or comprising a Cas12 (e.g., Cas12a) or an mRNA encoding a Cas12 (e.g., Cas12a).
  • the payload further consists of or comprises a gRNA.
  • the mRNA component of a gene modifying system comprises a recombinant nuclease, or a nucleic acid encoding the nuclease, for example a restriction endonuclease, meganuclease, homing endonuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).
  • a restriction endonuclease for example a restriction endonuclease, meganuclease, homing endonuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • the therapeutic agent (i.e., payload) delivered by the LNP can be a gene modifying system, or one or more nucleic acids (e.g., RNA molecules) comprising the gene modifying system (e.g., an mRNA encoding a gene modifying polypeptide and a template RNA), as described herein.
  • the gene modifying system e.g., an mRNA encoding a gene modifying polypeptide and a template RNA
  • a gene modifying system described herein comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises (i) a reverse transcriptase domain, and an endonuclease domain that contains DNA binding functionality; and (B) a template RNA.
  • a gene modifying polypeptide in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery.
  • the gene modifying protein may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain.
  • the DNA-binding function may involve an RNA component that directs the protein to a DNA sequence, e.g., a gRNA spacer.
  • the gene modifying polypeptide may comprise a reverse transcriptase domain and an endonuclease domain.
  • the RNA template element of a gene modifying system is typically heterologous to the gene modifying polypeptide element and provides an object sequence to be inserted (reverse transcribed) into the host genome.
  • the gene modifying polypeptide is capable of target primed reverse transcription.
  • a gene modifying polypeptide includes one or more domains that, collectively, facilitate: 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) integration of the at least a portion of the template nucleic acid into the target DNA.
  • the gene modifying polypeptide is an engineered polypeptide that comprises one or more amino acid substitutions to a corresponding naturally occurring sequence.
  • the gene modifying polypeptide comprises two or more domains that are heterologous relative to each other, e.g., through a heterologous fusion (or other conjugate) of otherwise wild-type domains, or well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain.
  • the RT domain is heterologous to the DBD; the DBD is heterologous to the endonuclease domain; or the RT domain is heterologous to the endonuclease domain.
  • the gene modifying polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., RNA) writing, e.g., reverse transcription.
  • each functions is contained within a distinct domain.
  • a function may be attributed to two or more domains (e.g., two or more domains, together, exhibit the functionality).
  • two or more domains may have the same or similar function (e.g., two or more domains each independently have DNA-binding functionality, e.g., for two different DNA sequences).
  • one or more domains may be capable of enabling one or more functions, e.g., a Cas9 domain enabling both DNA binding and target site cleavage.
  • the domains are all located within a single polypeptide.
  • a gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide that comprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain, wherein the RT domain is C- terminal of the Cas domain; and a linker disposed between the RT domain and the Cas domain.
  • the endonuclease domain is N-terminal relative to the RT domain. In some embodiments, the endonuclease domain is C-terminal relative to the RT domain.
  • the gene modifying polypeptide comprises a GG amino acid sequence between the Cas domain and the linker, an AG amino acid sequence between the RT domain and the second NLS, and/or a GG amino acid sequence between the linker and the RT domain.
  • the gene modifying polypeptide comprises a sequence of SEQ ID NO:171 which comprises the first NLS and the Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • the gene modifying polypeptide comprises a sequence of SEQ ID NO:167 which comprises the second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.
  • N-terminal NLS-Cas9 domain MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLT LLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLV KLNRED
  • the DNA binding domain is a CRISPR-associated protein that recognizes the structure of a template nucleic acid (e.g., template RNA) comprising a gRNA.
  • a gene modifying polypeptide comprises a DNA-binding domain comprising a CRISPR-associated protein that associates with a gRNA scaffold that allows the DNA-binding domain to bind a target genomic DNA sequence.
  • the gRNA scaffold and gRNA spacer is comprised within the template nucleic acid (e.g., template RNA), thus the DNA-binding domain is also the template nucleic acid binding domain.
  • the polypeptide possesses RNA binding function in multiple domains, e.g., can bind a gRNA structure in a CRISPR- associated DNA binding domain and an additional sequence, or structure in a reverse transcriptase domain.
  • Writing domain RT Domain
  • the writing domain of the gene modifying system possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (a RT domain).
  • the RT domain comprises an RT catalytic portion and RNA-binding region (e.g., a region that binds the template RNA).
  • a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, HTLV2, JSRV, MLVF5, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV.
  • a gene modifying polypeptide described herein comprises an RT domain having an amino acid sequence according to Table 6 of International Application WO/2023/039440 (which Table is incorporated herein by reference in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • a nucleic acid described herein encodes an RT domain having an amino acid sequence according to Table 6 of International Application WO/2023/039440, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
  • an RT domain (e.g., as listed in Table 6 of International Application WO/2023/039440) comprises one or more mutations as listed in Table 2A of International Application WO/2023/039440, which Table is herein incorporated by reference in its entirety.
  • an RT domain as listed in Table 6 of International Application WO/2023/039440 comprises one, two, three, four, five, or six of the mutations listed in the corresponding row of Table 2A of International Application WO/2023/039440.
  • a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.
  • a gene modifying polypeptide comprises an amino acid sequence according to Table A1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. Table T1. Selection of exemplary gene modifying polypeptides
  • a gene modifying polypeptide possesses the function of DNA target site cleavage via an endonuclease domain.
  • a gene modifying polypeptide comprises a DNA binding domain, e.g., for binding to a target nucleic acid.
  • a domain e.g., a Cas domain
  • the gene modifying polypeptide comprises two or more smaller domains, e.g., a DNA binding domain and an endonuclease domain.
  • a DNA binding domain e.g., a Cas domain
  • the binding is mediated by a gRNA.
  • a domain has two functions.
  • the endonuclease domain is also a DNA-binding domain.
  • the endonuclease domain is also a template nucleic acid (e.g., template RNA) binding domain.
  • a polypeptide comprises a CRISPR- associated endonuclease domain that binds a template RNA comprising a gRNA, binds a target DNA sequence (e.g., with complementarity to a portion of the gRNA), and cuts the target DNA sequence.
  • a target DNA sequence e.g., with complementarity to a portion of the gRNA
  • an endonuclease domain or endonuclease/DNA-binding domain from a heterologous source can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein.
  • the gene modifying polypeptide comprises a Cas domain
  • the Cas domain comprises an endonuclease domain and a DNA-binding domain.
  • the Cas domain comprises a Cas9 or a mutant or variant thereof (e.g., as described herein).
  • the Cas domain is associated with a guide RNA (gRNA), e.g., as described herein.
  • the Cas domain is directed to a target nucleic acid (e.g., DNA) sequence of interest by the gRNA.
  • the endonuclease domain has nickase activity and cleaves one strand of a target DNA.
  • nickase activity reduces the formation of double-stranded breaks at the target site relative to an endonuclease domain with double strand break activity.
  • the heterologous endonuclease is derived from a CRISPR-associated protein, e.g., Cas9.
  • the heterologous endonuclease is engineered to have only nickase activity, e.g., be a Cas9 nickase, e.g., SpCas9 with D10A, H840A, or N863A mutations.
  • the endonuclease domain has nickase activity and does not form double-stranded breaks.
  • the endonuclease domain forms single-stranded breaks at a higher frequency than double-stranded breaks, e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% of the breaks are single-stranded breaks, or less than 10%, 5%, 4%, 3%, 2%, or 1% of the breaks are double-stranded breaks.
  • the endonuclease forms substantially no double-stranded breaks. In some embodiments, the endonuclease does not form detectable levels of double-stranded breaks.
  • the endonuclease domain has nickase activity that nicks the target site DNA of the first strand; e.g., in some embodiments, the endonuclease domain cuts the genomic DNA of the target site near to the site of alteration on the strand that will be extended by the writing domain. In some embodiments, the endonuclease domain has nickase activity that nicks the target site DNA of the first strand and does not nick the target site DNA of the second strand.
  • a polypeptide comprises a CRISPR- associated endonuclease domain having nickase activity
  • said CRISPR-associated endonuclease domain nicks the target site DNA strand containing the PAM site (e.g., and does not nick the target site DNA strand that does not contain the PAM site).
  • said CRISPR-associated endonuclease domain nicks the target site DNA strand not containing the PAM site (e.g., and does not nick the target site DNA strand that contains the PAM site).
  • a gene modifying polypeptide described herein comprises a Cas domain.
  • the Cas domain can direct the gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis.”
  • a reverse transcriptase domain of a gene modifying polypeptide is fused to a Cas domain.
  • CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e.g., Cas9 or Cpf1) to cleave foreign DNA.
  • an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences.
  • a target nucleotide sequence e. g., a site in the genome that is to be sequence-edited
  • sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences.
  • Three classes (I-III) of CRISPR systems have been identified.
  • the class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins).
  • One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”).
  • the crRNA contains a “spacer” sequence, a typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence (“protospacer”).
  • spacer a typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence (“protospacer”).
  • crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure that is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid molecule.
  • a crRNA/tracrRNA hybrid then directs the Cas endonuclease to recognize and cleave a target DNA sequence.
  • a target DNA sequence is generally adjacent to a “protospacer adjacent motif” (“PAM”) that is specific for a given Cas endonuclease and required for cleavage activity at a target site matching the spacer of the crRNA.
  • CRISPR endonucleases identified from various prokaryotic species have characteristic PAM sequence requirements; examples of PAM sequences include 5 ⁇ -NGG (Streptococcus pyogenes), 5 ⁇ -NNAGAA (Streptococcus thermophilus CRISPR1), 5 ⁇ -NGGNG (Streptococcus thermophilus CRISPR3), and 5 ⁇ - NNNGATT (Neisseria meningiditis).
  • Some endonucleases e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e.g., 5 ⁇ -NGG), and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5 ⁇ from) the PAM site.
  • Another class II CRISPR system includes the type V endonuclease Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.).
  • Cpf1-associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpf1 system, in some embodiments, comprises only Cpf1 nuclease and a crRNA to cleave a target DNA sequence.
  • Cpf1 endonucleases are typically associated with T-rich PAM sites, e. g., 5 ⁇ -TTN.
  • Cpf1 can also recognize a 5 ⁇ -CTA PAM motif.
  • Cpf1 typically cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5 ⁇ overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3 ⁇ from) from a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al.
  • Cas protein A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species.
  • a particular Cas protein e.g., a particular Cas9 protein
  • a DNA-binding domain or endonuclease domain includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9.
  • a Cas protein e.g., a Cas9 protein
  • a Cas protein may be obtained from a bacteria or archaea or synthesized using known methods.
  • a Cas protein may be from gram-positive bacteria or gram-negative bacteria.
  • a Cas protein may be from Streptococcus (e.g., S.
  • pyogenes or S. thermophilus
  • Francisella e.g., F. novicida
  • Staphylococcus e.g., S. aureus
  • Acidaminococcus e.g., Acidaminococcus sp. BV3L6
  • Neisseria e.g., N. meningitidis
  • Cryptococcus Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter.
  • a gene modifying polypeptide may comprise a Cas domain as listed in Table 7 or 8 of International Application WO/2023/039440 (which Tables are incorporated herein by reference in their entireties), or a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
  • a gene modifying polypeptide comprises a Cas domain according to SEQ ID NO:226, or a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
  • a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or
  • the PAM is or comprises, from 5 ⁇ to 3 ⁇ , NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G.
  • a Cas protein is a protein listed in Table 7 or 8 of International Application WO/2023/039440.
  • a Cas protein comprises one or more mutations altering its PAM.
  • the Cas protein is modified to deactivate or partially deactivate the nuclease, e.g., nuclease-deficient Cas9.
  • a DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9.
  • Many catalytically inactive Cas9 proteins are known in the art.
  • dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A or N863A mutations.
  • a catalytically inactive or partially inactive CRISPR/Cas domain comprises a Cas protein comprising one or more mutations, e.g., one or more of the mutations listed in Table 7 of International Application WO/2023/039440.
  • a Cas protein described on a given row of Table 7 of International Application WO/2023/039440 comprises one, two, three, or all of the mutations listed in the same row of Table 7 of International Application WO/2023/039440.
  • a Cas protein e.g., not described in Table 7 of International Application WO/2023/039440, comprises one, two, three, or all of the mutations listed in a row of Table 7 of International Application WO/2023/039440 or a corresponding mutation at a corresponding site in that Cas protein.
  • an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9 or SpyCas9) or a functional fragment or variant thereof.
  • the endonuclease domain or DNA binding domain comprises a modified SpCas9.
  • the modified SpCas9 comprises a modification that alters protospacer-adjacent motif (PAM) specificity.
  • the PAM has specificity for the nucleic acid sequence 5 ⁇ -NGT-3 ⁇ .
  • the endonuclease domain or DNA binding domain comprises a Cas domain, e.g., a Cas9 domain.
