WO2024229251A2 - Édition génique ciblée de cellules souches hématopoïétiques (hsc) et leurs procédés d'utilisation - Google Patents

Édition génique ciblée de cellules souches hématopoïétiques (hsc) et leurs procédés d'utilisation Download PDF

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WO2024229251A2
WO2024229251A2 PCT/US2024/027460 US2024027460W WO2024229251A2 WO 2024229251 A2 WO2024229251 A2 WO 2024229251A2 US 2024027460 W US2024027460 W US 2024027460W WO 2024229251 A2 WO2024229251 A2 WO 2024229251A2
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lnp
another embodiment
lipid
composition
cells
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WO2024229251A3 (fr
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Hamideh Parhiz
Tyler PAPP
Stefano Rivella
Laura BREDA
Michael Triebwasser
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Childrens Hospital of Philadelphia CHOP
University of Pennsylvania Penn
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Childrens Hospital of Philadelphia CHOP
University of Pennsylvania Penn
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • 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

  • the present application hereby incorporates by reference the entire contents of the XML file named “046483-6267-00WO_SequenceListing.xml” which was created on April 30, 2024, and is 18,261 bytes in size.
  • HSCs Hematopoietic stem cells
  • SCF stem cell factor
  • CD117 CD117 is expressed on both short- and long-term HSCs and some hematopoietic progenitors (Kent, D., et al., 2008, Clin Cancer Res, 14:1926-1930).
  • NMHD non-malignant hematopoietic disorders
  • NMHD can be cured by allogeneic HSCT, but not all patients have a suitable immunologic match to minimize graft versus host disease (GVHD), a morbid, and potentially fatal, complication.
  • Gene therapy can eliminate the risk of GVHD and cure NMHD by using autologous HSCs with the genetic defect corrected, either by gene addition or editing.
  • Current hematopoietic gene therapy requires isolation of HSC from the body and ex vivo lentiviral transduction for gene addition or electroporation with purified reagents for genome editing.
  • HSC elimination i.e. conditioning
  • chemotherapy or radiation is required to prepare a patient for HSCT.
  • These conditioning procedures carry significant acute and chronic systemic toxicities, including infertility and secondary malignancies due to accumulated DNA damage.
  • Some NMHD are due to DNA repair pathway mutations, such as radiosensitive severe combined immunodeficiency (SCID) or Fanconi anemia.
  • SCID radiosensitive severe combined immunodeficiency
  • Fanconi anemia are due to excessive toxicity with alkylating chemotherapy or radiation, as well as increased rates of malignancy long-term.
  • the invention relates to a composition for targeted delivery of gene editing agents to a target hematopoietic stem cell (HSC), the composition comprising a first delivery vehicle comprising an mRNA molecule encoding a Cas9 base editor and a second delivery vehicle comprising a short guide RNA (sgRNA), wherein each of the first and second delivery vehicle comprises a targeting moiety specific for CD117.
  • HSC target hematopoietic stem cell
  • each of the first and second delivery vehicles comprises a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • gene editing agents are encapsulated within the LNPs.
  • the mRNA molecule encoding a Cas9 base editor encodes an adenine base editor (ABE) or a cytidine base editor (CBE).
  • the mRNA encoding the ABE is transcribed from a nucleic acid molecule comprising SEQ ID NO: 7.
  • the mRNA molecule is an isolated nucleoside- modified mRNA molecule.
  • at least one isolated nucleoside- modified RNA comprises pseudouridine or 1-methyl-pseudouridine.
  • the targeting moiety of each of the first and second delivery vehicle comprises a CD117 antibody.
  • the sgRNA is administered in excess of the Cas9 base editor.
  • the invention relates to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a composition of composition for targeted delivery of gene editing agents to a target hematopoietic stem cell (HSC), the composition comprising a first delivery vehicle comprising an mRNA molecule encoding a Cas9 base editor and a second delivery vehicle comprising a short guide RNA (sgRNA), wherein each of the first and second delivery vehicle comprises a targeting moiety specific for binding to the HSC to the subject.
  • HSC target hematopoietic stem cell
  • sgRNA short guide RNA
  • the disease or disorder is a blood monogenic disorder, genetic defect, bone marrow genetic defect, cancer, platelet disorder, immunodeficiency, metabolic disease or autoimmune disease.
  • the disease or disorder is a sickle cell disease.
  • the sickle cell disease is sickle cell anemia.
  • the sgRNA targets a hemoglobin subunit beta (HBB) gene.
  • the sgRNA comprises the nucleotide sequence of SEQ ID NO:8.
  • the composition is administered by intravenous (IV), intraosseous infusion (IO), intraperitoneal (IP), intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery.
  • IV intravenous
  • IO intraosseous infusion
  • IP intraperitoneal
  • intradermal subcutaneous, inhalation, intranasal, or intramuscular delivery.
  • Figure 1 depicts representative in vitro targeting of whole bone marrow or hematopoietic progenitors (Lin') cells incubated with LNPs encapsulating luciferase (CD117/LNP-Luc) or Cre recombinase (CD117/LNP-Cre) mRNAs.
  • Figure 1C depicts representative assessment of ZsGreen + reporter induction after 6-hour CD 117/LNP-Cre treatment and 12-hour culture in Ai6 bone marrow (BM) cells triggered by removal of loxP flanked stop cassette by Cre.
  • Figure ID depicts representative assessment of ZsGreen + reporter induction after 6-hour CD 117/LNP-Cre treatment and 12 hour culture in a Lin'Scal cKit 1 (LKS) subset of Lin' BM cells triggered by removal of loxP flanked stop cassette by Cre.
  • Figure IE depicts representative assessment of ZsGreen + reporter induction after 18-hour CD 117/LNP-Cre treatment in Ai6 bone marrow (BM) cells triggered by removal of loxP flanked stop cassette by Cre.
  • Figure IF depicts representative assessment of ZsGreen + reporter induction after 18-hour CD 117/LNP-Cre treatment in a Lin'Scal + cKit + (LKS) subset of Lin' BM cells triggered by removal of loxP flanked stop cassette by Cre.
  • Figure 1G depicts representative assessment of ZsGreen + reporter induction after 18-hour CD 117/LNP-Cre treatment and 72-hour culture in Ai6 bone marrow (BM) cells triggered by removal of loxP flanked stop cassette by Cre.
  • BM bone marrow
  • Figure 2 depicts representative delivery to antigen positive cells in whole bone marrow and viability in LNP-Cre treated bone marrow cells.
  • Figure 2A depicts representative luciferase activity normalized to protein in cell lysate and to the frequency of antigen positive cells in whole bone marrow (WBM) cells (anti-CD45 [85%], antiCDl 17 [2.8%], or unnormalized for control IgG/LNP.
  • Figure 2B depicts representative viability of Ai6 WBM cells after 6-hour treatment, and 12-hour culture, with in vitro exposure to increasing doses of LNP (up to 1 pg) and assessed by AO/PI staining.
  • Figure 2C depicts representative viability of Ai6 WBM cells after 18-hour treatment with in vitro exposure to increasing doses of LNP (up to 1 pg) and assessed by AO/PI staining.
  • Figure 3 depicts representative results demonstrating CD 117/LNP-Cre treatment ex vivo leads to superior tdTomato marking upon transplantation.
  • Figure 3C depicts representative kinetic analysis of erythroid editing measured up to 16 weeks post HSCT. Mean and SEM shown.
  • Figure 3D depicts representative tdTomato marking in in the bone marrow (BM) and BM subsets: c-Kit + (Lin'c-Kit + ), LSK (Lin'c-Kit + Scal + ), and LT-HSC (LSK CD150 + CD48’).
  • Figure 3E depicts a representative colony forming unit assay from Ail4 bone marrow treated ex vivo with 0.1 pg or 1 pg of control IgG/LNP-Cre or CD 117/LNP- Cre formulations or untreated.
  • Figure 3F depicts representative semi-quantitative PCR of bone marrow genomic DNA isolates from the groups in Figure 1A through Figure 1C at 4-months post-BMT.
  • Figure 3F depicts representative semi-quantitative PCR of spleen genomic DNA isolates from the groups in Figure 1A through Figure 1C at 4-months post- BMT. **271bp Cre-recombinase edited gDNA region, *1142bp unedited region are indicated.
  • Figure 3 A and Figure 3B mean and SEM are shown.
  • P-values are Tukey’s multiple comparison test after one-way ANOVA (p ⁇ 0.05). *** p ⁇ 0.001, **** p ⁇ 0.0001.
  • Figure 4 depicts representative results demonstrating ex vivo CD117-LNP/Cre treated HSC retain multi-lineage engraftment.
  • Figure 4A depicts representative peripheral blood donor chimerism in lethally irradiated congenic (CD45.1) recipients of ex vivo treated of bone marrow cells from Ail4 donor mice (CD45.2) with CD117/LNP-Cre (left) or Control IgG/LNP-Cre (right) across a 100-fold range in dose.
  • CD45.1 lethally irradiated congenic
  • CD45.2 recipients of ex vivo treated of bone marrow cells from Ail4 donor mice
  • CD117/LNP-Cre left
  • Control IgG/LNP-Cre right
  • Figure 4B depicts representative tdTomato+ cell frequency in red blood cells (RBC), white blood cells (CD45.2+), and granulocytes (Grl+) cells from 8-16 weeks post-transplant after ex vivo treatment of Ail4 bone marrow cells with CD 117/LNP-Cre.