  • the endonuclease domain or DNA binding domain comprises a nuclease- active Cas domain, a Cas nickase (nCas) domain, or a nuclease-inactive Cas (dCas) domain.
  • the endonuclease domain or DNA binding domain comprises a nuclease- active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-inactive Cas9 (dCas9) domain.
  • the endonuclease domain or DNA binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpfl, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i.
  • the endonuclease domain or DNA binding domain comprises a Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpfl, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i.
  • the endonuclease domain or DNA binding domain comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof.
  • the endonuclease domain or DNA binding domain comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference.
  • the endonuclease domain or DNA binding domain comprises spCas9, SpCas9-SpRY, spCas9-VRQR, spCas9-VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.
  • exemplary Gene Modifying Polypeptides for use in the gene modifying systems may comprise additional components as described herein.
  • Linkers [0440]
  • a gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 10 of International Application WO/2023/039440 (which Table is incorporated herein by reference in its entirety).
  • a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain of Table 8 of International Application WO/2023/039440), a linker of Table 10 of International Application WO/2023/039440 (or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and an RT domain (e.g., an RT domain of Table 6 of International Application WO/2023/039440).
  • a Cas domain e.g., a Cas domain of Table 8 of International Application WO/2023/039440
  • a linker of Table 10 of International Application WO/2023/039440 or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto
  • an RT domain e.g., an RT domain of Table 6 of International Application WO/2023/039440.
  • a gene modifying polypeptide comprises a flexible linker between the endonuclease and the RT domain, e.g., a linker comprising the amino acid sequence AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO:221), or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.
  • an RT domain of a gene modifying polypeptide may be located C-terminal to the endonuclease domain.
  • an RT domain of a gene modifying polypeptide may be located N-terminal to the endonuclease domain.
  • a gene modifying polypeptide comprises: (i) a Cas domain comprising a Cas sequence as listed in a row of Table T2 of International Application WO/2023/039440 (which Table is incorporated herein by reference in its entirety), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; (ii) a linker comprising a linker sequence as listed in the same row of Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; and (iii) an RT domain comprising an RT domain sequence as listed in the same row of Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.
  • a gene modifying system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence (NLS).
  • a gene modifying polypeptide or a nucleic acid (e.g., RNA) encoding a gene modifying polypeptide comprises an NLS.
  • a polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS).
  • the NLS is a bipartite NLS.
  • an NLS facilitates the import of a protein comprising an NLS into the cell nucleus.
  • the NLS is fused to the N-terminus of a gene modifying polypeptide as described herein. In some embodiments, the NLS is fused to the C-terminus of the gene modifying polypeptide. In some embodiments, the NLS is fused to the N-terminus or the C- terminus of a Cas domain. In some embodiments, a linker sequence is disposed between the NLS and the neighboring domain of the gene modifying polypeptide. [0444] In some embodiments, an NLS comprises an amino acid sequence as disclosed in Table 11 of International Application WO/2023/039440 (which Table is incorporated herein by reference in its entirety), or a sequence having at least 80%, 85%, 90%, or 95% identity thereto.
  • An NLS may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3, or more copies of an NLS in an N-terminal domain, between peptide domains, in a C-terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus.
  • Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. [0445] In some embodiments, the NLS is a bipartite NLS.
  • a bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length).
  • a monopartite NLS typically lacks a spacer.
  • the gene modifying polypeptide comprises, in N-terminal to C-terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS), a DNA binding domain, a linker, an RT domain, and/or a second NLS.
  • NLS nuclear localization signal
  • a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a first NLS, a DNA binding domain, a linker, an RT domain, and a second NLS.
  • a gene modifying polypeptide comprises: (i) an N-terminal NLS comprising an NLS sequence of PAAKRVKLDGG (SEQ ID NO:168), or a functional fragment thereof (e.g., an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); (ii) a Cas domain comprising a Cas sequence of Table 7 or 8 of International Application WO/2023/039440, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; (iii) a linker comprising a linker sequence of AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO:168), or a functional fragment
  • the gene modifying polypeptide comprises the amino acid sequence of SEQ ID NO:222, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. [0448] In some embodiments, the gene modifying polypeptide comprises an N-terminal methionine residue. [0449] In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the first NLS and the DNA binding domain. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises the amino acid sequence GG.
  • the gene modifying polypeptide further comprises a spacer sequence between the DNA binding domain and the linker. In certain embodiments, the spacer sequence between the DNA binding domain and the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the DNA binding domain and the linker comprises the amino acid sequence GG. [0451] In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the linker and the RT domain. In certain embodiments, the spacer sequence between the linker and the RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the linker and the RT domain comprises the amino acid sequence GG.
  • the gene modifying polypeptide further comprises a spacer sequence between the RT domain and the second NLS.
  • the spacer sequence between the RT domain and the second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the spacer sequence between the RT domain and the second NLS comprises the amino acid sequence AG.
  • Nucleic Acids encoding gene modifying polypeptides [0453] Provided herein are nucleic acids encoding gene modifying polypeptides for use in the gene modifying systems disclosed herein. In some embodiments, the nucleic acid is mRNA.
  • a nucleic acid e.g., mRNA
  • a nucleic acid encoding a gene modifying polypeptide is flanked by untranslated regions (UTRs) that modify protein expression levels.
  • UTRs untranslated regions
  • a nucleic acid e.g., an RNA
  • encoding a gene modifying polypeptide further comprises a poly(A) tail for enhancing expression of the polypeptide.
  • Template nucleic acids [0454]
  • the gene modifying systems described herein can modify a host target DNA site using a template nucleic acid sequence.
  • the gene modifying systems described herein transcribe an RNA sequence template into host target DNA sites by target- primed reverse transcription (TPRT).
  • TPRT target- primed reverse transcription
  • the gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step.
  • the gene modifying system can also delete a sequence from the target genome or introduce a substitution using an object sequence. Therefore, the gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and/or function information.
  • the template nucleic acid comprises one or more sequence (e.g., 2 sequences) that binds the gene modifying polypeptide.
  • a system or method described herein comprises a single template nucleic acid (e.g., template RNA).
  • a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs).
  • a template RNA can comprise a gRNA sequence, e.g., to direct the gene modifying polypeptide to a target site of interest.
  • a template RNA comprises (e.g., from 5 ⁇ to 3 ⁇ ): (i) a gRNA spacer that binds a target site (e.g., a second strand of a site in a target genome), (ii) a gRNA scaffold that binds a polypeptide described herein (e.g., a gene modifying polypeptide, e.g., the Cas domain of the gene modifying polypeptide), (iii) a heterologous object sequence comprising a mutation region (optionally the heterologous object sequence comprises, from 5 ⁇ to 3 ⁇ , a first homology region, a mutation region, and a second homology region), and (iv) a primer binding site (PBS) sequence comprising a 3 ⁇ target homology domain.
  • a target site e.g., a second strand of a site in a target genome
  • a gRNA scaffold that binds a polypeptide described herein (e.g., a gene modifying
  • a template RNA molecule for use in the system comprises, from 5 ⁇ to 3 ⁇ : (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence; and (4) a primer binding site (PBS) sequence.
  • PBS primer binding site
  • Is a gRNA spacer of ⁇ 18-22 nt e.g., is 20 nt.
  • Is a gRNA scaffold comprising one or more hairpin loops, e.g., 1, 2, of 3 loops for associating the template with a Cas domain, e.g., a nickase Cas9 domain.
  • the gRNA scaffold comprises the sequence, from 5 ⁇ to 3 ⁇ , GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGGACCGAGTCGGTCC (SEQ ID NO:170).
  • the heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20- 30, 30-40, 40-50, 50-60, 60-70, or 70-80 nt or, 80-90 nt in length.
  • the PBS sequence that binds the target priming sequence after nicking occurs is e.g., 3-20 nt, e.g., 7-15 nt, e.g., 12-14 nt. In some embodiments, the PBS sequence has 40-60% GC content.
  • the template nucleic acid (e.g., template RNA) component of a genome editing system described herein typically is able to bind the gene modifying polypeptide of the system.
  • the template nucleic acid e.g., template RNA
  • the binding region may also provide DNA target recognition, e.g., a gRNA hybridizing to the target DNA sequence and binding the polypeptide, e.g., a Cas9 domain.
  • the template RNA has a poly-A tail at the 3 ⁇ end. In some embodiments the template RNA does not have a poly-A tail at the 3 ⁇ end.
  • the template RNA comprises one or more chemically modified nucleotides. In some embodiments, the template RNA comprises a backbone modification, e.g., a modification to a sugar or phosphate group in the backbone. In some embodiments, the template RNA comprises a nucleobase modification.
  • a system described herein comprises two nucleic acids which together comprise the sequences of a template RNA described herein.
  • the two nucleic acids are associated with each other non-covalently, e.g., directly associated with each other (e.g., via base pairing), or indirectly associated as part of a complex comprising one or more additional molecule.
  • an RNA sequence e.g., a template RNA sequence or an RNA sequence encoding a gene modifying polypeptide
  • T thymine
  • U uracil
  • the RNA sequence may comprise U at every position shown as T in the sequence. More specifically, the present disclosure provides an RNA sequence according to every nucleic acid sequence herein that comprises a T, wherein the RNA sequence has a U in place of each T. Additionally, it is understood that terminal Us and Ts may optionally be added or removed from tracrRNA sequences and may be modified or unmodified when provided as RNA.
  • a template RNA described herein may comprise, from 5 ⁇ to 3 ⁇ : (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence; and (4) a primer binding site (PBS) sequence. Each of these components is now described in more detail.
  • a template RNA described herein may comprise a gRNA spacer that directs the gene modifying system to a target nucleic acid, and a gRNA scaffold that promotes association of the template RNA with the Cas domain of the gene modifying polypeptide.
  • the systems described herein can also comprise a gRNA that is not part of a template nucleic acid.
  • a gRNA that comprises a gRNA spacer and gRNA scaffold, but not a heterologous object sequence or a PBS sequence can be used, e.g., to induce second strand nicking, e.g., as described in the section herein entitled “Second Strand Nicking”.
  • the gRNA is a short synthetic RNA composed of a scaffold sequence that participates in CRISPR-associated protein binding and a user-defined ⁇ 20 nucleotide targeting sequence for a genomic target.
  • the template nucleic acid e.g., template RNA
  • the template nucleic acid has at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 bases of at least 80%, 85%, 90%, 95%, 99%, or 100% sequence homology to the target site, e.g., at the 5 ⁇ end, e.g., comprising a gRNA spacer sequence of length appropriate to the Cas9 domain of the gene modifying polypeptide.
  • a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5 ⁇ to 3 ⁇ direction, a crRNA of Table 12 of International Application WO/2023/039440, a tetraloop from the same row of Table 12, and a tracrRNA from the same row of Table 12, or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.
  • the gRNA or template RNA comprising the scaffold further comprises a gRNA spacer having a length within the Spacer (min) and Spacer (max) indicated in the same row of Table 12.
  • the gRNA or template RNA having a sequence according to Table 12 of International Application WO/2023/039440 is comprised by a system that further comprises a gene modifying polypeptide, wherein the gene modifying polypeptide comprises a Cas domain described in the same row of Table 12.
  • Heterologous object sequence A template RNA described herein may comprise a heterologous object sequence that the gene modifying polypeptide can use as a template for reverse transcription, to write a desired sequence into the target nucleic acid.
  • the heterologous object sequence comprises, from 5 ⁇ to 3 ⁇ , a post-edit homology region, the mutation region, and a pre-edit homology region.
  • an RT performing reverse transcription on the template RNA first reverse transcribes the pre-edit homology region, then the mutation region, and then the post-edit homology region, thereby creating a DNA strand comprising the desired mutation with a homology region on either side.
  • the heterologous object sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 nt in length, e.g., 10-80, 10-50, or 10-20 nt in length, e.g., about10-20 nt in length.
  • the heterologous object sequence is 8-30, 9-25, 10-20, 11-16, or 12-15 nucleotides in length, e.g., is 11-16 nt in length.
  • the heterologous object sequence can be designed to result in insertions, substitutions, or deletions at the target DNA locus.
  • the pre-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
  • the post-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
  • a template nucleic acid (e.g., template RNA) comprises a PBS sequence.
  • a PBS sequence is disposed 3 ⁇ of the heterologous object sequence and is complementary to a sequence adjacent to a site to be modified by a system described herein, or comprises no more than 1, 2, 3, 4, or 5 mismatches to a sequence complementary to the sequence adjacent to a site to be modified by the system/gene modifying polypeptide.
  • the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in the target nucleic acid molecule.
  • binding of the PBS sequence to the target nucleic acid molecule permits initiation of target-primed reverse transcription (TPRT), e.g., with the 3 ⁇ homology domain acting as a primer for TPRT.
  • TPRT target-primed reverse transcription
  • the PBS sequence is 5-20, 8-16, 8-14, 8- 13, 9-13, 9-12, or 10-12 nucleotides in length, e.g., 9-12 nucleotides in length.