  • Figure 4C depicts representative tdTomato+ cell frequency in red blood cells (RBC), white blood cells (CD45.2+), and granulocytes (Grl+) cells from 8-16 weeks post-transplant after ex vivo treatment of Ail4 bone marrow cells with control IgG/LNP-Cre.
  • Figure 4D depicts representative tdTomato 1 cell frequency in peripheral blood myeloid (Grl+) and lymphoid cells (CD3+ [T-cells], B220+ [B-cells]) and in bone marrow (BM) subsets (c-Kit, Lin'c-Kit + subset, LSK, Lin'c- Kit + Scal + , SLAM, LSK CD150 4 CD48 ) at 4 months after 0.01 or 0.05 mg of CD 117/LNP-Cre or control IgG/LNP-Cre.
  • Figure 4E depicts a representative colony forming unit assay from bone marrow at 4 months after transplantation with ex vivo treated Ail4 BM at the doses shown.
  • Figure 5 depicts representative results demonstrating that ex vivo CD 117/LNP-Cre edited HSC persist upon secondary transplantation.
  • Figure 5A depicts representative tdTomato + cell frequency in peripheral red blood cells four months after transplantation with bone marrow from primary chimeras of ex vivo CD117 or control IgG/LNP-Cre (0.01 mg mRNA) treated bone marrow.
  • Figure 5 A depicts representative tdTomato cell frequency in peripheral blood myeloid cells (Grl+) four months after transplantation with bone marrow from primary chimeras of ex vivo CD117 or control IgG/LNP-Cre (0.01 mg mRNA) treated bone marrow.
  • Figure 5 A depicts representative tdTomato + cell frequency in peripheral blood lymphoid cells (CD3+ [T-cells], B220+ [B-cells]) four months after transplantation with bone marrow from primary chimeras of ex vivo CD117 or control IgG/LNP-Cre (0.01 mg mRNA) treated bone marrow.
  • Figure 5D depicts representative donor chimerism in peripheral blood at 4 months in secondary transplants.
  • Figure 5E depicts representatitve frequency of gene edited cells in whole bone marrow (BM) and bone marrow subsets (c- Kit, Lin'c-Kit + subset, LSK, Lin’c-Kit + Scal + , SLAM, LSK CD150 + CD48 ) in secondary chimeras at 16 weeks posttransplant.
  • BM bone marrow
  • FIG. 5E depicts representatitve frequency of gene edited cells in whole bone marrow (BM) and bone marrow subsets (c- Kit, Lin'c-Kit + subset, LSK, Lin’c-Kit + Scal + , SLAM, LSK CD150 + CD48 ) in secondary chimeras at 16 weeks posttransplant.
  • Figure 6 comprising Figure 6A through Figure 6L, CD117/LNP-Cre formulations lead to over 50% tdTomato marking in LT-HSC after in vivo injection.
  • Figure 6A depicts representative biodistribution of i.v. injection of 1 pg of targeted LNP- mRNA expression in vivo by luminescence imaging at 24 hours.
  • Figure 6B depicts a representative sample set of dissected mouse organs were analyzed 5 min after the administration of D-luciferin. tdTomato + cell frequency in peripheral blood myeloid (Grl+) cells at 4 months after 5 pg of CD 117/LNP-Cre.
  • Figure 6C depicts a representative sample set of dissected mouse organs were analyzed 5 min after the administration of D-luciferin.
  • tdTomato + cell frequency in peripheral blood lymphoid cells CD3+ [T-cells], B220+ [B-cells]) at 4 months after 5 pg of CD 117/LNP-Cre.
  • Figure 6B depicts a representative sample set of dissected mouse organs were analyzed 5 min after the administration of D-luciferin.
  • tdTomato + cell frequency in bone marrow (BM) subsets (c-Kit, Lin c-Kit + subset, LSK, Lin c-Kit + Scal + , SLAM/LT-HSC, LSK CD1 0 + CD48 ) at 4 months after 5 pg of CD 117/LNP-Cre.
  • Figure 6E depicts representative tdTomato cell frequency in peripheral blood myeloid cells at 4 months after 5 or 1 pg of CD117/LNP-Cre.
  • Figure 6E depicts representative tdTomato + cell frequency in peripheral lymphoid cells at 4 months after 5 or 1 pg of CD 117/LNP-Cre.
  • Figure 6E depicts representative tdTomato + cell frequency in bone marrow subsets at 4 months after 5 or 1 pg of CD 117/LNP-Cre.
  • Figure 6J depicts a representative colony forming unit assay from bone marrow at 4 months after in vivo treatment with 5 pg control-IgG/LNP-Cre (top), no treatment (middle), or 5 pg CD117/LNP-Cre (bottom).
  • Figure 6K depicts representative semi-quantitative PCR of bone marrow genomic DNA isolates from the groups in A-C at
  • Figure 6L depicts representative semi-quantitative PCR of spleen genomic DNA isolates from the groups in A-C at 4 months post BMT. **271bp Cre- recombinase edited gDNA region, *1142bp unedited region are indicated.
  • Figure 6B through Figure 6G mean and SEM of the shown P-values are reported from paired t-test. *** p ⁇ 0.001.
  • Figure 7 depicts representative in vivo editing after CD117/LNP treatment.
  • Figure 7A depicts representative percentage of total CFU that are tdTomato+ at 4 months post IV treatment .
  • Figure 7B depicts representative gene editing frequency in non-hematopoietic organs/cells of the liver 16 weeks post in vivo treatment with CD117/LNP-Cre (1 pg and 5 pg) and control IgG/LNP-Cre (5 pg) assessment by flow cytometry.
  • Figure 7C depicts representative gene editing frequency in non-hematopoietic organs/cells of the lung 16 weeks post in vivo treatment with CD 117/LNP-Cre (1 pg and
  • Figure 7E depicts representative gene editing frequency in non-hematopoietic organs/cells of the testis 16 weeks post in vivo treatment with CD 117/LNP-Cre (5 pg and 1 pg) and control IgG/LNP-Cre (5 pg) assessment by flow cytometry.
  • Figure 8 depicts representative results demonstrating CD 117/LNP-Cre edited HSC persist upon primary transplantation of BM from in vivo treated mice.
  • Figure 8 A depicts representative tdTomato + cell frequency in peripheral blood red blood cells 4 months after transplantation with bone marrow from Ai9 mice injected in vivo with control IgG/LNP-Cre or CD117/LNPCre formulations.
  • Figure 8B depicts representative tdTomato + cell frequency in peripheral myeloid cells (Grl+) 4 months after transplantation with bone marrow from Ai9 mice injected in vivo with control IgG/LNP-Cre or CD 117/LNPCre formulations.
  • Figure 8C depicts representative tdTomato + cell frequency in peripheral blood lymphoid cells (CD3+ [T-cells], B220+ [B-cells]) 4 months after transplantation with bone marrow from Ai9 mice injected in vivo with control IgG/LNP-Cre or CD117/LNPCre formulations.
  • Figure 7D depicts representative donor chimerism in the primary chimeras at 16 weeks posttransplant.
  • Figure 8E depicts representative frequency of gene edited cells in whole bone marrow (BM) and bone marrow subsets (c-Kit, Lin‘c-Kit + subset, LSK, Lin'c- Kit + Scal + , SLAM, LSK CD150 + CD48 ) in primary chimeras at 16 weeks posttransplant.
  • BM bone marrow
  • FIG. 8E depicts representative frequency of gene edited cells in whole bone marrow (BM) and bone marrow subsets (c-Kit, Lin‘c-Kit + subset, LSK, Lin'c- Kit + Scal + , SLAM, LSK CD150 + CD48 ) in primary chimeras at 16 weeks posttransplant.
  • Figure 9 depicts representative analyses of genome editing, cell viability and proliferation in human erythroid cells treated with CD117/LNP-ABE and CD117/LNP-sgRNA.
  • Figure 9A depicts representative sequences of C editing of control unedited (top) and edited (bottom) genomic DNA extracted from SCD cells after treatment with anti-human CD117/LNP formulations carrying an adenine base editor and a sgRNA aimed at converting the pathogenic codon 6 (highlighted in blue, GAG,) to non-pathogenic variant (->GCG) named HBB G ’ A/otorar .
  • Figure 9B depicts quantification of C editing in as in Figure 9A.
  • Figure 9D depicts proliferation rate of specimens in Figure 9C calculated by measuring the cell count fold increase from DO to D7/8 in differentiation media.
  • Figure 10 depicts representative base editing of the E6V sickle cell mutation with Human CD117 targeted LNP.
  • Figure 10A depicts representative reverse-phase (RP) HPLC of in vitro differentiated sickle cell disease affected erythroid progenitors after treatment with anti- human CD117 (hCD 1 17)/LNP-NRCH cas9 ABE-8e mRNA and hCDl 17/LNP gRNA.
  • Base editing yields non-pathogenic Hbb G- akassar (b G ), which elutes before Hbb s (pathogenic, b s ) and the a-globin protein (a).
  • % shown is b G / (b G +b s ) *100.
  • Figure 10B depicts representative images of sickling of in vitro differentiated erythroid progenitors under hypoxic conditions at the treatments in (A). Arrowheads indicate sickled morphology. Scale bar 20 microns.
  • Figure 10D depicts representative correlation of %b G by RP- HPLC (protein) to base edited allele frequency (DNA).
  • Figure 11 depicts representative imaging of mice and livers upon i.v. administration of CD117/LNP-Luc versus CD117/LNP-Luc-miRt formulations.
  • Figure 11A depicts representative biodistribution upon i.v. injection of 1 pg of targeted LNP-mRNA expression in mice in vivo by luminescence imaging at 24 hours.
  • Figure 1 IB depicts representative biodistribution in livers dissected from animals of Figure 11A analyzed 5 min after the administration of D-luciferin.
  • Figure 11C depicts representative quantification of bioluminescence.
  • the present invention relates to compositions comprising CD117 targeted LNP molecules comprising at least one RNA molecule for genomic editing of Hematopoietic stem cells (HSCs).
  • the composition relates to a combination of a first CD117-targed LNP comprising an mRNA molecule encoding a base-editing molecule and a second CD 117-targed LNP comprising a guide RNA for targeting of the base editing molecule to a target base.
  • the target base is a disease-associated single nucleotide polymorphism (SNP), and the base editing method of the invention serves to revert the disease-associated mutation to a “wild-type,” or non-disease associated, base.
  • SNP disease-associated single nucleotide polymorphism
  • the present invention also relates to methods of use of the compositions described herein for genetic editing of HSCs as well as methods of treating diseases or disorders in subjects including, but not limited to, monogenic disorders, non- hematopoietic diseases and bone marrow genetic defects.
  • monogenic disorders include, but are not limited to, non-malignant hematopoietic disorder (NMHD) such as hemoglobinopathies, congenital anemias or thrombocytopenias, and immunodeficiencies.
  • NMHD non-malignant hematopoietic disorder
  • non-hematopoietic diseases include, but are not limited to, cystic fibrosis, metabolic disorders, and myopathies.
  • Exemplary bone marrow genetic defects include, but are not limited to, leukemia, aplastic anemia, myeloproliferative disorders, an inherited bone marrow failure syndrome (IBMFS) such as Fanconi anemia, dyskeratosis congenital, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, severe congenital neutropenia, a primary immunodeficiency such as Xl-SCID and Wiskott-Aldrich syndrome, an erythroid disorder such as sickle cell disease (SCD), pyruvate kinase deficiency, or a lysosomal storage diseases such as Fabry disease and Pompe disease.
  • IBMFS inherited bone marrow failure syndrome
  • Fanconi anemia dyskeratosis congenital, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, severe congenital neutropenia
  • a primary immunodeficiency such as Xl-SCID and Wiskott-Aldrich syndrome
  • an element means one element or more than one element.
  • adjuvant means an agent that modifies or boosts the strength and longevity of a desired therapeutic response, and/or broadens the therapeutic response to a concomitantly administered agent.
  • antibody refers to an immunoglobulin molecule, which specifically binds with an antigen or epitope.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic-specificity determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.
  • an “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, k and 1 light chains refer to the two major antibody light chain isotypes.
  • synthetic antibody as used herein, is meant an antibody, which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • the term should also be construed to mean an antibody, which has been generated by the synthesis of an RNA molecule encoding the antibody.
  • the RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned) or other technology, which is available and well known in the art.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • an “effective amount” as used herein means an amount which provides a therapeutic or prophylactic benefit.
  • physiologically effective dosage refers to an amount of an agent that produces a measurable biologic or physiologic effect in the recipient subject that is related to the activity of the agent(s).
  • the physiologically effective dosage will vary depending on the compound, the age, weight, etc., of the subject being administered the agent, and the biologic or physiologic effect being measured.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • nucleosides nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage
  • A refers to adenosine
  • C refers to cytidine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • moduleating mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translation by translational machinery in a cell. For example, an mRNA where all of the uridines have been replaced with pseudouridine, 1 -methyl pseudouridine, or another modified nucleoside.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • nucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside.
  • a “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).
  • “pseudouridine” refers, in another embodiment, to m 1 acp 3 Y (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine.
  • the term refers to m J Y (1-methylpseudouridine).
  • the term refers to Ym (2'-O-methylpseudouridine.
  • the term refers to m 5 D (5-methyldihydrouridine).
  • the term refers to m 3 Y (3- methylpseudouridine).
  • the term refers to a pseudouridine moiety that is not further modified.
  • the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines.
  • the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.
  • an antibody By the term “specifically binds,” as used herein with respect to an affinity ligand, in particular, an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • terapéutica as used herein means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.
  • therapeutically effective amount refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.
  • the therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • under transcriptional control or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double and/or triple bonds), having from one to twenty-four carbon atoms (C1-C24 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-Ce alkyl) and which is attached to the rest of the molecule by a single bond, e g., methyl, ethyl, n propyl, 1 -methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-l-enyl, pent-l-enyl, pen
  • Alkylene or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (z.c., contains one or more double (alkenylene) and/or triple bonds (alkynylene)), and having, for example, from one to twenty-four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (Ci-Cs alkylene), one to six carbon atoms (Ci-Ce alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, //-butylene, ethenylene, propenylene, zz-buteny
  • the alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond.
  • the points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.
  • Cycloalkyl or “carbocyclic ring” refers to a stable non aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond.
  • Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic radicals include, for example, adamantyl, norbomyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise, a cycloalkyl group is optionally substituted.
  • Cycloalkylene is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.
  • Heterocyclyl or “heterocyclic ring” refers to a stable 3- to 18-membered non-aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.
  • the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated.
  • heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thio
  • a non-hydrogen atoms such
  • the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is a oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.
  • Optional or “optionally substituted” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
  • optionally substituted alkyl means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.
  • ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the CD117-LNP comprises an mRNA molecule encoding a base editing protein.
  • base editing proteins that can be encoded by the mRNA molecule and delivered to HSC according to the methods of the invention include, but are not limited to, an adenine base editor (ABE), a cytidine base editor (CBE), a CRISPR based nucleic acid editor (e.g., Cas9), and other nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes.
  • ABE adenine base editor
  • CBE cytidine base editor
  • Cas9 CRISPR based nucleic acid editor
  • other nucleic acid editing proteins or protein domains e.g., deaminase domains, polymerase domains, and/or base excision enzymes.
  • compositions of the invention provide a CD 117-LNP comprising an mRNA molecule encoding a base editor, wherein the base editor when in association with a guide RNA (gRNA) specifically edits a target base.
  • the target base is a disease-associated base. Therefore, in some embodiments, the compositions of the invention provide a combination of a CD 117-LNP comprising an mRNA molecule encoding a base editor and a CD117-LNP comprising a gRNA for targeted base editing.
  • mRNA molecules include nucleotide oligomers containing modified backbones or non-natural inter-nucleoside linkages.
  • Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleotide oligomers.
  • Nucleotide oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriest- ers, and boranophosphates.
  • Various salts, mixed salts and free acid forms are also included.
  • the composition of the invention comprises in vitro transcribed (IVT) RNA. In some embodiments, the composition of the invention comprises IVT RNA encoding a therapeutic protein. In some embodiments, the composition of the invention comprises IVT RNA encoding a plurality of therapeutic proteins.
  • IVT in vitro transcribed
  • an IVT RNA can be introduced to a cell as a form of transient transfection.
  • the RNA is produced by in vitro transcription using a plasmid DNA template generated synthetically.
  • DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired template for in vitro transcription is a therapeutic protein, as described elsewhere herein.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the DNA is a full-length gene of interest of a portion of a gene.
  • the gene can include some or all of the 5' and/or 3' untranslated regions (UTRs).
  • the gene can include exons and introns.
  • the DNA to be used for PCR is a human gene.
  • the DNA to be used for PCR is a human gene including the 5' and 3' UTRs.
  • the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi.
  • the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein.
  • the portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • Genes that can be used as sources of DNA for PCR include genes that encode a gene editing protein or a variant thereof.
  • the gene editing protein is a CRISPR based nucleic acid editor (e.g., a Cas9 adenine base editor (ABE) or a Cas9 cytidine base editor (CBE), or other nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes.
  • a plasmid is used to generate a template for in vitro transcription of RNA which is used for transfection.
  • the RNA has 5' and 3' UTRs.
  • the 5' UTR is between zero and 3000 nucleotides in length.
  • the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 RNA polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized RNA which is effective in eukaryotic transfection when it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).
  • polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E- PAP) or yeast polyA polymerase.
  • E- PAP E. coli polyA polymerase
  • yeast polyA polymerase E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase RNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods to include a 5' capl structure can be generated using Vaccinia capping enzyme and 2 ’-O-methyl transferase enzymes (CellScript, Madison, WI).
  • 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958-966 (2005)).
  • the composition of the present invention comprises a nucleoside-modified nucleic acid. In some embodiments, the composition of the invention comprises a nucleoside-modified RNA encoding a protein for genetic modification. In some embodiments, the composition of the invention comprises a nucleoside-modified RNA encoding a protein for base editing. In some embodiments, the composition of the invention comprises a gRNA molecule specific for binding to a target nucleic acid molecule.
  • the composition comprises one or more nucleoside-modified RNA.
  • the composition comprises a nucleoside-modified mRNA encoding a protein for genetic modification.
  • Nucleoside- modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation.
  • Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent No. 8,278,036, which is incorporated by reference herein in its entirety.
  • expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors.
  • the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins.
  • the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA.
  • using mRNA rather than the protein also has many advantages.
  • the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine.
  • inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16: 1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23: 165-175).
  • RNA containing pseudouridines suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23: 165-175).
  • protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16: 1833-1840).
  • the present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside.
  • the composition comprises an isolated nucleic acid, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.
  • the composition comprises a vector, comprising an isolated nucleic acid, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.
  • the nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein.
  • the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase.
  • the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase.
  • the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.
  • the modified nucleoside is nfacp 3 '? (l-methyl-3- (3-amino-3-carboxypropyl) pseudouridine.
  • the modified nucleoside is m 1 'P (1 -methylpseudouridine).
  • the modified nucleoside is Tm (2'-O-methylpseudouridine.
  • the modified nucleoside is m 5 D (5-methyldihydrouridine).
  • the modified nucleoside is m 3v P (3 -methylpseudouridine).
  • the modified nucleoside is a pseudouridine moiety that is not further modified.
  • the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines.
  • the modified nucleoside is any other pseudouridine-like nucleoside known in the art.
  • the nucleoside that is modified in the nucleoside- modified RNA the present invention is uridine (U).
  • the modified nucleoside is cytidine (C).
  • the modified nucleoside is adenosine (A).
  • the modified nucleoside is guanosine (G).
  • the modified nucleoside of the present invention is m 5 C (5-methylcytidine). In another embodiment, the modified nucleoside is m 5 U (5- methyluridine). In another embodiment, the modified nucleoside is m 6 A (N 6 - methyladenosine). In another embodiment, the modified nucleoside is s 2 U (2- thiouridine). In another embodiment, the modified nucleoside is T (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).
  • the modified nucleoside is m'A (1- m ethyl adenosine); m 2 A (2-methyladenosine); Am (2'-O-methyladenosine); ms 2 m 6 A (2- methylthio-N 6 -methyladenosine); i 6 A (N 6 -isopentenyladenosine); ms 2 i6A (2-methylthio- N 6 isopentenyladenosine); io 6 A (N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A (2- methylthio-N 6 -(cis-hydroxyisopentenyl) adenosine); g 6 A (N 6 - glycinylcarbamoyladenosine); t 6 A (N 6 -threonylcarbamoyladenosine); ms 2 t 6 A (2- methylthi
  • a nucleoside-modified RNA of the present invention comprises a combination of 2 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.
  • the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%.
  • the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.
  • the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.
  • 0.1% of the residues of a given nucleoside are modified.
  • the fraction of the given nucleotide that is modified is 0.2%.
  • the fraction is 0.3%.
  • the fraction is 0.4%.
  • the fraction is 0.5%.
  • the fraction is 0.6%.
  • the fraction is 0.8%.
  • the fraction is 1%.
  • the fraction is 1.5%.
  • the fraction is 2%.
  • the fraction is 2.5%.
  • the fraction is 3%.
  • the fraction is 4%.
  • the fraction is 5%.
  • the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.
  • the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.
  • a nucleoside-modified RNA of the present invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence.
  • the nucleoside-modified RNA exhibits enhanced ability to be translated by a target cell.
  • translation is enhanced by a factor of 2-fold relative to its unmodified counterpart.
  • translation is enhanced by a 3 -fold factor.
  • translation is enhanced by a 5-fold factor.
  • translation is enhanced by a 7-fold factor.
  • translation is enhanced by a 10-fold factor.
  • translation is enhanced by a 15-fold factor.
  • translation is enhanced by a 20-fold factor.
  • translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100- fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10- 1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000- fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.
  • the delivery vehicle is a colloidal dispersion system, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e g., an artificial membrane vesicle).
  • the use of lipid formulations is contemplated for the introduction of the at least one agent into a host cell (in vitro, ex vivo or in vivo).
  • the at least one agent may be associated with a lipid.
  • the at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/nucleic acid or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long- chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-agent complexes are also contemplated.
  • delivery of the at least one agent comprises any suitable delivery method, including exemplary delivery methods described elsewhere herein.
  • delivery of the at least one agent to a subject comprises mixing the at least one agent with a transfection reagent prior to the step of contacting.
  • a method of the present invention further comprises administering the at least one agent together with the transfection reagent.
  • the transfection reagent is a cationic lipid reagent.
  • the transfection reagent is a lipid-based transfection reagent.
  • the transfection reagent is a protein-based transfection reagent.
  • the transfection reagent is a polyethyleneimine based transfection reagent.
  • the transfection reagent is calcium phosphate.
  • the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®.
  • the transfection reagent is any other transfection reagent known in the art.
  • the transfection reagent forms a liposome.
  • Liposomes in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity.
  • liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane.
  • the liposomes comprise an internal aqueous space for entrapping water-soluble compounds.
  • liposomes can deliver the at least one agent to cells in an active form.
  • the composition comprises a lipid nanoparticle (LNP) and at least one agent.
  • LNP lipid nanoparticle
  • lipid nanoparticle refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids.
  • the particle includes a lipid of Formula (I), (II) or (III).
  • lipid nanoparticles are included in a formulation comprising at least one agent as described herein.
  • such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or (III)) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid such as a pegylated lipid of structure (IV), such as compound IVa).
  • the at least one agent is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.
  • the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 n
  • the lipid nanoparticles have a mean diameter of about 83 nm. In some embodiments, the lipid nanoparticles have a mean diameter of about 102 nm. In some embodiments, the lipid nanoparticles have a mean diameter of about 103 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, the at least one agent, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation by intra- or intercellular enzymes.
  • the LNP may comprise any lipid capable of forming a particle to which the at least one agent is attached, or in which the at least one agent is encapsulated.
  • lipid refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.
  • the LNP comprises a cationic lipid.
  • cationic lipid refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
  • the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)- N-2-(sperminecarboxamido)ethyl)-N
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3- phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.);
  • LIPOFECT AMINE® commercially available cationic liposomes comprising N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).
  • DOSPA dioctadecylamidoglycyl carboxyspermine
  • DOGS dioctadecylamidoglycyl carboxyspermine
  • lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), N,N-dimethyl-2,3-bis(((9Z, 12Z, 15Z)-octadeca-9, 12, 15-trien-l - yl)oxy)propan-l -amine (DLenDMA).
  • DODAP DODAP
  • DODMA DODMA
  • DMDMA l,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA N,N-dimethyl-2,3-bis(((9Z, 12Z, 15Z)-octadeca-9, 12, 15-trien-l - yl)oxy)propan-l -amine
  • the cationic lipid is an amino lipid.
  • Suitable amino lipids useful in the invention include those described in WO 2012/016184, incorporated herein by reference in its entirety.
  • Representative amino lipids include, but are not limited to, 1,2-dilinoley oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 - morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-
  • Suitable amino lipids include those having the formula: wherein Ri and R2 are either the same or different and independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted Cio-C24 alkynyl, or optionally substituted Cio-C24acyl;
  • R3 and R4 are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2- Ce alkynyl or R3 and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
  • R5 is either absent or present and when present is hydrogen or Ci-Ce alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and
  • Y and Z are either the same or different and independently O, S, or NH.
  • Ri and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In some embodiments, the amino lipid is a dilinoleyl amino lipid.
  • a representative useful dilinoleyl amino lipid has the formula:
  • n 0, 1, 2, 3, or 4.
  • the cationic lipid is a DLin-K-DMA. In some embodiments, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).
  • the cationic lipid component of the LNPs has the structure of Formula (I):
  • R la and R lb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R la is H or C1-C12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 2a is H or C1-C12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 3a is H or C1-C12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R? and R 6 are each independently methyl or cycloalkyl
  • R 7 is, at each occurrence, independently H or C1-C12 alkyl
  • R 8 and R 9 are each independently C1-C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2.
  • R la and R lb are not isopropyl when a is 6 or n-butyl when a is 8.
  • R la and R lb are not isopropyl when a is 6 or n-butyl when a is 8.
  • R 8 and R 9 are each independently unsubstituted C1-C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;
  • one of L 1 or L 2 is a carboncarbon double bond. In other embodiments, both L 1 and L 2 are a carbon-carbon double bond.
  • carbon-carbon double bond refers to one of the following structures: wherein R a and R b are, at each occurrence, independently H or a substituent.
  • R a and R b are, at each occurrence, independently H, Ci- C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.
  • the lipid compounds of Formula (I) have the following structure (la):
  • the lipid compounds of Formula (I) have the following structure
  • the lipid compounds of Formula (I) have the following structure (
  • a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.
  • b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.
  • c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.
  • d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.
  • a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.
  • the sum of a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties.
  • a and b are chosen such that their sum is an integer ranging from 14 to 24.
  • c and d are chosen such that their sum is an integer ranging from 14 to 24.
  • the sum of a and b and the sum of c and d are the same.
  • the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24.
  • a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.
  • e is 1. In other embodiments, e is 2.
  • R la , R 2a , R 3a and R 4a of Formula (I) are not particularly limited.
  • R la , R 2a , R 3a and R 4a are H at each occurrence.
  • at least one of R la , R 2a , R 3a and R 4a is C1-C12 alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is Ci-Cs alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is Ci-Ce alkyl.
  • the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R la , R lb , R 4a and R 4b are C1-C12 alkyl at each occurrence.
  • At least one of R lb , R 2b , R 3b and R 4b is H or R lb , R 2b , R 3b and R 4b are H at each occurrence.
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 5 and R 6 of Formula (I) are not particularly limited in the foregoing embodiments.
  • one or both of R 5 or R 6 is methyl.
  • one or both of R 5 or R 6 is cycloalkyl for example cyclohexyl.