  • Exemplary template RNA sequences [0474]
  • the template RNA comprises a gRNA spacer comprising a gRNA spacer sequence of Table X1.
  • a template RNA described herein may comprise a single nucleotide polymorphism (SNP) relative to a reference sequence.
  • a cell line used for screening may contain one or more additional SNPs in the HBB locus relative to a patient or reference sequence, e.g., the hg38 human genome reference sequence, and a landing pad containing the target mutation is optionally designed to carry the one or more non-pathogenic SNPs to match the endogenous cell line HBB locus, e.g., designed to carry a mutation that recapitulates a SNP present in the endogenous HBB locus in HEK293T cells.
  • template RNA sequences found to successfully edit a target mutation at a site containing an additional SNP relative to a reference sequence would differ from a therapeutic template RNA in any region overlapping the additional SNP.
  • a successful template RNA in a HEK293T- based screening assay where a genomic landing pad contains the target mutation (corresponding to the endogenous E6V mutation caused by DNA substitution NC_000011.10:g.5227002T>A) and an additional substitution relative to hg38 (corresponding to the NC_000011.10:g.5227013T>C mutation at the endogenous HBB locus in HEK293T cells) in the protospacer may provide a candidate composition where the corresponding therapeutic template RNA would thus have a substitution (C>T) in the spacer region relative to the corresponding spacer region of the screening template RNA, in order to enable therapeutic correction of the E6V mutation at a target site lacking the additional substitution, e.g., at a target site
  • a screening cell line containing a target site landing pad comprising the pathogenic mutation with an additional T>C substitution in the protospacer region might be corrected using a screening template RNA comprising the spacer sequence 5 ⁇ -CATGGTGCACCTGACTCCTG-3 ⁇ (SEQ ID NO:215) , whereas the corresponding therapeutic template RNA might comprise the spacer sequence 5 ⁇ - CATGGTGCATCTGACTCCTG-3 ⁇ (SEQ ID NO:216), where the underlined nucleotides indicate the position that is altered to match either the screening cell target sequence or the hg38 target sequence.
  • the spacer, PBS, and/or RT template regions may need to be adjusted in this manner to account for any discrepancies between screening and reference target sequences.
  • a given patient or patient population may possess one or more SNPs relative to hg38 at the target locus in addition to the pathogenic E6V mutation and thus a similar adaptation of candidate template RNA molecules could be used to generate template RNA sequences specific for the patient or patient population.
  • a gene modifying system used for correcting the pathogenic E6V mutation in HBB is described in WO2023039440, which is hereby incorporated by reference in its entirety.
  • Table X1 provides a plurality of gRNAs for correcting the pathogenic EV6 mutation in HBB.
  • a payload described herein comprises a template RNA comprising a spacer of Table X1, or a sequence with no more than 1, 2, or 3 sequence differences relative thereto.
  • Table X1 Exemplary gRNA spacer Cas pairs scaffold that binds SpyCas9.
  • the gRNA scaffold that binds SpyCas9 comprises a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO:429) or a sequence having at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity thereto, or a fragment thereof.
  • the heterologous object sequence comprises an RT template sequence from Table X2.
  • the RT sequences of Table X2 are compatible with, for example, the spacer HBB5 with SNP or HBB5 no SNP.
  • a payload described herein comprises a template RNA comprising an RT of Table X2, or a sequence with no more than 1, 2, or 3 sequence differences relative thereto.
  • Table X2 Exemplary RT sequence (heterologous object sequence)
  • the PBS comprises an PBS template sequence from Table X3.
  • the RT sequences of Table X3 are compatible with, for example, the spacer HBB5 with SNP or HBB5 no SNP.
  • a payload described herein comprises a template RNA comprising an PBS of Table X3, or a sequence with no more than 1, 2, or 3 sequence differences relative thereto.
  • the heterologous object sequence comprises an RT template sequence from Table X4.
  • the RT sequences and PBS sequences of Table X4 are compatible with the spacers listed in the first column of Table X4.
  • the primer binding site (PBS) sequence has a sequence comprising the PBS sequence from the same row of Table X4 as the RT template sequence.
  • Table X4 provides exemplified PBS sequences and heterologous object sequences (reverse transcription template regions) of a template RNA for correcting the pathogenic EV6 mutation in HBB.
  • gRNA spacers (e.g., from Table X1) were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme.
  • PBS sequences and heterologous object sequences were designed relative to the nick site directed by the cognate gRNA from Table X1, as described in this application. For exemplification, these regions were designed to be 8-17 nt (priming) and 1-50 nt extended beyond the location of the edit (RT).
  • sequences are provided that use the maximum length parameters and comprise all templates of shorter length within the given parameters.
  • the system further comprises a second strand-targeting gRNA that directs a nick to the second strand of the human HBB gene.
  • the second strand-targeting gRNA comprises a gRNA spacer sequence, gRNA scaffold sequence, or both, from Table X5.
  • Table X5 provides exemplified second-nick gRNA species for optional use for correcting the pathogenic E6V mutation in HBB.
  • Table X5 Exemplary second nick gRNAs
  • the systems and methods provided herein may comprise a template sequence listed in Table X6.
  • Table X6 shows the full-length sequences of various template RNAs described herein, e.g., comprising spacers of Table X1.
  • a template RNA comprises a sequence according to Table X6, or a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.
  • the template RNA according to Table X6 herein comprises one or more chemical modification, e.g., chemical modifications as described in Table X4 of International Application WO/2023/039440 (which Table is incorporated herein by reference in its entirety).
  • Table X6 Exemplary template RNA sequences
  • the heterologous object sequences and PBS sequences may be designed to correct the SCD mutation by replacing a “T” nucleotide with an “A” nucleotide (wild type) or with a “C” (Makassar installation) at the mutation site using a gene modifying system described herein.
  • the template RNA sequences shown herein may be customized depending on various factors. For example, in some embodiments it is desired to inactivate a PAM sequence upon editing (e.g., using a “PAM-kill” modification) to decrease the potential for further gene editing (e.g., by Cas retargeting) following the initial edit.
  • a mutation region within the heterologous object sequence of the template RNA may comprise a PAM-kill sequence.
  • a PAM-kill sequence prevents re-engagement of the gene modifying polypeptide upon completion of a genetic modification, or decreases re-engagement relative to a template RNA lacking a PAM-kill sequence.
  • a PAM-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the PAM-kill sequence results in a silent mutation. In other embodiments, it is desired to leave the PAM sequence intact (no PAM-kill). [0487] In some embodiments it is desired to inactive a PAM sequence upon editing (“PAM-kill”) and in other embodiments it is preferred to leave the PAM sequence intact (no PAM-kill).
  • the RT template can be designed as a “PAM-kill” or “no PAM-kill” version, for example, as shown below.
  • HBB5 Spacer (no SNP): CATGGTGCATCTGACTCCTG (SEQ ID NO:216)
  • HBB5 PBS (no SNP): GAGTCAGAtgcaccatg (SEQ ID NO:218)
  • HBB5 RT template (no PAM-kill): aacggcagactTCTCGTCAG (SEQ ID NO:219)
  • HBB8 RT template (no SNP): tggtgcatctgACTCCTGAG (SEQ ID NO:220)
  • a mutation region within the heterologous object sequence of the template RNA may comprise a seed-kill sequence.
  • a seed-kill sequence prevents re-engagement of the gene modifying polypeptide upon completion of genetic modification, or decreases re- engagement relative to an otherwise similar template RNA lacking a seed-kill sequence.
  • a seed-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the seed-kill sequence results in a silent mutation. In other embodiments, it is desired to leave the seed region intact, and a seed-kill sequence is not used. [0489] In further embodiments, to optimize or improve gene editing efficiency, it may be desirable to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand. In some embodiments, multiple silent mutations (for example, silent substitutions) may be introduced within the RT template sequence to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand.
  • a gene modifying system described herein comprises a nickase activity (e.g., in the gene modifying polypeptide) that nicks the first strand, and a nickase activity (e.g., in the same gene modifying polypeptide or a polypeptide separate from the gene modifying polypeptide) that nicks the second strand of target DNA.
  • nicking of the first strand of the target site DNA is thought to provide a 3 ⁇ OH that can be used by an RT domain to reverse transcribe a sequence of a template RNA, e.g., a heterologous object sequence.
  • a template RNA e.g., a heterologous object sequence.
  • introducing an additional nick to the second strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence.
  • the additional nick to the second strand is made by the same endonuclease domain (e.g., nickase domain) as the nick to the first strand.
  • the same gene modifying polypeptide performs both the nick to the first strand and the nick to the second strand.
  • the gene modifying polypeptide comprises a CRISPR/Cas domain and the additional nick to the second strand is directed by an additional nucleic acid, e.g., comprising a second gRNA directing the CRISPR/Cas domain to nick the second strand.
  • the additional second strand nick is made by a different endonuclease domain (e.g., nickase domain) than the nick to the first strand.
  • second strand nicking may occur in two general orientations: inward nicks and outward nicks. Inward nick and outward nick orientations are described in more detail in International Application WO/2023/039440.
  • Exemplary gRNAs suitable for introducing second strand nicks are provided, e.g., in Table X5 herein.
  • the systems or methods provided herein can be used to upregulate the expression of fetal hemoglobin by introducing a regulatory edit at the promoter of bcl11a, thereby treating sickle cell disease.
  • Table 4 provides exemplary indications (column 1), genes (column 2), and regulatory edits that can be introduced using the systems or methods described herein (column 3). Table 4. Indications, genes, and compensatory regulatory edits.
  • multiple components of a gene modifying system may be prepared as a single LNP formulation, e.g., an LNP formulation comprises mRNA encoding for the gene modifying polypeptide and an RNA template. Ratios of nucleic acid components may be varied in order to maximize the properties of a therapeutic.
  • the ratio of RNA template to mRNA encoding a gene modifying polypeptide is about 1:1 to 100:1, e.g., about 1:1 to 20:1, about 20:1 to 40:1, about 40:1 to 60:1, about 60:1 to 80:1, or about 80:1 to 100:1, by molar ratio.
  • a system of multiple nucleic acids may be prepared by separate formulations, e.g., one LNP formulation comprising a template RNA and a second LNP formulation comprising an mRNA encoding a gene modifying polypeptide.
  • the system may comprise more than two nucleic acid components formulated into LNPs.
  • the system may comprise a protein, e.g., a gene modifying polypeptide, and a template RNA formulated into at least one LNP formulation.
  • a protein e.g., a gene modifying polypeptide
  • a template RNA formulated into at least one LNP formulation.
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is an mRNA encoding a gene modifying polypeptide, as described herein.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • RT reverse transcriptase
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9-RT fusion protein.
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is a guide RNA (gRNA).
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is a template RNA encoding a heterologous nucleic acid for use with a gene modifying polypeptide to insert the heterologous nucleic acid sequence into a DNA sequence, e.g., the genomic DNA of a cell.
  • the components of a system for modifying or altering genomic DNA e.g., a gene modifying system
  • have nuclease activity e.g., nickase activity.
  • the components of a system for modifying or altering genomic DNA do not have nuclease activity. In some embodiments, the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) do not elicit a double-stranded break in the genomic DNA. In such embodiments, the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA. In some embodiments, system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous sequence into the genomic DNA.
  • TPRT target-primed reverse transcription
  • the targets on the surface of the HSC are selected from CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184 (CXCR4).
  • at least one of the targeting moieties binds to CD117.
  • at least one of the targeting moieties binds to CD34.
  • at least one of the targeting moieties binds to CD34.
  • the two targeting moieties bind to CD117 and CD34.
  • the two targeting moieties bind to CD117 and CD71.
  • the two targeting moieties bind to CD34 and CD71.
  • the ionizable lipid is V003 or a lipid from Table 1, Table 2, or Table 3.
  • the ionizable lipid is Lipid 093.
  • the ionizable lipid is Lipid 092.
  • the ionizable lipid is Lipid 154.
  • the helper lipid of the targeted LNP is DSPC or sphingomyelin.
  • the mo1% of the DSPC or sphingomyelin in the targeted LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the targeted LNP further comprises a pegylated lipid.
  • the pegylated lipid comprises at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated is DPPE-PEG2000 or DPG- PEG2000.
  • the targeted delivery of a system for modifying or altering genomic DNA results in synergistic enhancement of gene editing or modification of the target HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 5% of the genomes of the HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 10% of the genomes of the HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 15% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 20% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 25% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the HSCs or LT-HSCs.
  • the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 20% to about 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 20% of the genomes of the HSCs or LT-HSCs.
  • system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA.
  • system for modifying or altering genomic DNA includes a gene modifying polypeptide and a template RNA for a nucleic acid sequence to be inserted at a specific location of the genomic DNA, hence resulting in modification of the genomic DNA.
  • the gene modifying system comprises a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • RT reverse transcriptase
  • system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous nucleic acid sequence into a DNA sequence, e.g., genomic DNA. V.