  • the cycloalkyl may be substituted or not substituted.
  • the cycloalkyl is substituted with C1-C12 alkyl, for example tert-butyl.
  • R 7 are not particularly limited in the foregoing embodiments of Formula (I). In certain embodiments at least one R 7 is H. In some other embodiments, R 7 is H at each occurrence. In certain other embodiments R 7 is C1-C12 alkyl. In certain other of the foregoing embodiments of Formula (I), one of R 8 or R 9 is methyl. In other embodiments, both R 8 and R 9 are methyl.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.
  • exemplary lipid of Formula (I) can include
  • the LNPs comprise a lipid of Formula (I), at least one agent, and one or more excipients selected from neutral lipids, steroids and pegylated lipids.
  • the lipid of Formula (I) is compound 1-5. In some embodiments the lipid of Formula (I) is compound 1-6.
  • the cationic lipid component of the LNPs has the structure of Formula ( or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
  • G 3 is Ci-Ce alkylene
  • R a is H or C1-C12 alkyl
  • R la and R lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R la is H or C1-C12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 2a is H or C1-C12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 3a is H or C1-C12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 5 and R 6 are each independently H or methyl
  • R 7 is C4-C20 alkyl
  • R 8 and R 9 are each independently C1-C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.
  • the lipid compound has one of the following structures (IIA) or (IIB):
  • the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).
  • one of L 1 or L 2 is a direct bond.
  • a “direct bond” means the group (e.g., L 1 or L 2 ) is absent.
  • each of L 1 and L 2 is a direct bond.
  • R la is H or C1-C12 alkyl
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4a is H or C1-C12 alkyl
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 2a is H or C1-C12 alkyl
  • R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 3a is H or C1-C12 alkyl
  • R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • the lipid compound has one of the following structures (IIC) or (IID): wherein e, f, g and h are each independently an integer from 1 to 12.
  • the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).
  • structures (IIC) or (IID) are each independently an integer from 4 to 10.
  • a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.
  • b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.
  • c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.
  • d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.
  • e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.
  • f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.
  • g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.
  • h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.
  • a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.
  • the sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties.
  • a and b are chosen such that their sum is an integer ranging from 14 to 24.
  • c and d are chosen such that their sum is an integer ranging from 14 to 24.
  • the sum of a and b and the sum of c and d are the same.
  • the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24.
  • a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.
  • R la , R 2a , R 3a and R 4a of Formula (II) are not particularly limited.
  • at least one of R la , R 2a , R 3a and R 4a is H.
  • R la , R 2a , R 3a and R 4a are H at each occurrence.
  • at least one of R la , R 2a , R 3a and R 4a is C1-C12 alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is Ci-Cs alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is Ci-Ce alkyl.
  • the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R la , R lb , R 4a and R 4b are C1-C12 alkyl at each occurrence.
  • At least one of R lb , R 2b , R 3b and R 4b is H or R lb , R 2b , R 3b and R 4b are H at each occurrence.
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 3 and R 6 of Formula (II) are not particularly limited in the foregoing embodiments.
  • one of R 5 or R 6 is methyl.
  • each of R 3 or R 6 is methyl.
  • R b is branched C1-C15 alkyl.
  • R b has one of the following structures:
  • one of R 8 or R 9 is methyl. In other embodiments, both R 8 and R 9 are methyl.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.
  • G 3 is C2-C4 alkylene, for example C3 alkylene.
  • the lipid compound has one of the following structures:
  • the LNPs comprise a lipid of Formula (II), at least one agent, and one or more excipient selected from neutral lipids, steroids and pegylated lipids.
  • the lipid of Formula (II) is compound II-9.
  • the lipid of Formula (II) is compound II- 10.
  • the lipid of Formula (II) is compound IT- 1 1 .
  • the lipid of Formula (II) is compound 11-12.
  • the lipid of Formula (II) is compound 11-32.
  • G 1 and G 2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R a is H or C1-C12 alkyl
  • R 1 and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl
  • R 4 is C1-C12 alkyl
  • R 3 is H or Ci-Ce alkyl; and x is 0, 1 or 2.
  • the lipid has one of the following structures (IIIA) or (IIIB): wherein:
  • A is a 3 to 8-membered cycloalkyl or cycloalkylene ring
  • R 6 is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
  • the lipid has one of the following structures wherein y and z are each independently integers ranging from 1 to 12.
  • the lipid has one of the followin In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ) :
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is
  • n is 5. In some embodiments, n is 6.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H. In other of the foregoing embodiments, R 6 is C1-C24 alkyl. In other embodiments, R 6 is OH.
  • G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C1-C24 alkylene or linear C1-C24 alkenylene.
  • R 1 or R 2 is C6-C24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: wherein:
  • R 7a and R 7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • At least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is Ci-Cs alkyl.
  • Ci-Cs alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of
  • R 3 is OH
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has
  • the LNPs comprise a lipid of Formula (III), at least one agent, and one or more excipient selected from neutral lipids, steroids and pegylated lipids.
  • the lipid of Formula (III) is compound III-3.
  • the lipid of Formula (III) is compound III-7.
  • the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount of about 50 mole percent. Tn some embodiments, the LNP comprises only cationic lipids.
  • the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.
  • Suitable stabilizing lipids include neutral lipids and anionic lipids.
  • neutral lipid refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH.
  • Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.
  • Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoylol eoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE),
  • the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
  • the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to the neutral lipid ranges from about 2:1 to about 8: 1.
  • the LNPs further comprise a steroid or steroid analogue.
  • a “steroid” is a compound comprising the following carbon skeleton:
  • the steroid or steroid analogue is cholesterol.
  • the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to cholesterol ranges from about 2: 1 to 1 : 1.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N- dodecanoylphosphatidylethanolamines N-succinylphosphatidylethanolamines
  • N- glutarylphosphatidylethanolamines N- glutarylphosphatidylethanolamines
  • the LNP comprises glycolipids (e.g., monosialoganglioside GMi). In certain embodiments, the LNP comprises a sterol, such as cholesterol.
  • the LNPs comprise a polymer conjugated lipid.
  • polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion.
  • An example of a polymer conjugated lipid is a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.
  • the LNP comprises an additional, stabilizing - lipid which is a polyethylene glycol-lipid (pegylated lipid).
  • Suitable polyethylene glycollipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
  • Representative polyethylene gly col-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.
  • the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2ooo)carbamyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the polyethylene glycol-lipid is PEG-c-DOMG).
  • the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-l-O-(D- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG- cer), or a PEG dialkoxypropylcarbamate such as ⁇ -methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3
  • the LNPs comprise a pegylated lipid having the following structure (IV): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has mean value ranging from 30 to 60.
  • R 10 and R 11 are not both n-octadecyl when z is 42.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R 10 is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R 11 is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.
  • z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g/mol. In some embodiments, the average z is about 45.
  • the pegylated lipid has one of the following structures: wherein n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g/mol.
  • the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In some embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In some embodiments, the additional lipid is present in the LNP in about 1 mole percent or about 1 .5 mole percent.
  • the LNPs comprise a lipid of Formula (I), a nucleoside-modified RNA, a neutral lipid, a steroid and a pegylated lipid.
  • the lipid of Formula (I) is compound 1-6.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the pegylated lipid is compound IVa.
  • the LNP comprises one or more targeting moi eties that targets the LNP to a stem cell or stem cell population.
  • the targeting domain is a ligand which directs the LNP to a receptor found on a stem cell surface.
  • Exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al
  • Embodiments of the lipid of Formula (I) can be prepared according to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24.
  • Method A General Reaction Scheme 1
  • compounds of structure A-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • a mixture of A-l, A-2 and DMAP is treated with DCC to give the bromide A-3.
  • a mixture of the bromide A-3, a base (e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessarily workup and or purification step.
  • a base e.g., N,N-diisopropylethylamine
  • N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessarily workup and or purification step.
  • Compound B-5 can be prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24.
  • Method B General Reaction Scheme 2
  • compounds of structure B-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • a solution of B-l (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine).
  • the crude product is treated with an oxidizing agent (e.g., pyridinum chlorochromate) and intermediate product B-3 is recovered.
  • an oxidizing agent e.g., pyridinum chlorochromate
  • a solution of crude B-3, an acid e.g., acetic acid
  • N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and/or purification.
  • a reducing agent e.g., sodium triacetoxyborohydride
  • starting materials A-l and B-l are depicted above as including only saturated methylene carbons, starting materials which include carbon-carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds.
  • Method C9 can be prepared according to General Reaction Scheme 3 (“Method C”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24.
  • Method C General Reaction Scheme 3
  • compounds of structure C-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • Embodiments of the compound of Formula (II) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein R la , R lb , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 8 , R 9 , L 1 , L 2 , G 1 , G 2 , G 3 , a, b, c and d are as defined herein, and R 7 represents R 7 or a C3-C19 alkyl.
  • Method D General Reaction Scheme 4
  • D-l and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • a solution of D-l and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up.
  • a solution of D-3 and a base e.g. trimethylamine, DMAP
  • acyl chloride D-4 or carboxylic acid and DCC
  • D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary work up and/or purification.
  • Embodiments of the lipid of Formula (II) can be prepared according to General Reaction Scheme 5 (“Method E”), wherein R la , R lb , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , G 3 , a, b, c and d are as defined herein.
  • General Reaction Scheme 2 compounds of structure E-1 and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • E-1 in excess
  • E-2 e.g., potassium carbonate
  • a base e.g., potassium carbonate
  • E-3 e.g. trimethylamine, DMAP
  • E-4 acyl chloride
  • DCC carboxylic acid and DCC
  • General Reaction Scheme 6 provides an exemplary method (Method F) for preparation of Lipids of Formula (III).
  • G 1 , G 3 , R 1 and R 3 in General Reaction Scheme 6 are as defined herein for Formula (III), and Gl’ refers to a one-carbon shorter homologue of Gl.
  • Compounds of structure F-l are purchased or prepared according to methods known in the art. Reaction of F-l with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester/alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).
  • lipids of Formula (III) are available to those of ordinary skill in the art.
  • other lipids of Formula (III) wherein L 1 and L 2 are other than ester can be prepared according to analogous methods using the appropriate starting material.
  • General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G 1 and G 2 are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G 1 and G 2 are different.
  • Suitable protecting groups include hydroxy, amino, mercapto and carboxylic acid.
  • Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, /-butyldiphenyl silyl or trimethyl silyl), tetrahydropyranyl, benzyl, and the like.
  • Suitable protecting groups for amino, amidino and guanidino include -butoxycarbonyl, benzyloxycarbonyl, and the like.
  • Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), /2-methoxybenzyl, trityl and the like.
  • Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters.
  • Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley.
  • the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.
  • the targeting domain specifically binds to CD117.
  • the targeting domain may comprise a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, and the like that targets the particle to a site in particular need of the therapeutic agent.
  • the particle comprises multivalent targeting, wherein the particle comprises multiple targeting mechanisms described herein.
  • the targeting domain may be co-polymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to the composition comprising the delivery vehicle, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle.
  • Targeting domains of the instant invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.
  • the composition comprises a targeting domain that directs the delivery vehicle to CD 117.
  • the targeting domain is an affinity ligand which specifically binds to CD117.
  • the present invention relates to composition
  • composition comprising a delivery vehicle conjugated to a CD117 targeting domain.
  • the targeting domain binds to CD117 expressed on the surface of a target stem cell, thereby directing the composition to the target stem cell.
  • the targeting domain of the invention comprises a peptide.
  • the peptide targeting domain specifically binds to marker of a cell type of interest.
  • the targeting domain directs the vehicle to an endothelial cell, an immune cell, a stem cell, or another specific cell type of interest.
  • the targeting domain directs the vehicle to a CD117 expressing stem cell.
  • the peptide of the present invention may be made using chemical methods.
  • peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • the peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide.
  • the composition of a peptide may be confirmed by amino acid analysis or sequencing.
  • the variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag).
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • the fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence.
  • Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
  • the “similarity” between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide to a sequence of a second peptide.
  • Variants are defined to include peptide sequences different from the original sequence. In some embodiments, variants are different from the original sequence in less than 40% of residues per segment of interest. In some embodiments, variants are different from the original sequence in less than 25% of residues per segment of interest.
  • variants are different by less than 10% of residues per segment of interest. In some embodiments, variants are different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence.
  • the present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
  • the identity between two amino acid sequences is determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403- 410 (1990)].
  • the peptides of the invention can be post-translationally modified.
  • post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc.
  • Some modifications or processing events require introduction of additional biological machinery.
  • processing events such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.
  • the peptides of the invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.
  • the targeting domain of the invention comprises an isolated nucleic acid, including for example a DNA oligonucleotide and a RNA oligonucleotide.
  • the nucleic acid targeting domain specifically binds to CD117.
  • the nucleic acid comprises a nucleotide sequence that specifically binds to CD117.
  • nucleotide sequences of a nucleic acid targeting domain can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and/or deletions of one or more nucleotides, with the condition that the resulting nucleic acid functions as the original and specifically binds to CD117.
  • nucleotide sequence is “substantially homologous” to any of the nucleotide sequences describe herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%. In some embodiments, the degree of identity is at least 70%. In some embodiments, the degree of identity is at least 85%. In some embodiments, the degree of identity is at least 95%. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence.
  • the degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
  • the identity between two amino acid sequences is determined by using the BLASTN algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].
  • the targeting domain of the invention comprises an antibody, or antibody fragment.
  • the antibody targeting domain specifically binds to CD117.
  • Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.
  • the antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No.
  • a chimeric antibody for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin.
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.
  • Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals.
  • the choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost.
  • Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species.
  • Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity.
  • the delivery vehicle e.g., LNP
  • the delivery vehicle is conjugated to the CD117 targeting domain.
  • exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic (“van der Waals”) interactions.
  • the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain upon exposure to certain conditions or chemical agents.
  • the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate the targeting domain.
  • the conjugation comprises a covalent bond between an activated polymer conjugated lipid and the targeting domain.
  • the term “activated polymer conjugated lipid” refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group.
  • the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group.
  • the activated polymer conjugated lipid is an activated pegylated lipid.
  • the first coupling group is bound to the lipid portion of the pegylated lipid.
  • the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid.
  • the second functional group is covalently attached to the targeting domain.
  • the first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions.
  • the first coupling group or second coupling group are selected from the group consisting of maleimides, N-hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups.
  • the first coupling group or second coupling group is selected from the group consisiting of free amines (-NH2), free sulfhydryl groups (-SH), free hydroxide groups (-OH), carboxylates, hydrazides, and alkoxyamines.
  • the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl.
  • the first coupling group is a maleimide.
  • the second coupling group is a sulfhydryl group.
  • the sulfhydryl group can be installed on the targeting domain using any method known to those of skill in the art.
  • the sulfhydryl group is present on a free cysteine residue.
  • the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2-mercaptoethylamine.
  • the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S- acetylthioacetate (SATA).
  • the polymer conjugated lipid and the targeting domain are functionalized with groups used in “click” chemistry.
  • Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles.
  • Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cy cl cooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone
  • the composition of the present invention comprises a combination of agents described herein (e.g., a combination of a CD117-LNP comprising a mRNA encoding a protein for genetic editing and a CD 117-LNP comprising a gRNA molecule).
  • a composition comprising a combination of agents described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual agent.
  • a composition comprising a combination of agents described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual agent.
  • a composition comprising a combination of agents comprises individual agents in any suitable ratio.
  • the composition comprises a 1 : 1 ratio of two individual agents.
  • the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • the invention provides methods for genetic editing of patient HSCs.
  • the method allows for the treatment of a monogenic HSC disease or disorder without depletion/ablation of the patient’s HSC population, and with minimal or no hospitalization.
  • Exemplary diseases or disorders that can be treated using the methods of the invention include, but are not limited to, blood monogenic disorders, genetic defects, bone marrow genetic defects, cancers, platelet disorders, immunodeficiencies, non- hematologic diseases (e.g., cystic fibrosis), metabolic disease and autoimmune diseases.
  • Exemplary bone marrow genetic defects include, but are not limited to, leukemia, aplastic anemia, myeloproliferative disorders, an inherited bone marrow failure syndrome (IBMFS) such as Fanconi anemia, dyskeratosis congenital, Shwachman- Diamond syndrome, Diamond-Blackfan anemia, severe congenital neutropenia, a primary immunodeficiency such as Xl-SCID and Wiskott-Aldrich syndrome, an erythroid disorder such as sickle cell disease (SCD), pyruvate kinase deficiency, or a lysosomal storage diseases such as Fabry disease and Pompe disease.
  • the SCD is sickle cell anemia.
  • Exemplary inflammatory conditions and autoimmune diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, anklosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac spruedermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, Dego's disease, dermatomyositis, dermatomyositis - juvenile, discoid lupus, essential mixed cryoglob
  • the method comprises delivery of one or more CD117 targeted LNP delivery vehicles, comprising at least one mRNA encoding a base editor and a guide RNA for site-specific editing of a target nucleobase, wherein each delivery vehicle is conjugated to a CD117 targeting domain.
  • the invention is not limited to treatment of diseases or disorders that are already established.
  • the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of diseases or disorders do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing diseases or disorders, in that a composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of diseases or disorders, thereby preventing diseases or disorders.
  • the prevention of a disease or disorder encompasses administering to a subject a composition as a preventative measure against the development of, or progression of, a disease or disorder.
  • compositions of the invention can be administered singly or in any combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and/or after each other.
  • compositions of the invention can be used to prevent or to treat a disease or disorder, and that a composition can be used alone or in any combination with another composition to affect a therapeutic result.
  • any of the compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with diseases or disorders.
  • the invention includes a method comprising administering a combination of compositions described herein.
  • the method has an additive effect, wherein the overall effect of the administering a combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor.
  • the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.
  • the method comprises administering a combination of composition in any suitable ratio.
  • the method comprises administering two individual compositions at a 1 : 1 ratio.
  • the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • a pharmaceutical composition of the invention may further comprise one or more additional adjuvants.
  • additional adjuvants include, but are not limited to, aluminum-based adjuvant and monophosphoryl lipid A.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers. In some embodiments, the diameter is in a range from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure.
  • the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent.
  • the propellant comprise particles with a particle size of the same order as particles comprising the active ingredient).
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.