  • TPRT target-primed reverse transcription
  • an LNP conjugate
  • HSC hematopoietic stem cell
  • an LNP e.g., targeted LNP, disclosed herein is delivered ex vivo to isolated HSC cells of a subject (e.g., human patient) in need thereof.
  • HSCS are first collected from the bone marrow of a subject.
  • the LNPs of the disclosure can be mixed with the HSCs, thereby resulting in effective transduction of the payload (e.g., gene modifying system) into the HSCs.
  • the modified HSCs can be administered to the patient.
  • an LNP, e.g., targeted LNP, as disclosed herein can be introduced into cells, tissues and/or multicellular organisms.
  • the LNPs, e.g., targeted LNPs are delivered to the cells via mechanical means or physical means.
  • Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).
  • the LNP described herein e.g., targeted LNPs
  • the high level of transduction and protein expression following in vivo administration allows for sufficient levels of transduction and expression of proteins (e.g., gene modifying proteins), and hence can be used to treat various diseases including but not limited to the diseases listed in Table 5.
  • an LNP, e.g., targeted LNP, described herein is delivered to a tissue or cell from or in the bone marrow.
  • the LNP e.g., targeted LNP
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous, intradermal, epidural, intracerebral, intracerebroventricular, epicutaneous, nasal, intra-arterial, intra-articular, intracavernous, intraocular, intraosseous infusion, intraperitoneal, intrathecal, intrauterine, intravaginal, intravesical, perivascular, or transmucosal administration).
  • an LNP e.g., targeted LNP
  • a therapeutic agent as described above, to HSCs (e.g., LT-HSCs) of a patient following in vivo administration.
  • an LNP, e.g., targeted LNP, of the disclosure delivers a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, to HSCs (e.g., LT-HSCs) of a patient following in vivo administration.
  • an LNP e.g., targeted LNP
  • a gene modifying system to HSCs (e.g., LT-HSCs) of a patient following in vivo administration.
  • an LNP, e.g., targeted LNP, of the disclosure delivers gene editing components to HSCs (e.g., LT-HSCs) of a patient following in vivo administration. Exemplary gene editing systems are described above. Following in vivo administration, the nucleic acids encoding components of a system for modifying or altering a genome can be expressed, resulting in in vivo gene editing of the HSC (e.g., LT-HSC).
  • LNP formulations e.g., comprising particular amounts of helper lipid
  • HSCs e.g., LT-HSCs
  • HSCs e.g., LT-HSCs
  • LT-HSCs e.g., LT-HSCs
  • the LNPs described herein result in enhanced levels of modification or alteration of the genomic DNA of HSCs, e.g., enhanced levels of gene editing in the HSCs.
  • the percentage of HSCs exhibiting modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs is at least 20%.
  • the LNPs described herein result in enhanced levels of modification or alteration of the genomic DNA of HSCs, e.g., enhanced levels of gene editing in the HSCs.
  • the percentage of HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs is at least 30%.
  • the percentage of HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs is at least 40%. In some embodiments, the percentage of HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 50%. In some embodiments, the percentage of HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 60%.
  • the percentage of HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs is at least 70%. In some embodiments, the percentage of HSCs (e.g., LT-HSCs) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is from about 20% to about 50%, from about 20% to about 40%, or from about 20% to about 30%.
  • 10% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • a baseline LNP e.g., an LNP comprising V003
  • 15% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 20% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 25% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • a baseline LNP e.g., an LNP comprising V003
  • 30% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 35% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • a baseline LNP e.g., an LNP comprising V003
  • 40% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 45% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • a baseline LNP e.g., an LNP comprising V003
  • 50% more HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., an LNP comprising V003) comprising the same therapeutic agent.
  • the number of HSCs comprising modified or altered (e.g., edited) genomic DNA can be determined by DNA sequencing.
  • the LNPs described herein can deliver greater amounts of a payload, e.g., a therapeutic agent or a reporter, to an HSC (e.g., an LT-HSC) when administered in vivo to a subject compared to a baseline LNP (e.g., V003).
  • the targeted LNPs described herein can deliver greater amounts of a payload, e.g., a therapeutic agent or a reporter, to an HSC (e.g., an LT-HSC) when administered in vivo to a subject compared to a baseline LNP (e.g., an LNP comprising V003) comprising the same targeting moieties as the targeted LNP.
  • the LNPs described herein can deliver greater amounts of a nucleic acid, e.g., a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide or one or more nucleic acids comprising a gene modifying system), to an HSC (e.g., an LT-HSC) when administered in vivo to a subject compared to a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid e.g., a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide or one or more nucleic acids comprising a gene modifying system
  • HSC e.g., an LT-HSC
  • a baseline LNP e.g., an LNP comprising V003
  • a nucleic acid payload e.g., a therapeutic agent such as a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • HSCs e.g., LT-HSCs
  • an LNP as described herein e.g., a targeted LNP
  • a baseline LNP e.g., an LNP comprising V003
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 10% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 15% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 20% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 25% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 30% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 35% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 40% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 45% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 50% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • a nucleic acid payload that is delivered to HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits between 10% to 50%, between 20% to 40%, between 10% to 30%, or between 30% to 50% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • the level of expression of the payload can be determined by measuring protein levels, e.g., by flow cytometry.
  • the expression level of the payload can be measured by determining the level of modification or alteration of the genomic DNA of the HSCs, e.g., by DNA sequencing.
  • the LNPs, e.g., targeted LNPs, of the disclosure can be used to treat sickle cell diseases.
  • the LNP, e.g., targeted LNPs, of the disclosure can be used to treat beta thalassemia.
  • the LNPs, e.g., targeted LNPs, following in vivo administration are capable of delivering the therapeutic agent (e.g., a gene modifying polypeptide or gene modifying system, e.g., gene editing components) to long-term HSC (LT-HSC) subpopulations.
  • the therapeutic agent e.g., a gene modifying polypeptide or gene modifying system, e.g., gene editing components
  • the LNPs are capable of delivering the therapeutic agent (e.g., a gene modifying polypeptide or gene modifying system, e.g., gene editing components) to HSC progenitors.
  • the disclosure provides a method of treating sickle cell disease in a human patient in need of, said method comprising administering to the patient a targeted lipid nanoparticle (LNP) comprising: i. an ionizable lipid; ii. a helper lipid; iii.
  • each of the two different targeting moieties binds to a target on the surface of the HSCs (e.g., LT-HSCs); and iv. one or more nucleic acids (e.g., two or more nucleic acids) encoding components of a system for modifying or altering genomic DNA (e.g., a gene modifying system), wherein the two or more nucleic acids are RNA molecules.
  • the nucleic acid components do not introduce double stranded breaks into the genomic DNA.
  • greater than 5% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • greater than 10% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, greater than 15% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, greater than 20% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, greater than 30% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • from about 5% to about 15% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 10% to about 30% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 10% to about 20% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 15% to about 20% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.
  • the HSCs or LT-HSCs maintain their hematopoietic potential and function to support multilineage development.
  • the gene modifying system corrects the E6V HBB mutation in the HSCs and LT-HSCs of the patient.
  • a lipid nanoparticle (LNP) for delivery of a therapeutic agent to hematopoietic stem cells (HSCs) in vivo comprising: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from the lipids in Table 1, Table 2 and Table 3; and a therapeutic agent encapsulated within the LNP.
  • LNP lipid nanoparticle
  • HSCs hematopoietic stem cells
  • the LNP of Embodiment 1, wherein the ionizable lipid has the structure: . 4. The LNP of Embodiment 1, wherein the ionizable lipid has the structure: . 5. The LNP of any one of Embodiments 1-4, wherein the therapeutic agent comprises at least one nucleic acid molecule. 6. The LNP of Embodiment 5, wherein the nucleic acid molecule is an mRNA molecule or a DNA molecule. 7. The LNP of Embodiment 5, wherein the nucleic acid molecule is an mRNA molecule. 8. The LNP of Embodiment 5, wherein the nucleic acid molecule is a siRNA or miRNA molecule. 9.
  • the LNP of Embodiment 5, wherein the therapeutic agent comprises two nucleic acid molecules.
  • the LNP of Embodiment 9, wherein the two nucleic acid molecules are RNA molecules.
  • the LNP of Embodiment 14 wherein the at least one component comprises an mRNA encoding a CRISPR-associated nuclease (Cas) and a guide RNA.
  • the Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).
  • helper lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1
  • the LNP of Embodiment 20, wherein the helper lipid is DSPC. 22.
  • the LNP of Embodiment 20, wherein the helper lipid is sphingomeylin.
  • the LNP of Embodiment 22, wherein the sphingomeylin has a head group selected from phosphocholine, phosphoethanolamine and ceramide.
  • the LNP of Embodiment 23, wherein the sphingomeylin is egg sphingomeylin.
  • the LNP of any one of Embodiments 20-24, wherein the mol% of helper lipid is from about 18% to about 32%. 26.
  • the LNP of Embodiment 25, wherein the mol% of helper lipid is from about 20% to about 25%.
  • a method of treating and/or delaying the progression of a disease in a subject comprising administering to the subject a therapeutically effective amount of a LNP of any one of Embodiments 1-27. 29.
  • Embodiment 28 wherein the disease is selected from the group consisting of adenosine deaminase severe combined immune deficiency (ADA-SCID), adrenoleukodystrophy (CALD), alpha-mannosidosis, chronic granulomatous disease, common variable immunodeficiency, Gaucher disease, globoid cell leukodystrophy, hemophagocytic lymphohistiocytosis, interleukin-7 receptor (IL-7R) SCID, janus kinase 3 (JAK-3) SCID, malignant infantile osteopetrosis, metachromatic leukodystrophy, Scheie syndrome, mucopolysaccharidosis type II (MPS2), MPS7, mucolipidosis II, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, paroxysmal nocturnal hemoglobinuria, Pompe disease, pyruvate kinase defic
  • a conjugate for targeted delivery of a therapeutic agent to hematopoietic stem cells (HSCs) in vivo comprising: a lipid nanoparticle (LNP) comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from the lipids in Table 1, Table 2 and Table 3; a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties is configured to target HSCs; and a therapeutic agent encapsulated within the LNP.
  • the conjugate of Embodiment 31, wherein the ionizable lipid has one of the structures listed in Embodiment 2. 33. The conjugate of Embodiment 31, wherein the ionizable lipid has the structure: ⁇ ⁇ ⁇ 34. The conjugate of Embodiment 31, wherein the ionizable lipid has the structure: . 35. The conjugate of Embodiment 31, wherein the ionizable lipid has the structure: . 36. The conjugate of Embodiment 31, wherein the ionizable lipid has the structure:
  • the conjugate of any one of Embodiments 31-47, wherein the plurality of targeting moieties comprises antibodies or fragments thereof. 49. The conjugate of any one of Embodiments 31-47, wherein the plurality of targeting moieties comprises Fab fragments. 50. The conjugate of any one of Embodiments 31-47, wherein the plurality of targeting moieties comprises single chain variable fragments (scFv). 51. The conjugate of any one of Embodiment 31-50, wherein the therapeutic agent comprises at least one nucleic acid molecule. 52. The conjugate of Embodiment 51, wherein the nucleic acid molecule is an mRNA molecule or a DNA molecule. 53.
  • the conjugate of Embodiment 51, wherein the nucleic acid molecule is an mRNA molecule. 54. The conjugate of Embodiment 51, wherein the nucleic acid molecule is a siRNA or miRNA molecule. 55. The conjugate of Embodiment 51, wherein the therapeutic agent comprises two nucleic acid molecules. 56. The conjugate of Embodiment 55, wherein the two nucleic acid molecules are RNA molecules. 57. The conjugate of Embodiment 55, wherein one nucleic acid molecule comprises an mRNA molecule and one nucleic acid molecule comprises an RNA molecule. 58.
  • the conjugate of Embodiment 55 wherein one nucleic acid molecule comprises an mRNA molecule and one nucleic acid molecule comprises a guide RNA.
  • 59 The conjugate of Embodiment 55, wherein one nucleic acid molecule is an mRNA molecule and one nucleic acid molecule is a DNA molecule.
  • 60 The conjugate of any one of Embodiments 55-59, wherein at least one of the nucleic acid molecules encodes at least one component for altering a genome.
  • the at least one component comprises an mRNA encoding a CRISPR-associated nuclease (Cas) and a guide RNA.
  • the Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).
  • the guide RNA is a single guide RNA.
  • 64. The conjugate of any one of Embodiments 31-63, wherein the mol% of ionizable lipids in the LNP ranges from about 35% to about 60%.
  • 65. The conjugate of Embodiment 64, wherein the mol% of ionizable lipids in the LNP ranges from about 40% to about 50%. 66.
  • Embodiment 64 wherein the mol% of ionizable lipids in the LNP ranges from about 45% to about 50%.
  • the helper lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatid
  • the conjugate of Embodiment 72, wherein the mol% of helper lipid in the conjugate is from about 20% to about 25%.
  • the conjugate of Embodiment 72, wherein the mol% of helper lipid in the conjugate is from about 21% to about 23%.
  • the conjugate of Embodiment 72, wherein the mol% of helper lipid in the conjugate is from about 22% to about 28%.
  • 76. The conjugate of any one of Embodiments 31-75, wherein the LNP further comprises one or more PEGylated lipids.
  • 77. The conjugate of Embodiment 76, wherein the targeting moieties are conjugated to the LNP through the PEGylated lipids. 78.
  • Embodiment 76 or Embodiment 77 wherein the PEGylated lipids each comprise a lipid component, wherein the lipid component of the PEGylated lipids is selected from the group consisting of DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, stearic acid, and any combination thereof.
  • the lipid component of the PEGylated lipids is DSPE. 80.
  • the conjugate of Embodiment 84, wherein the plurality of targeting moieties comprises more than 100 targeting moieties.
  • the conjugate of Embodiment 94, wherein the tetrazine ring is 6-methyltetrazine.
  • the conjugate of Embodiment 92, wherein the tetrazine ring has one of the following structures:
  • a method of treating and/or delaying the progression of a disease in a subject comprising administering to the subject a therapeutically effective amount of a conjugate of any one of Embodiments 31-113.
  • the disease is selected from the group consisting of adenosine deaminase severe combined immune deficiency (ADA-SCID), adrenoleukodystrophy (CALD), alpha-mannosidosis, chronic granulomatous disease, common variable immunodeficiency, Gaucher disease, globoid cell leukodystrophy, hemophagocytic lymphohistiocytosis, interleukin-7 receptor (IL-7R) SCID, janus kinase 3 (JAK-3) SCID, malignant infantile osteopetrosis, metachromatic leukodystrophy, Scheie syndrome, mucopolysaccharidosis type II (MPS2), MPS7, mucolipidosis II
  • MPS2 mucopolysaccharidosis type
  • Embodiment 115 The method of Embodiment 115, wherein the disease is Sickle Cell Disease. 117. The method of Embodiment 115, wherein the disease is thalassemia. 118. A method of altering the genome of HSCs in vivo in a subject, the method comprising administering to the subject a conjugate of any one of Embodiments 31-113. 119. The method of Embodiment 118, wherein the genomes of at least 2% of the HSCs in the subject are altered after administration of the conjugate. 120. The method of Embodiment 118, wherein the genomes of at least 5% of the HSCs in the subject are altered after administration of the conjugate. 121.
  • the method of Embodiment 118 wherein the genomes of at least 10% of the HSCs in the subject are altered after administration of the conjugate. 122. The method of Embodiment 118, wherein the genomes of at least 15% of the HSCs in the subject are altered after administration of the conjugate. 123. The method of Embodiment 118, wherein the genomes of at least 20% of the HSCs in the subject are altered after administration of the conjugate. 124. The method of Embodiment 118, wherein the genomes of at least 5% to 20% of the HSCs in the subject are altered after administration of the conjugate. 125. A pharmaceutical composition comprising the conjugate of any one of Embodiments 31-124 and a pharmaceutically acceptable excipient. 126.
  • Embodiments 31-113 for treating a ⁇ - hemoglobinopathy.
  • ⁇ -hemoglobinopathy is Sickle Cell Disease. 128.
  • Embodiment 126, wherein the ⁇ -hemoglobinopathy is thalassemia.
  • the LNP of Embodiment 11, wherein the mRNA encodes a gene modifying polypeptide and the RNA molecule is a template RNA comprising a gRNA scaffold. 130.
  • B2M Beta 2 Microglobulin
  • a method of editing a human B2M locus in human cells comprising administering to a human subject an LNP of any one of Embodiments 129-136.
  • the method of Embodiment 136, wherein the method comprises in vivo administration to a subject.
  • a template RNA comprising from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence.
  • Example 1 Transfection of LNPs modified site-specifically in CD34 cells
  • the ethanol phase was prepared with five lipids, containing ionizable lipid (V003) , DSPC, cholesterol, DMG-PEG and DSPE-PEG2K-TCO with a molar ratio of 47%: 8%: 43.5%: 1%: 0.5% respectively.
  • the aqueous phase was composed of mRNA (encoding EGFP) dissolved in 25 mM acetate buffer.
  • storage buffer 50mM Tris-HCl, 50mM NaCl, 9% Sucrose, pH 7.2
  • LNP with anti-CD117 Fab site specific conjugation: Anti- CD117 Fab was produced with an additional sortase tag (LPETG, SEQ ID NO:5) on the C terminus of the heavy chain. 10uM anti-CD117 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm.
  • sortase buffer 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4
  • the resulting meTz-modified anti-CD117 Fab was added to the TCO modified-LNP solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding EGFP) of 1:1. The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD117 Fab was removed by a 300K MPES TFF membrane. The resulting antibody-LNP product was further concentrated using an amicon column. [0522] Transfection Experiments: Human CD34+ cells were treated with anti-CD117 IgG-LNP, anti-CD117 Fab-LNP, or naked LNPs (no antibody), after an 8hr treatment, the efficacy of mRNA transfection was quantified.
  • LNPs with site-specific conjugation of anti-CD117 Fab induced stronger transfection and higher MFI in the CD34+ cells compared to LNPs with randomly-conjugated anti-CD117 IgG.
  • Both types of LNPs with conjugated anti-CD117 (site-specific and randomly conjugated) induced stronger transfection and higher MFI levels in the CD34+ cells compared to LNPs with no conjugated anti-CD117 targeting moiety.
  • Example 2 Comparison of LNPs decorated with randomly modified and site-specifically modified (sortase method) Fab fragments
  • LNP-TCO Production of LNP without Fab
  • the ethanol phase was prepared with five lipids, containing ionizable lipid (V003), DSPC, cholesterol, DMG-PEG and DSPE- PEG2K-TCO with a molar ratio of 47%: 8%: 43.5%: 1%: 0.5% respectively.
  • the aqueous phase was composed of mRNA (encoding EGFP) dissolved in 25 mM acetate buffer.
  • storage buffer 50mM Tris-HCl, 50mM NaCl, 9% Sucrose, pH 7.2
  • LNP with anti-CD117 Fab site specific conjugation: Anti- CD117 Fab was produced with an additional sortase tag (LPETG, SEQ ID NO:5) on the C terminus of the heavy chain.10uM anti-CD117 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm.
  • sortase buffer 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4
  • the resulting meTz-modified anti-CD117 Fab was added to the TCO modified-LNP solution described above for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding EGFP) of 1:1. The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD117 Fab was removed by a 300K MPES TFF membrane. The resulting antibody-LNP product was further concentrated using an amicon column. As shown in FIG.19A, the site-specific sortase method for conjugating a targeting moiety to an LNP produced LNPs where only the C- terminal residue of the conjugated Fab fragments is bound to the LNP.
  • DOL was measured by absorbances at A280 and A590 using DOL calculator.
  • the resulting meTz-modified anti-CD117 Fab was added to the TCO modified-LNP solution described above for antibody-LNP conjugation reaction, with a mass ratio between antibody and mRNA (encoding EGFP) of 1.5:1.
  • the solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD117 Fab was removed by a 300K MPES TFF membrane, the resulting antibody-LNP product was further concentrated using an amicon column.
  • the random modification method for conjugating a targeting moiety to an LNP produces LNPs with different amino acid residues of the Fab fragments conjugated to the LNP.
  • Kasumi-1 cells (a CD117+ cell line) were treated with LNPs conjugated with randomly modified Fab, site-specifically modified Fab or no Fab. After an 8hr treatment, the efficacy of mRNA transfection was quantified. Specifically, flow cytometry was used to assess the percentage of cells expressing GFP (% GFP+ cells) (FIG. 20A) and median fluorescent intensity (MFI) of GFP (FIG.20B). LNPs with site-specific conjugation of anti-CD117 Fab induced stronger transfection and higher MFI in the Kasumi- 1 cells compared to LNPs with randomly-conjugated anti-CD117 Fab.
  • Nonspecific Targeted LNP Delivery of GFP to HSPCs [0527] Production of LNP without Fab (LNP-TCO): The ethanol phase was prepared with five lipids, containing ionizable lipid (V003), DSPC, cholesterol, DMG-PEG and DSPE- PEG2K-TCO with a molar ratio of 47%: 8%: 43.5%: 1%: 0.5% respectively. The aqueous phase was composed of mRNA encoding GFP dissolved in 25 mM acetate buffer.
  • storage buffer 50mM Tris-HCl, 50mM NaCl, 9% Sucrose, pH 7.2
  • HSC and early progenitor cells were defined as hCD45+/Lin-/CD34+/CD38-, while long-term HSCs (LT-HSCs) were defined as hCD45+/Lin-/CD34+/CD38-/CD90+/CD45RA-.
  • HSPCs early progenitor cells
  • LT-HSCs long-term HSCs
  • FIG.21A shows that up to 58% of HSCs plus early progenitors received the LNPs with the sortase-modified Fab fragments compared to about 20% of HSCs plus early progenitors for the base LNP, as determined by the percentages of cells expressing GFP (% GFP+ cells). Control mice that were not exposed to LNPs showed no GFP expression.
  • FIG.21B shows that up to 62% of long-term (LT)-HSCs received the LNPs with the sortase- modified Fab fragments compared to a little over 20% of LT-HSCs exposed to the base LNP.
  • LNPs with sortase-modified Fab fragments exhibited significantly higher in vivo transfection levels in both HSCs plus early progenitors and in LT-HSCs relative to LNPs that did not have the site-specifically conjugated anti-CD117 Fab fragments.
  • FIG.21C there is a good correlation between in vitro transduction of primary HSPCs and in vivo targeting in HSPCs.
  • Example 4 In vivo Experiments (Mice) - Specific targeted delivery of LNP (Sortase Method) with increased percentage of helper lipid to HSPCs
  • LNP-TCO LNP without Fab
  • helper lipid DSPC The ethanol phase was prepared with five lipids, containing ionizable lipid (V003), DSPC, cholesterol, DMG-PEG and DSPE-PEG2K-TCO with a molar ratio of 47%: 22%: 28.5%: 2%: 0.5% respectively.
  • the LNP comprised an increased percentage of helper lipid (DSPC) and a decreased percentage of cholesterol relative to the LNP prepared in Example 3 (22% vs.8% DSPC, respectively).
  • LNP B anti-CD117 Fab
  • helper lipid DSPC The LNP conjugated to anti-CD117 Fab was produced as described in Example 3, but with a molar ratio of 47%: 22%: 28.5%: 2%: 0.5% ionizable lipid (V003), DSPC, cholesterol, DMG-PEG and DSPE-PEG2K-TCO, respectively.
  • mice NBSGW mice (NOD.Cg- Kit W41J Tyr + Prkdc scid Il2rg tm1Wjl /ThomJ) 8-10 weeks of age were engrafted with 0.5 X 10 6 CD34+ human bone marrow hematopoietic stem cells via tail vein injection.
  • the HSPCs were allowed to engraft for 12 weeks and then cohorts of 3 animals were treated with 2.0 mg/kg of LNP A (without a conjugated Fab fragment) or LNP B (with a conjugated anti- CD117 Fab) via tail vein injection.
  • FIG.22 demonstrates that LNP A (without anti-CD117 Fab) delivers GFP mRNA to ⁇ 38% of human HSPCs in the mice, whereas LNP B (conjugated to anti- CD117 Fab) delivers GFP mRNA to ⁇ 82% of human HSPCs in the mice.
  • Example 5 In vivo Experiments (Mice) - Specific Targeted Delivery of LNP (Sortase Method) with sphingomyelin as helper lipid [0535] An experiment similar to that described in Example 4 was conducted but with LNPs comprising egg sphingomyelin as the helper lipid rather than DSPC.
  • LNPs conjugated to an anti-CD117 Fab were produced as described in Example 4 (as for LNP B), but were prepared with five lipids, containing ionizable lipid (V003), egg sphingomyelin, cholesterol, DMG-PEG and DSPE-PEG2K-TCO with a molar ratio of 47%: 8%: 42.5%: 2%: 0.5%; 47%: 22%: 28.5%: 2%: 0.5%; or 30%: 40%: 27.5%: 2%: 0.5%, respectively.
  • FIGs.23A and 23B demonstrate that targeted LNPs comprising ionizable lipid and 8% DSPC provide similar levels of GFP transduction and expression levels in HSPCs when compared to targeted LNPs comprising ionizable lipid and 8% (molar) sphingomyelin.
  • Example 6 In vivo Experiments (Non-Human Primates) - Specific targeted LNP (sortase method) delivery of mRNA encoding GFP to HSPCs [0537]
  • This Example demonstrates that a conjugate of the disclosure is capable of delivering a GFP mRNA payload to HSPCs in cynomolgus macaques.
  • anti-CD117 Fab was produced with an additional sortase tag (LPETG, SEQ ID NO:5) on the C- terminus of the heavy chain.10uM anti-CD117 Fab-LPETG and 1mM Triglycine meTz were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at room temperature for 4 hours with shaking at 800 rpm.