  • Example 1 In vivo modification of hematopoietic stem cells by targeted lipid nanoparticles encapsulating mRNA
  • LNP HSC-targeted lipid nanoparticles
  • This method utilizes antibodies against CD117 conjugated to LNP (CD117/LNP-mRNA). CD117 is internalized after binding of SCF, which facilitates or augments LNP internalization (Yee, N. S., et al., 1994, J Biol Chem, 269:31991-31998). Nucleoside-modified and purified mRNA is non-immunogenic, stable, extensible, and can be used to express virtually any protein of interest (Weissman, D.
  • Lipid nanoparticles are thus far the most promising delivery systems to fulfill the therapeutic potential of mRNA molecules (Cullis, P. R. and Jope, M. J., 2017, Mol Ther, 25: 1467-1475; Samaridou, E., et al., 2020, Adv Drug Deliv Rev, 154-155:37-63).
  • LNP contain ionizable lipids (positively charged at pH ⁇ 6.4), which aid in packaging the mRNA and endosomal escape.
  • ionizable lipids positively charged at pH ⁇ 6.4
  • Such LNP were first approved in 2018 for siRNA, but became widely utilized in 2020, due to the LNP -mRNA platform for Modema and Pfizer COVID-19 vaccines (Akinc, A., et al., 2010, Mol Ther, 18: 1357- 1364).
  • the LNP-mRNA in these FDA-approved vaccines drive antigen expression, but do not actively target specific cells or organs.
  • LNP loaded with diverse mRNA cargos can access HSC in the BM niche in situ, with a single systemic injection. Delivery efficacy to long-term HSC in the BM niche is greatly increased by conjugation of a targeting moiety (anti-CDl 17 antibody).
  • a targeting moiety anti-CDl 17 antibody.
  • LNP loaded with a Cre mRNA cargo can induce durable genome edits in long-term HSC ex vivo and in vivo at, or above, the level required for cure of most NMHD. This approach can be translated to primary human cells, where it was able to achieve high rates of therapeutic base editing in hematopoietic cells from individuals with sickle cell disease.
  • Firefly Luciferase luc2
  • Cre Recombinase ere
  • eGFP enhanced Green Fluorescent Protein
  • IVT-mRNA production template plasmid carrying a T7 promoter, 5’ and 3’ UTR elements, Kozak consensus sequence, and 101 poly(A) tail.
  • DNA synthesis, cloning and industrial grade endotoxin-free plasmid preparation service was provided by GenScript (Piscataway, NJ, USA).
  • IVT-mRNA was produced using linearized IVT template plasmid and the MEGAScript T7 kit (Thermo Fisher Scientific, AMB 13345, Waltham, MA, USA) and formulated with nucleoside-modified m IT*- 5 ’-triphosphate (TriLink, N-1081, San Diego, CA) instead of UTP.
  • IVT-mRNAs were performed co-transcriptionally using the trinucleotide capl analog, CleanCap® Reagent AG (3’ OMe) (TriLink, N-7413, San Diego, CA, USA). Single-stranded IVT-mRNA was purified by cellulose purification, as previously described (Baiersdorfer, M., et al., 2019, Mol Ther Nucleic Acids, 15:26-35). All mRNAs were analyzed by agarose gel electrophoresis and were stored at -20 °C.
  • RNA-loaded particles were characterized by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and a Ribogreen assay.
  • LNP- mRNAs The mean hydrodynamic diameter of these LNP- mRNAs was approximately 80 nm with a poly dispersity index of 0.02-0.06 and an encapsulation efficiency of -95%.
  • LNP used in this study are proprietary to Acuitas Therapeutics (Vancouver, BC, Canada).
  • the ionizable cationic lipid and LNP composition are described in US patent US10,221,127.
  • LNP-mRNA were conjugated with purified rat antimouse CD117 (c-kit), clone 2B8 (BioLegend, 93235, San Diego, CA, USA) or mouse anti -human CD117 (c-kit), clone 104D2 (BioLegend, 95747, San Diego, CA, USA), mouse anti -mouse CD45.2, clone 104 (Biolegend, 92176, San Diego, CA, USA), and control isotype-matched IgG (Thermo Fisher Scientific, Rat IgG Isotype 31933, Mouse IgG Isotype 10400C, Waltham, MA, USA) via SATA-maleimide chemistry, as described previously (Parhiz, H., et al., 2018, J Control Release, 291: 106-115).
  • LNP was modified with maleimide functioning groups (DSPE-PEG-mal) by a post-insertion technique.
  • the antibody was functionalized with SATA (N-succinimidyl S- acetylthioacetate, 26102) from Thermo Fisher (Burlington, MA, USA) to introduce sulfhydryl groups allowing conjugation to maleimide.
  • SATA was deprotected using 0.5 M hydroxylamine followed by removal of the unreacted components by Zeba spin desalting columns (Thermo Fisher Scientific, 89890, Waltham, MA, USA). The reactive sulfhydryl group on the antibody was then conjugated to maleimide moieties using thioether conjugation chemistry.
  • LNP carrying reporter luciferase IVT-mRNA were added at increasing concentrations to the cells and incubated for 24 h. Plates were then washed with PBS, lysed in luciferase cell culture lysis reagent (Promega, E1531, Madison, WI, USA). The cell lysate was mixed with Firefly Luciferase Assay System substrate (Promega, El 500, Madison, WI, USA) and measured on a MiniLumat LB 9506 luminometer (Berthold/EG&G; Wallac, Bad Wildbad, Germany).
  • C57BL/6J mice were i.v. injected with control IgG/LNP-Luc, CD117/LNP-Luc or CD 117/LNP-Luc-miRts formulations.
  • bioluminescence imaging was carried out as described previously using an IVIS Spectrum imaging system (Caliper Life Sciences, Waltham, MA, USA) (Kariko, K., et al., 2011, Nucleic Acids Res, 29:el42).
  • D-luciferin sodium salt (Regis Technologies, 1- 360243-200, Morton Grove, IL, USA) dissolved in PBS was administered to mice intraperitoneally at a dose of 150 mg/kg.
  • mice After 5 min, the mice were euthanized; desired tissues were harvested, washed with PBS, and immediately placed on the imaging platform. Harvested femurs were slightly crushed by spatula to expose the bone marrow for imaging. Tissue luminescence was measured on the IVIS imaging system using an exposure time of 5s or longer to ensure that the signal obtained was within operative detection range.
  • Specimens from patients with sickle cell disease were collected. Deidentified apheresis waste product from patients with SCD and CD34 + progenitor cells were isolated with the MACS MicroBead kit (Miltenyi).
  • Bone marrow cells were isolated from femurs of animals, after removal of muscle and connective tissues, by mechanical crushing, which maximizes cell recovery.
  • BM cells were resuspended in 4% FBS (SH3007103) from Hyclone (Logan, UT) and PBS (10010023) from Gibco (Waltham, MA) and RBC were lysed using ACK lysis buffer (A1049201) from Gibco (Waltham, MA) at room temperature, according to manufacturer protocol, fdtered through a 40 mm sterile strainer (431750) from Coming (Glendale, AZ) and washed with 4% FBS PBS solution.
  • CS2-0106 After lysis, cells were counted and assessed for viability by AOPI staining (CS2-0106), using a Cellometer Auto 2000 cell viability counter (Nexcelom Bioscience, Lawrence, MA) and seeded at a 1.5*10 6 /mL concentration in Stemspan SFEM (096000) from Stem Cell Technologies (Vancouver, BC, Canada) supplemented with 50 ng/mL mSCF (250-03), 6 ng/mL mIL-3 (213-13), and 10 ng/mL mIL-6 (216-16), all supplied by Peprotech (Cranbury, NJ), LNP formulations were added at the time of seeding and for up to 18 hours, depending upon assay.
  • Human erythroid progenitor cells (ErPC) editing and differentiation Human CD34+ cells were isolated from blood products by immunomagnetic-separation using the CD34 MicroBead Kit (130-046-702) from Miltenyi Biotec Inc. (Auburn, CA). ErPC were obtained through expansion of CD34+ cells, using a culture system previously described and frozen after 6-10 days.
  • the degree of cell sickling was measured using a modified version of a method already described (Breda, L., et al., 2016, Blood, 128:113-1143). Briefly, l * 10 6 differentiated erythroblasts were suspended in lOOpL of isotonic TES, supplemented with 10 mM glucose and 0.2% bovine serum albumin, in individual wells of a Costar polystyrene 96-well microplate (N° 9017; Coming, Corning, NY).
  • the microplate was then transferred to a Thermomixer R shaker-incubator (Eppendorf, Enfield, CT), and maintained under hypoxia (2.5% Oxygen gas, balance Nitrogen gas), with continuous agitation at 900 rpm, at 37 °C for 2 hours. At conclusion, aliquots ( ⁇ 20 pL) of each sample were collected in 2% glutaraldehyde solution for immediate fixation without exposure to air.
  • Hbb A-F-S-C Helena Laboratories, Beaumont, TX
  • Hbb A-F-S-C Helena Laboratories, Beaumont, TX
  • CFU assay was conducted using reagents from Stem Cell Technologies. Bone marrow harvested from animals was seeded using a at 30,000 cells/well in complete Methocult media (M3434), in meniscus-free 6-well SmartDish plates (27371), using 16- gauge blunt end needles (28110) per directions from Stem Cell Technologies. Colonies were incubated for 2 weeks at 37 °C in CO2 incubators. Colonies were imaged using Evos FL Auto (AMAFD1000) manufactured by Life Technologies (Waltham, MA) microscope and analyzed using superimposed bright field and Texas Red filter images.