  • sortase buffer 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4
  • the ethanol phase was prepared with five lipids, containing ionizable lipid (V003), DSPC, cholesterol, DMG-PEG and DSPE- PEG2K-TCO with a molar ratio of 47%: 8%: 42.5%: 2%: 0.5% respectively.
  • the aqueous phase was composed of EGFP mRNA dissolved in 25 mM acetate buffer.
  • FIG.24 demonstrates that the exemplified LNP delivers GFP mRNA to >20% of HSPCs.
  • Example 7 Site-specific conjugation by lipoic acid ligase
  • Anti-CD117 Fab was produced with an additional LplA acceptor peptide (LAP) tag on C terminus of the heavy chain.
  • LAP LplA acceptor peptide
  • a solution containing 20uM anti-CD117 Fab-LAP tag, 2 uM LplA enzyme lipoic acid ligase W37V, 200 ⁇ M 10-azidodecanoic acid, 1 mM ATP, and 5 mM magnesium acetate in PBS pH 7.4 was incubated at 37 °C for 1.5 hours with shaking at 800 rpm.
  • the modified Fab fragment which now includes a terminal click handle can be reacted with a complementary Click handle covalently bonded to an LNP, as described herein, to produce a site-specifically modified conjugate.
  • the azide group can be substituted with another Click handle on the Fab fragment (e.g., a Tz or meTz group) and reacted with a TCO group covalently bonded to an LNP.
  • Example 8 Synthesis of ionizable lipids [0544] A number of ionizable lipids were synthesized for formulation in LNPs that were tested for delivery to HSPCs.
  • LC-MS Liquid Chromatography-Mass Spectrum
  • ESI* Liquid Chromatography-Mass Spectrum
  • Method A (20 min run): 60-100AB-20MIN_Phenyl-Hexyl-ELSD-3 LC/MS (The gradient was 60% B in 0-0.4 min, 60-100% B in 0.4-17 min, 100% B in 17-20 min, 60% B in 0.01, the flow rate was 0.8 ml/min.
  • Mobile phase A was 0.05% Trifluoroacetic Acid in water
  • mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile.
  • the column used for chromatography was a Waters XSelect CSH Phenyl-Hexyl 150*4.6mm, 3.5 um.
  • Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.
  • Method B (22 min run): 50-100AB-22MIN-CSH_Phenyl-Hexyl-ELSD-3M LC/MS (The gradient was 50% B in 0-0.4 min, 50-100% B in 0.4-17 min, 100% B in 17-20 min, 50% B in 0.01, the flow rate was 0.8 ml/min.
  • Mobile phase A was 0.05% Trifluoroacetic Acid in water
  • mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile.
  • the column used for chromatography was a Waters XSelect CSH Phenyl-Hexyl 150*4.6mm, 3.5 um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000. [0548] Method C (10 min run): WUXIAB60-Phenyl-Hexyl-10MIN-1: LCMS (The gradient was 60% B in 0.4 min and 60-100% B at 0.4-8.0 min, hold at 100% B for 1 min, 100-0% B in 0.01 min, and then held at 60% B for 2min.
  • Mobile phase A was 0.05% Trifluoroacetic Acid in water
  • mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile.
  • the column used for chromatography was a Waters XSelect CSH Phenyl-Hexyl 50*2.1mm, 3.5 um. Detection methods are ELSD. The ESI MS range was 100-2000. [0549] Method D (10 min run): WUXIAB40-Phenyl -Hexyl_10min LC/MS (The gradient was 40% B in 0-0.4 min, 40-100% B in 0.4-8 min, 100% B in 8-9 min, 40% B in 9.01-10 min, the flow rate was 0.8 ml/min.
  • Mobile phase A was 0.05% Trifluoroacetic Acid in water
  • mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile.
  • the column used for chromatography was a Waters XSelect CSH Phenyl-Hexyl 50*2.1mm, 3.5 um.
  • Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.
  • DCM dichloromethane
  • DIEA N,N-diisopropylethylamine.
  • reaction mixture was diluted with H2O 20 mL and extracted with DCM (30mL * 2). The combined organic layers were washed with brine (30 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue (40 g). DCM (100 mL) and 80 g of silica was added to the residue. Solvent was concentrated under reduced pressure again.
  • Lipid 092 was prepared according to the synthetic scheme shown in FIG.25F. In a 100 mL round-bottom flask equipped with a stir bar, compound 10 (3.21 g, 20.14 mmol, 2.0 eq) was dissolved in DCM (70 mL).
  • Lipid 093 was prepared according to the synthetic scheme shown in FIG.26C.
  • compound 14 (10 g, 13.87 mmol, 1.0 eq) was dissolved in DCM (100 mL).
  • Py (1.76 g, 22.19 mmol, 1.79 mL, 1.6 eq)
  • DMAP 508.26 mg, 4.16 mmol, 0.3 eq
  • (4-nitrophenyl) carbonochloridate 4.47 g, 22.19 mmol, 1.6 eq
  • Lipid 162 was prepared according to the synthetic scheme shown in FIG.27C.
  • Lipid153 was prepared according to the synthetic scheme shown in FIG.28C.
  • Compound 10 was prepared according to the synthetic scheme shown in FIG.25E.
  • DIEA 182.8 mg, 1.41 mmol, 246.3 ⁇ L, 2.0 eq
  • DMAP 43.19 mg, 353.6 ⁇ mol, 0.5 eq
  • EDCI 271.1 mg, 1.41 mmol, 2.0 eq
  • compound 25 500 mg, 707.1 ⁇ mol, 1.0 eq
  • the mixture was stirred at 20°C for 12 hr under N 2 atmosphere.
  • Lipid 154 was prepared according to the synthetic scheme shown in FIG.29F. To a mixture of 4-(diethylamino)butanoic acid (2.48 g, 15.56 mmol, 3.0 eq) in DCM (40 mL) was added DIEA (2.01 g, 15.56 mmol, 2.71 mL, 3.0 eq), DMAP (316.8 mg, 2.59 mmol, 0.5 eq) and EDCI (2.98 g, 15.56 mmol, 3.0 eq). Then compound 32 (4 g, 5.19 mmol, 1.0 eq) was added to the mixture.
  • Lipid155 was prepared according to the synthetic scheme shown in FIG.30E.
  • compound 37 600 mg, 807.4 ⁇ mol, 1.0 eq
  • DCM 10 mL
  • DIEA 156.5 mg, 1.21 mmol, 211.0 ⁇ L, 1.5 eq
  • DMAP 201.5 ⁇ mol, 0.2 eq
  • EDCI 232.2 mg, 1.21 mmol, 1.5 eq
  • 4-(diethylamino)butanoic acid 385.7 mg, 2.42 mmol, 3.0 eq
  • Lipid 176 was prepared according to the synthetic scheme shown in FIG.31F.
  • the paradigm is as follows: a) screen “base LNPs” that each have an identical formulation except for the ionizable lipid (selected from the ionizable lipids in Table 1, Table 2, or Table 3) incorporated in the formulation in C57BL/6 mice to identify those that exhibit enhanced delivery of a reporter GFP mRNA to mouse LSK cells (HSCs) and LT-HSCs relative to a base LNP comprising a baseline control ionizable lipid (the V003 lipid); b) conjugate a targeting moiety (as described herein) that can bind to a protein on the surface of human HSPCs/LT-HSCs to those base LNPs exhibiting higher HSC delivery in C57BL/6 mice relative to the baseline control LNP to create and screen for targeted LNPs (tLNPs comprising the base LNPs plus targeting moiety) exhibiting higher delivery of a reporter GFP mRNA to human HSPCs/LT-HSCs engrafted in NBSGW mice; c
  • Ionizable Lipid Synthesis Ionizable lipids were produced according to or by methods similar to those described in Example 8.
  • Base LNP Production Base LNPs were formulated with an ionizable lipid from Table 1, Table 2, or Table 3 (above) or with the V003 ionizable lipid. LNPs were prepared using molar ratios of either 47% ionizable lipid: 42.5% cholesterol: 8.0% DSPC: 2.0% DMG- PEG2000: 0.5% DSPE-PEG2000- using a N:P of 6. LNPs were formulated using microfluidic mixing using a Precision Nanosystems NanoAssemblr Ignite according to the manufacturer’s protocol.
  • a ratio of aqueous: organic solvent of 3:1 was maintained during mixing by using differential flow rates.
  • each LNP formulated was characterized by dynamic light scattering (DLS) to measure the size (e.g., diameter) and polydispersity (PDI).
  • DLS dynamic light scattering
  • PDI polydispersity
  • each LNP formulated was characterized by DLS to measure the size and PDI.
  • Each LNP formulation was concentrated using 100kDa Amicon Filters at 4 ⁇ and 1500 RCF. The concentrated solution was then sterile filtered using a 0.2 ⁇ m syringe filter and measured using DLS. The filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) assay.
  • TMS 2-(p-toluidinyl)naphthalene-6-sulfonic acid
  • LNP hydrodynamic diameter and polydispersity index were measured using single cell dynamic light scattering (DLS) (Malvern Zetasizer Pro Red Label), high throughput DLS (Wyatt DynaPro III), or high resolution DLS (Malvern NanoSight NS300). Samples were prepared by mixing LNP mixture with 1X PBS (GIBCO 10010-023) at a 1:5 ratio before LNP concentration or a 1:20 ratio after LNP concentration and then analyzed.
  • DLS single cell dynamic light scattering
  • 1X PBS GIC 10010-023
  • Nucleic acid concentration for unlysed and lysed LNPs were found as per manufacturer’s instruction with the encapsulated mRNA used as the known standard. The concentration of the mRNA in the lysed LNP was used as the concentration of the LNP, and the encapsulation was given as the ratio of the difference in lysed and unlysed LNPs to the lysed LNPs.
  • Characterization by TNS To determine the pKa of the LNPs, stock buffers of 25 mM PBS and 125 mM sodium chloride were prepared. The pH of the various buffers was adjusted to roughly pH 4 – 9.5 using hydrogen chloride and sodium hydroxide.
  • Base Lipid (LNP) Screening in C57BL/6 mice A series of studies was conducted to compare base LNPs comprising different ionizable lipids selected from Table 1, Table 2, or Table 3 (above) to a base LNP comprising the V003 ionizable lipid (baseline LNP).
  • the V003 ionizable lipid was used as a standard control to set a baseline delivery level to C57BL/6 LSK cells and LT-HSCs by which to compare the delivery levels of the test ionizable lipids in each study.
  • Those base LNPs comprising an ionizable lipid from Table 1, Table 2, or Table 3 that demonstrated an increase in the percent delivery to and/or GFP expression in LSK cells and/or LT-HSCs relative to the baseline LNP comprising the V003 ionizable lipid were selected for further testing in humanized NBSGW mice.
  • Female C57BL/6 mice 6-8 weeks of age were purchased from Jackson Labs (Bar Harbor, ME; Catalog #000664). Following acclimation to the vivarium for 3-5 days, animals were randomized into groups of 4-5 and baseline weights were obtained. Animals were then treated via intravenous tail vein injection with 200 ul of candidate base lipids formulated with GFP mRNA at a dose of 2 mg/kg.
  • HSCs and early progenitor cells were defined as hCD45+/Lin-/CD34+/CD38-, while long-term HSCs (LT-HSCs) were defined as hCD45+/Lin-/CD34+/CD38-/CD90+/CD45RA-.
  • Table 6 Flow cytometry Antibodies
  • Base LNPs containing Lipid 092, Lipid 093, Lipid 153, Lipid 154, Lipid 155, Lipid 162, Lipid 163, Lipid 169, Lipid 176, Lipid 178, and Lipid 183 on average exhibited increased delivery to and/or increased GFP expression in LSK cells and/or LT- HSCs in C57BL/6 mice relative to a base LNP containing the V003 ionizable lipid (baseline LNP).
  • FIGs.32A and 32B show a subset of the results for Base LNPs containing high performing ionizable lipids.
  • FIG.32A shows enhanced transduction of GFP in LNPs using ionizable Lipid 092 or ionizable Lipid 093 relative to ionizable Lipid V003 (Study 1 in Table 7).
  • FIG.32B shows GFP expression levels about equivalent to V003 LNP in this mouse model.
  • FIGs.32C to 32F show enhanced transduction of GFP and/or enhanced GFP expression in LSK cells and in LT-HSCs using LNPs containing ionizable Lipid 154 or ionizable Lipid 155 relative to an LNP containing ionizable Lipid V003 (Study 6 in Table 7).
  • LNPs formulated with these ionizable lipids could exhibit enhanced delivery of a payload gene to HSCs in vivo for therapeutic applications.
  • a subset of these ionizable lipids were selected for testing in a humanized NBSGW mouse model harboring human HSPCs to determine whether they are capable of enhancing delivery to human cells (see Example 10).
  • Table 7 Results of Base LNP screening in C57BL/6 Mice.
  • Example 10 Screening of targeted LNPs (tLNPs) comprising novel ionizable lipids for HSPC delivery in a humanized mouse model
  • tLNPs targeted LNPs
  • a subset of the base LNPs exhibiting enhanced delivery to HSCs relative to the baseline control LNP in Example 9 were modified to try to increase their ability to target human HSPCs in vivo, and then evaluated for their delivery GFP mRNA to HSPCs/LT-HSCs in a humanized mouse model in which human HSPCs were engrafted.
  • Targeted LNP (tLNP) production Human HSCs express CD117 on their surface.
  • a targeting moiety comprising an antibody Fab that binds specifically to CD117 was conjugated to the surface of the best performing base LNPs of Example 9 to further enhance their delivery potential for human HSPCs in vivo.
  • the anti- CD117 targeting moiety was conjugated to the surface of the base LNPs of Example 9 formulated with the Lipid092, Lipid093, Lipid097, Lipid153, Lipid154, Lipid155, and Lipid162 ionizable lipids to produce targeted LNPs (tLNPs) to be tested in an NBSGW mouse model engrafted with human HSPCs.
  • Base LNPs were prepared using molar ratios of either 47% ionizable lipid: 42.5% cholesterol: 8.0% DSPC: 2.0% DMG-PEG2000: 0.5% DSPE-PEG2000-TCO using a N:P of 6.
  • each LNP formulated was characterized by dynamic light scattering (DLS) to measure the size (e.g., diameter) and polydispersity (PDI).
  • DLS dynamic light scattering
  • PDI polydispersity
  • each LNP formulated was characterized by DLS to measure the size and PDI.
  • Each LNP formulation was concentrated using 100kDa Amicon Filters at 4 ⁇ and 1500 RCF.
  • the concentrated solution was then sterile filtered using a 0.2 ⁇ m syringe filter and measured using DLS.
  • the filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p-toluidinyl)naphthalene-6- sulfonic acid (TNS) assay.
  • LNP solutions were diluted to the desired concentration and stored at -80°C for future use.
  • An anti-CD117 Fab was produced with an additional sortase tag (LPETG, SEQ ID NO:5) on the C terminus of the heavy chain.
  • 10uM CD117 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with gentle mixing. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 10K MWCO Amicon centrifugal UF device.
  • sortase modified anti-CD117 Fab was allowed to react with 5eq AF594 containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed via Zeba desalting columns. DOL was determined by first measuring absorbances at A280 and A590 using a Nanodrop, and then calculating the DOL using extinction coefficients and correction factors. The resulting meTz-modified anti-CD117 Fab was added to the TCO modified-LNP solution described above for antibody-LNP conjugation, with a 1:1 mass ratio between antibody and mRNA (eGFP). The solution was incubated at room temperature for 2 hours and then at 4°C overnight.
  • eGFP mRNA
  • Targeted Lipid (tLNP) screening in “humanized” NBSGW mice A series of studies was conducted to compare anti-CD117 tLNPs formulated with the Lipid 092, Lipid 093, Lipid 097, Lipid 153, Lipid 154, Lipid 155, or Lipid 162 ionizable lipid to a targeted LNP (tLNP) formulated with the V003 ionizable lipid.
  • the V003 ionizable lipid was used as a standard control to set a baseline delivery level to human HSPCs/LT-HSCs engrafted in NBSGW mice by which to compare the delivery levels of the test ionizable lipids in each study.
  • tLNPs demonstrating an increase in the percent delivery to and/or GFP expression in human HSPCs and/or LT-HSCs relative to the tLNP comprising the V003 ionizable lipid (baseline tLNP) were selected for further testing in nonhuman primates (NHPs) (see Example 12).
  • NBSGW mice 6-8 weeks of age were purchased from Jackson Labs (Bar Harbor, ME).
  • mice Following acclimation to the vivarium for 3-5 days, animals received a tail vein injection of 2 X 10 6 human CD34+ HSPCs (HemaCare Division of Charles River Labs, Wilmington, MA) and the human cells were allowed to engraft for 12 weeks before the animals were used in screening studies.
  • “humanized” NBSGW mice were randomized into groups of 4-5 animals and baseline weights were obtained. Animals were then treated via intravenous tail vein injection with 100 ul of candidate tLNPs formulated with GFP mRNA at a dose of 2 mg/kg.
  • HSC and early progenitor cells were defined as hCD45+/Lin-/CD34+/CD38-, while long-term HSCs (LT-HSCs) were defined as hCD45+/Lin-/CD34+/CD38-/CD90+/CD45RA-.
  • Table 8 Flow cytometry antibodies [0600] Summary of Targeted LNP screening studies: Table 9 provides the results of the screening studies.
  • Anti-CD117 targeted LNPs containing Lipid 092, Lipid 093, Lipid 153, Lipid 154, Lipid 155, and Lipid 162 on average exhibited increased delivery to and/or increased GFP expression in human HSPCs and/or LT-HSCs in the humanized NBSGW mouse model relative to the targeted LNP containing the V003 ionizable lipid (baseline tLNP).
  • the results from Study 1 in Table 9 are depicted graphically in FIGs.33A and 33B.
  • targeted LNPs comprising Lipid 092 or 093 show significantly increased transduction (FIG.33A) and GFP expression (FIG.33B) relative to targeted LNPs comprising V003 in LT-HSPCs.
  • Lipid 092 and Lipid 093 tLNPs also exhibited increased GFP transduction and expression in the HSPCs. Surprisingly, Lipid 093 performed better in the humanized mouse model relative to the mouse model described in Example 9. [0602] The results from Study 2 in Table 9 are depicted graphically in FIGs.34A-D. These results show that tLNPs formulated with Lipid 153 or Lipid 154 exhibit higher GFP transduction and GFP expression levels in HSPCS and in LT-HSCs in the humanized mouse model relative to the baseline tLNP formulated with the V003 lipid. Lipid 155 tLNPs exhibited higher GFP transduction levels relative to the V003 baseline tLNP.
  • Example 11 Screening of targeted LNPs (tLNPs) comprising novel ionizable lipids for HSPC delivery in a humanized mouse model
  • tLNPs targeted LNPs
  • Another subset of the base LNPs comprising Lipid 093, Lipid 153 or Lipid 154) exhibiting enhanced delivery to HSPCs relative to the baseline control LNP in Example 9 were modified to try to increase their ability to target human HSPCs in vivo, and then evaluated for their delivery of GFP mRNA to HSPCs/LT-HSCs in a humanized mouse model in which human HSPCs were engrafted.
  • Experiments were conducted as described in Example 10, but with increased quantities of helper lipid (22% DSPC or sphingomyelin).
  • Results are shown in FIGs.35A-D. The results show that LNPs comprising 22% helper lipid deliver payload mRNA to a high percentage of HSPCs and LT-HSPCs, leading to GFP expression.
  • Example 12 Targeted LNPs exhibiting enhanced delivery to LT-HSPCs in nonhuman primates (NHPs) [0605] A subset of the tLNPs exhibiting enhanced delivery to HSPCs and LT-HSCs relative to the baseline control targeted LNPs were evaluated for their ability to deliver GFP mRNA to HSPCs/LT-HSCs in cynomolgus macaques.
  • tLNPs formulated with ionizable lipids Lipid 092, Lipid 093, and Lipid 154 were tested and compared to a tLNP formulated with the V003 ionizable lipid in a series of studies.
  • Targeted LNP (tLNP) production Base LNPs were prepared using the V003, Lipid 092, Lipid 093, or Lipid 154 ionizable lipid and either DSPC or egg sphingomyelin. LNPs were formulated using microfluidic mixing using a Precision Nanosystems NanoAssemblr Ignite according to the manufacturer’s protocol. A ratio of aqueous: organic solvent of 3:1 was maintained during mixing by using differential flow rates.
  • DLS dynamic light scattering
  • PDI polydispersity
  • each LNP formulated was characterized by DLS to measure the size and PDI.
  • Each LNP formulation was concentrated using 100kDa Amicon Filters at 4 ⁇ and 1500 RCF. The concentrated solution was then sterile filtered using a 0.2 ⁇ m syringe filter and measured using DLS. The filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p-toluidinyl)naphthalene-6- sulfonic acid (TNS) assay. LNP solutions were diluted to the desired concentration and stored at -80°C for future use.
  • TMS 2-(p-toluidinyl)naphthalene-6- sulfonic acid
  • a targeting moiety comprising an antibody Fab that binds specifically to CD117 was conjugated to the surface of the LNPs as described in prior examples. Specifically, an anti-CD117 Fab was produced with an additional sortase tag (LPETG, SEQ ID NO:5) on the C terminus of the heavy chain.10uM CD117 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with gentle mixing.
  • sortase buffer 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4
  • the resulting meTz-modified anti-CD117 Fab was added to the TCO modified-LNP solution described above for antibody-LNP conjugation, with a 1:1 mass ratio between antibody and mRNA (eGFP). The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD117 Fab was removed by a 300K mPES TFF membrane. The resulting antibody-LNP product was further concentrated using a 100k MWCO Amicon centrifugal UF device. A ribogreen assay was then performed to quantify mRNA content and encapsulation efficiency. [0608] Characterization: LNPs were characterized by DLS, RiboGreen, and TNS as described in Example 10.
  • tLNP screening in cynomolgus macaques Sexually mature male or female cynomolgus macaques (Biomere, Worcester, MA) were enrolled in the study and baseline weights and blood samples were collected (see Table 10). Animals were pre-treated with famotidine (0.5 mg/kg, IM) and diphenhydramine (5 mg/kg, IM) on Day -1, and on Day 1 at 30-60 minutes prior to start of treatment. Two of the animals administered tLNPs containing the V003 ionizable lipid pretreated with G-CSF and Plerixafor to mobilize HSCs prior to administering the tLNPs.
  • G-CSF was administered daily for 5 consecutive days (Day –4 to Day 0) before the tLNPs were administered.
  • Plerixafor was administered 2 hours before the tLNPs were administered.
  • tLNPs were administered intravenously through a saphenous vein, cephalic vein, or tail vein with a temporary IV catheter on Day 1 at a dose of 2 mg/kg via an approximate 60-minute infusion using an infusion pump, followed by about 0.5 mL of saline to flush the dose from the IV catheter. Additional blood samples were collected at 1 hour and 15 to 20 hours post infusion. Lymph node and bone marrow aspirate were also collected at 15-20 hours after the end of infusion.
  • Bone marrow cells (3 X 10 6 in 0.1 ml PBS) from each nonhuman primate in the studies were subsequently aliquoted to individual wells in 96 well plates and centrifuged at 500G for 5 min at RT.
  • a master mix composed of antibody fluorochrome conjugate mixtures (Table 11) was added to the cells in a total of 0.1 ml.
  • HSCs and early progenitor cells were defined as hCD45+/Lin-/CD34+/CD38-, while long-term HSCs (LT-HSCs) were defined as hCD45+/Lin-/CD34+/CD38-/CD90+/CD45RA-.
  • LT-HSCs long-term HSCs
  • Table 12 and FIG.36 provide the results of the NHP studies.
  • Targeted LNPs containing Lipid 092 deliver GFP mRNA to a greater percentage of HSPCs in NHPs relative to tLNPs containing the V003 ionizable lipid when these tLNPs were formulated with 8% helper lipid (DSPC) (compare Study 1 results to Study 2 results).
  • DSPC helper lipid
  • Increasing the percentage of helper lipid in the tLNP formulations enhanced the ability of targeted LNPs to deliver GFP mRNA to HSPCs and LT-HSCs across studies.
  • V003 targeted LNPs formulated with 22% DSPC exhibited slightly higher targeting of LT-HSCs relative to V003 tLNPs formulated with 8% DSPC (compare Study 1 results to Study 3 results).
  • Targeted LNPs containing Lipid 093 exhibited increased delivery of GFP mRNA to HSPCs and LT-HSCs relative to tLNPs containing the V003 ionizable lipid when the LNPs were formulated with 22% DSPC helper lipid (see Study 3 and Study 4 results).
  • Targeted LNPs containing Lipid 093 exhibited similarly elevated levels of delivery to HSPCs and LT-HSCs in NHPs when formulated with egg sphingomyelin, an alternative helper lipid (Study 4).
  • Table 12 shows that the HSPCs and/or LT-HSCs of NHPs that were transduced by the tLNPs formulated with Lipid 093 and 22% helper lipid (DSPC or sphingomyelin) in vivo generally expressed GFP at higher levels than the HSPCs and/or LT- HSCs of NHPs that were transduced with tLNPs formulated with the V003 ionizable lipid formulated with 22% helper lipid (DSPC).
  • DSPC helper lipid
  • Lipid 092 and Lipid 093 tLNPs can deliver a mRNA encoding a payload gene to more HSPCs and LT-HSCs in an NHP model relative to V003 tLNPs, and the cells transduced with Lipid 093 tLNPs can express higher levels of the payload gene relative to the cells transduced with V003 tLNPs.
  • the performance of the Lipid 093 tLNPs increased relative to the V003 tLNPs when the tLNPs are formulated with higher levels of helper lipids. Table 12. Screening results of targeted LNP (tLNP) screening in nonhuman primates.
  • Example 13 Screening targeting moieties that increase LNP delivery to HSPCs [0612] A screen was conducted to identify targeting moieties that can be conjugated to the surface of LNPs to create targeted LNPs (tLNPs) capable of enhanced delivery of a payload, such as one or more nucleic acids (e.g., comprising a gene modifying system), to human HSPCs and LT-HSPCs relative to LNPs lacking these targeting moieties.
  • tLNPs targeted LNPs
  • a payload such as one or more nucleic acids (e.g., comprising a gene modifying system)
  • Table 13 provides a list of target proteins that were identified as being present on the surface of HSPCs and LT-HSPCs.
  • Target proteins were selected based on an assessment of their expression levels on HSPCs, their internalization rate, the internalization pathway for the target protein, and the degree to which they are expressed on other cell types. Targeting moieties specific to each of the target proteins in Table 13 were conjugated site-specifically to the surface of LNPs as described in Example 1 and 10 and were then tested for their capacity to deliver an mRNA encoding GFP to human HSPCs in vitro.
  • Target proteins present on HSPCs/LT-HSPCs [0613] Targeted LNP (tLNP) production: Base LNPs were prepared using molar ratios of 47% ionizable lipid (V003): 42.5% cholesterol: 8.0% DSPC: 2.0% DMG-PEG2000: 0.5% DSPE-PEG2000-TCO. The aqueous phase was composed of mRNA encoding the eGFP in 25 mM acetate buffer. LNPs were formulated using microfluidic mixing using a Precision Nanosystems NanoAssemblr Ignite according to the manufacturer’s protocol. A ratio of aqueous:organic solvent of 3:1 was maintained during mixing by using differential flow rates.
  • DLS dynamic light scattering
  • PDI polydispersity
  • each LNP formulated was characterized by DLS to measure the size and PDI.
  • Each LNP formulation was concentrated using 100kDa Amicon Filters at 4 ⁇ and 1500 RCF. The concentrated solution was then sterile filtered using a 0.2 ⁇ m syringe filter and measured using DLS. The filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) assay. LNP solutions were diluted to the desired concentration and stored at -80°C for future use.
  • TMS 2-(p-toluidinyl)naphthalene-6-sulfonic acid
  • Each targeting moiety in Table 13 was produced with an additional sortase tag (LPETG, SEQ ID NO:5) on the C terminus of the heavy chain.
  • 10uM targeting moiety- LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with gentle mixing. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 10K MWCO Amicon centrifugal UF device.
  • sortase modified targeting moiety was allowed to react with 5eq AF594 containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed via Zeba desalting columns. DOL was determined by first measuring absorbances at A280 and A590 using a Nanodrop, and then calculating the DOL using extinction coefficients and correction factors. The resulting meTz-modified targeting moiety was added to the TCO modified-LNP solution described above for antibody- LNP conjugation, with a 1:1 mass ratio between antibody and mRNA (eGFP). The solution was incubated at room temperature for 2 hours and then at 4°C overnight.
  • eGFP a 1:1 mass ratio between antibody and mRNA
  • Targeted LNP Human primary CD34+ cells isolated from mobilized peripheral blood were cultured in Xvivo-10 media (Lonza, Basel) supplemented with 100ng/mL SCF, TPO, Flt3L (Peprotech, Rocky Hill, NJ) for 16 hours. 1.5 X 10 5 cells were subsequently transferred to individual wells of a 96 well plate in 80 uL of culture media.
  • each of the targeted LNPs were formulated with a GFP reporter mRNA as described above and stored at -80 ⁇ until use. After thawing for 20 min, the tLNPs were diluted to 20 ug/mL in Xvivo-10 media and then four additional sequential 1:2 dilutions were prepared (10, 5, 2.5, 1.25 ug/mL). In some instances, the tLNP were diluted in Xvivo-10 media, supplemented with 10% FBS. 20 uL of each tLNP dilution were added to each well of the 96 well plate containing the CD34+ cell culture to achieve a total volume of 100 uL/well.
  • a negative control was also prepared wherein base LNPs (no targeting moiety) encapsulating GFP mRNA were added to the CD34+ cells.
  • the cells were cultured in the presence of tLNP (or control) for 24h. In some instances, cells were cultured in the presence of tLNP for 4h, after which, the plate was centrifuged at 500G for 5 minutes, and the supernatants discarded. Cells were then resuspended in fresh Xvixo-10 media supplemented with cytokines and cultured for an additional 20h.
  • GFP expression was measured on an Agilent NovoCyte flow cytometer (Agilent, Santa Clara, CA).
  • FIG.37A shows that tLNPs containing targeting moieties specific to CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184 (CXCR4) delivered GFP mRNA more effectively to HSPCs as evidenced by higher numbers of cells expressing GFP (%GFP+ cells) relative to the negative control cells.
  • Targeted LNPs comprising anti-CD34, anti-CD43, anti-CD71, anti-CD117, anti-CD50, anti-CD49d, and anti-CD46 targeting moieties delivered GFP reporter to greater than 50% of HSPCs.
  • FIG.37B shows that HSPCs transduced with tLNPs containing anti-CD34, anti- CD43, anti-CD46, anti-CD71, anti-CD117, anti-CD50, anti-CD49d, and anti-CD184 targeting moieties exhibited the highest levels of GFP expression as evidenced by MFI levels relative to expression in negative control cells.
  • targeting moieties that bind to CD34, CD38, CD43, CD59, CD71, CD50, CD117, CD46, CD49d, and CD184 increase LNP delivery of mRNA to HSPCs. See Table 14 below for 4hr and 24hr timepoint summary of tLNP delivery data. Table 14. tLNP delivery data summary at 4hr and 24hr
  • Example 14 Conjugating multiple targeting moieties to the surface of an LNP [0617]
  • a methodology was developed to conjugate multiple targeting moieties to the surface of an LNP using a site-specific conjugation process described herein. This process can be used to efficiently conjugate two or more targeting moieties to the surface of an LNP at specific ratios.
  • Conjugating two targeting moieties on LNPs [0618] The ethanol phase was prepared with five lipids, containing ionizable lipid (in this case V003), DSPC, cholesterol, DMG-PEG and DSPE-PEG2K-TCO with a molar ratio of 47%: 8%: 42.5%: 2%: 0.5%, respectively.
  • Two methyltetrazine modified antibodies (anti-CD117 Fab-MeTz and anti-CD45 Fab-MeTz) were labeled by Alexa Fluor 647 and TAMRA NHS esters separately and then mixed at mass ratios of 1:3, 1:1, or 3:1 and then added to the LNP-TCO solution for targeting moiety-LNP conjugation. While targeting moiety ratios of 1:3, 1:1, and 3:1 were tested in this experiment, other ratios of two targeting moieties could also be used efficiently.
  • the mass ratio between the total targeting moieties and mRNA was 1:1. The solution was incubated at room temperature for 2 hours and then at 4°C overnight.
  • Example 15 Targeted LNPs with dual targeting moieties can exhibit enhanced delivery to HSPCs
  • Certain targeting moieties that enhance the delivery of an LNP payload to HSPCs when conjugated to the surface of LNPs were selected to create dual- targeted LNPs (dual tLNPs) that were conjugated to two targeting moieties that each bind to a different protein on the surface of an HSPC.
  • tLNPs were produced using the Lipid 093 ionizable lipid, discovered to be a high performing lipid for HSPC delivery, and then conjugated to one or two targeting moieties discovered to enhance delivery of a GFP mRNA to HSPCs when conjugated to an LNP (Example 13).
  • the targeting moieties conjugated to LNPs were as follows: an anti- CD34 Fab, an anti-CD117 Fab, an anti-CD71 Fab, anti-CD34 and anti-CD117 Fabs, anti- CD34 and anti-CD71 (low affinity) Fabs, anti-CD117 and anti-CD71 (low affinity) Fabs, anti-CD117 and anti-CD71 (high affinity) Fabs, anti-CD117 and anti-CD49d Fabs, or anti- CD117 and anti-CXCR4 Fabs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Optics & Photonics (AREA)
  • Nanotechnology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La divulgation concerne des nanoparticules lipidiques (LNPS) et des conjugués comprenant une fraction de ciblage et une nanoparticule lipidique (LNP) encapsulant un agent thérapeutique (par exemple, une charge utile) pour une administration à des cellules souches hématopoïétiques (CSH). Les conjugués peuvent être formulés dans une composition pharmaceutique et peuvent être directement administrés à un sujet ayant besoin d'un traitement (par exemple, par administration in vivo).
PCT/US2024/046812 2023-09-13 2024-09-13 Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules Pending WO2025059607A1 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US202363582456P 2023-09-13 2023-09-13
US202363582457P 2023-09-13 2023-09-13
US63/582,457 2023-09-13
US63/582,456 2023-09-13
US202363592196P 2023-10-22 2023-10-22
US202363592195P 2023-10-22 2023-10-22
US63/592,196 2023-10-22
US63/592,195 2023-10-22
US202463677892P 2024-07-31 2024-07-31
US63/677,892 2024-07-31

Publications (2)

Publication Number Publication Date
WO2025059607A1 true WO2025059607A1 (fr) 2025-03-20
WO2025059607A9 WO2025059607A9 (fr) 2025-05-01

Family

ID=95022190

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/046812 Pending WO2025059607A1 (fr) 2023-09-13 2024-09-13 Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules

Country Status (1)

Country Link
WO (1) WO2025059607A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220162521A1 (en) * 2020-11-25 2022-05-26 Akagera Medicines, Inc. Ionizable cationic lipids
US20220218614A1 (en) * 2020-12-04 2022-07-14 Tidal Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles, and methods of synthesis and use thereof
WO2022221695A1 (fr) * 2021-04-17 2022-10-20 Intellia Therapeutics, Inc. Compositions de nanoparticules lipidiques
WO2023039440A2 (fr) * 2021-09-08 2023-03-16 Flagship Pioneering Innovations Vi, Llc Compositions et procédés de modulation d'hbb

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220162521A1 (en) * 2020-11-25 2022-05-26 Akagera Medicines, Inc. Ionizable cationic lipids
US20220218614A1 (en) * 2020-12-04 2022-07-14 Tidal Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles, and methods of synthesis and use thereof
WO2022221695A1 (fr) * 2021-04-17 2022-10-20 Intellia Therapeutics, Inc. Compositions de nanoparticules lipidiques
WO2023039440A2 (fr) * 2021-09-08 2023-03-16 Flagship Pioneering Innovations Vi, Llc Compositions et procédés de modulation d'hbb

Also Published As

Publication number Publication date
WO2025059607A9 (fr) 2025-05-01

Similar Documents

Publication Publication Date Title
US20230235358A1 (en) Host defense suppressing methods and compositions for modulating a genome
JP2024045153A (ja) 改変ヒトｔ細胞受容体アルファ定常領域遺伝子を含む遺伝子改変細胞
US12018080B2 (en) Methods and compositions for treating cancer by targeting the CLEC2D-KLRB1 pathway
US20240200118A1 (en) Compositions and methods for optimizing tropism of delivery systems for rna
TW202425953A (zh) 脂質奈米顆粒 (lnp) 綴合物的製備
Damen et al. Transfection by cationic gemini lipids and surfactants
WO2025059596A1 (fr) Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules
US12311033B2 (en) Lipid nanoparticle formulations and compositions
US20200208177A1 (en) Methods and compositions for genome editing
JP2002316997A (ja) 目的とするアニオン性物質を細胞に導入するための複合体
WO2025076113A1 (fr) Lipides cationiques ionisables à espacement conservé et nanoparticules lipidiques
JP2024515880A (ja) 免疫応答を回避する脂質ナノ粒子治療薬
TW202448516A (zh) 接合hsc特異性抗體的脂質奈米粒子及其用途
TW202425952A (zh) 脂質奈米顆粒藥物綴合物
WO2025059607A1 (fr) Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules
WO2025076127A1 (fr) Lipides cationiques ionisables contraints et nanoparticules lipidiques
WO2025059599A1 (fr) Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules
KR20250153220A (ko) 변형된 시토신을 포함하는 dna 조성물
TW202428247A (zh) 表面修飾的脂質奈米顆粒
WO2025193861A2 (fr) Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des cellules
WO2025090525A1 (fr) Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des lymphocytes t
WO2025189161A1 (fr) Conjugaison spécifique à un site de fractions de ciblage à des nanoparticules lipidiques
CA2447548A1 (fr) Complexes utilises pour transferer des substances utiles dans une cellule
CN121219018A (zh) 脂质纳米颗粒药物缀合物
AU2024279278A1 (en) Lipid nanoparticle formulations and compositions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24866476

Country of ref document: EP

Kind code of ref document: A1