  • M3434 complete Methocult media
  • M343434 meniscus-free 6-well SmartDish plates
  • 28110 16- gauge blunt end needles
  • mice were induced to general anesthesia by injected by IP injection of a 200 mg/kg ketamine (NDC #13985-584-10 From Vet One, Boise, ID) and 20 mg/kg xylazine (NDA #139-236, from Akorn Inc., Lake Forest, IL) solution in PBS.
  • NDC #13985-584-10 From Vet One, Boise, ID
  • NDA #139-236 20 mg/kg xylazine
  • the heart was slowly infused with lOmL of 1% FBS PBS solution using a 27 G x U inch needle (305109) supplied by Beckton Dickinson (Franklin Lakes, NJ) upon interruption of portal vein flow.
  • tdTomato marking were carried out by direct measurement of tdTomato expression in whole blood for the RBC compartment, or after RBC lysis, using ACK lysis buffer at room temperature, for the WBCs analyses.
  • tdTomato expression in WBC was assessed using the following antibodies: violetFluorTM 450 CD3 (clone 17A2, 75-0032-U025) from Tonbo Biosciences (San Diego, CA), CD45R/B220-FITC (clone RA3-6B2, 103205) from BioLegend (San Diego, CA), Ly-6G/Grl PE- Cyanine7 (clone RB6-8C5, 565033) from BD Biosciences (Franklin Lakes, NJ), for detection of T, B cells and Granulocytes, respectively, while CD45.2 PerCP-Cyanine5.5 (clone 104, 45-0454-82) from eBioscience (Waltham, MA), CD45.1 APC (clone A20, 110713)
  • Bone marrow samples obtained after crushing were treated with ACK lysis buffer to remove RBC prior to analyses.
  • the following biotinylated antibodies were used to discriminate lineage committed cells: CD45R (13-0452-82)/CD8 (13-0081-82)/CD4 (3-0042-82)/CD127 (13- 1271-82)/Grl-Ly6G (13-593 l-82)/Terl 19 (13-5921-82) from eBiosciences.
  • An APC- eFluor 780 streptavidin (47-4317-82) from Invitrogen was used to bind lineage committed cells pre-incubated with biotinylated lineage antibody cocktail.
  • an APC anti-CDl 17 (clone 2B8, 17-1171-82) from Invitrogen was utilized along with a Ly6A/E ((ScaI)-PE-Cyanine7 antibody (clone D7, 25-5981-82) from eBioscience).
  • LT-HSC (LSK CD150+ CD48-) were gated using a Pacific Blue anti- CD48 antibody (clone HM48-1, 103418) and a BV650 anti-CDl 50 antibody (clone TC15-12F12.2, 115931), both from BioLegend.
  • C57BL/6 whole BM (WBM) or lineage depleted BM cells (Lin ) were incubated in vitro with either unconjugated LNP encapsulating 0.1, 1, or 3 pg of nucleoside-modified luciferase mRNA (unmodified LNP-Luc), anti-CD45 -conjugated LNP (CD45/LNP-Luc), anti-CDl 17-conjugated LNP (CD117/LNP-Luc), or isotype control IgG-conjugated LNP (control IgG/LNP-Luc).
  • CD45/LNP and CD117/LNP were hypothesized to bind all hematopoietic-derived cells or stem and progenitor cells, respectively.
  • CD117/LNP luciferase activity was 500 and 700-fold higher than CD45/LNP luciferase activity in WBM and Lin’, respectively, when normalized to the frequency of CD45 and CD117 positive cells in WBM and Lin’ ( Figure 2A). Normalized luciferase activity suggests that CD117 mediated targeting and delivery is superior to CD45 mediated targeting in vitro. This demonstrates efficient targeting and functional delivery of mRNA with CD 117/LNP.
  • CD 117/LNP encapsulating Cre recombinase mRNA (CD 117/LNP-Cre) was used to test LNP-mediated genetic recombination in HSCs and persistence of the editing in conjunction with three reporter murine models.
  • These murine models are engineered with a Cre-responsive reporter allele comprised of a loxP- flanked STOP cassette preventing transcription of a CAG promoter-driven green or red fluorescent reporter gene (ZsGreenl for Ai6 and tdTomato for Ai9 and Ail 4, respectively) inserted into the Gt(ROSA)26Sor locus (Madisen, L., et al., 2010, Nat Neurosci, 13: 133-140).
  • a Cre-responsive reporter allele comprised of a loxP- flanked STOP cassette preventing transcription of a CAG promoter-driven green or red fluorescent reporter gene (ZsGreenl for Ai6 and tdTomato for Ai9 and Ail 4, respectively) inserted into the Gt(ROSA)26Sor locus (Madisen, L., et al., 2010, Nat Neurosci, 13: 133-140).
  • CD117/LNP-Cre showed higher efficacy in LSK cells at lower concentrations: treatment with 0.1 pg CD 117/LNP-Cre was 2.5- fold more effective at targeting LSK cells compared to treatment with 0.1 pg CD45/LNP- Cre ( Figure ID). There was no significant difference between targeted cell frequency in the LSK subset with the 0.1 pg and 0.5 pg dose or 0.5 pg and 1 pg dose. The media of cells treated for 18 hours with LNP-Cre and was also replaced and the cells kept for 3 additional days in culture to assess the maximum targeting achieved after exposing WBM to LNP.
  • mice had durable editing in all lineages, specifically myeloid cells (Grl+, Figure 3A), lymphoid cells (CD3+ and B220+, Figure 3B), and erythroid cells (Figure 3C) at 4 months post-HSCT, consistent with genome editing of multipotent HSC.
  • RBC and leukocyte editing rates with CD 117/LNP-Cre were >99% at 0.05, 0.1, and 1 pg mRNA dose, and 91.8% at the 0.01 pg dose ( Figure 3A through Figure 3C and Figure 4D).
  • targeting mediated by control IgG/LNP-Cre was near 0% at 0.01 pg ( Figure 4C and Figure 4D).
  • tdTomato + Grl + cells had the fastest rise ( Figure 4B and Figure 4C), which is expected given their rapid turnover of 2-3 days.
  • BM cells harvested from these animals showed similar editing rates in colony forming assays, a functional assay for clonogenic potential, thus corroborating the flow cytometry results of LT-HSC ( Figure 3E, Figure 4E, and Figure 4F).
  • splenocytes had genome editing levels comparable to those in the WBM ( Figure 3F and Figure 3G), consistent with migration of edited BM-derived cells to the spleen.
  • LT-HSC could be targeted in vivo, as well (Parhiz, H., et al., 2018, J Control Release, 291 : 106-115; Marcos-Contreras, O. A., et al., 2020, Proc Natl Acad Sci USA 117:3405- 3414; Tombacz, I., et al., 2021, Mol Ther, 29:3293-33041 Rurik, J. G., et al., 2022, Science, 375:91-96).
  • Intravenous (i.v.) administration of CD117/LNP-Luc generated luciferase activity in the femur at 24 hours, whereas IgG/LNP-Luc did not ( Figure 6A).
  • both control IgG/LNP-Luc and CD 117/LNP-Luc showed comparable luciferase activity in the liver, as LNP bind ApoE and are non-specifically targeted to the LDL receptor, which is expressed on hepatocytes (Akinc, A., et al., 2010, Mol Ther, 18: 1357- 1364).
  • ⁇ n vivo multilineage editing was tested by quantifying tdTomato expression in peripheral blood cells of i.v.
  • CD117/LNP-Cre treated animals over time (up to 4 months) and tdTomato expression in bone marrow at 4 months.
  • Peripheral blood multilineage and BM editing was evaluated at 16 weeks and, specifically, in LT-HSC.
  • CD117/LNP-Cre treated mice had consistently higher editing in all peripheral blood lineages ( Figure 6B and Figure 6C), and importantly 3-fold higher editing in LT-HSC (55 vs 19%, respectively) compared to that observed in control IgG/LNP-Cre treated mice ( Figure 6D).
  • HSC editing after in vivo treatment with CD117/LNP-Cre was dose dependent in peripheral blood and bone marrow at 16 weeks with a 5.5-fold increase in the percentage of gene-edited LT-HSC with 5 pg versus 1 pg ( Figure 6E through Figure 6G).
  • LNP-Cre in vivo editing led to appearance of edited RBC and WBC (not shown) with similar kinetics to transplantation of ex vivo treated bone marrow ( Figure 6H and Figure 61).
  • tdTomato expression levels in lung and liver cells were compared 4-months after in vivo treatment with a single dose of CD 117/LNP-Cre (1 and 5 pg dose) or control IgG/LNP-Cre (5 pg dose).
  • liver editing was high (76-79% of cells), and editing was comparable between the two treatments (Figure 7B), consistent with known non-specific ApoE and LDL receptor axis mediated LNP mediated uptake (Akinc, A., et al., 2010, Mol Ther, 18: 1357-1364).
  • targeting was adapted to human CD117 and utilized LNP containing mRNA encoding a cas9 adenine base editor (ABE) and LNP carrying a single guide RNA (sgRNA) targeted to the beta-globin sickle cell mutation.
  • Adenine base editing of the A to G leads to conversion of the pathogenic E6V (HBB s 'j mutation to a non-pathogenic E6A variant ⁇ HBBP ⁇ Makassai ”) (Newby, G. A., et al., 2021, Nature, 595:295-302).

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  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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Abstract

La présente invention concerne des molécules LNP ciblées par CD117 pour l'administration de molécules d'édition de gènes à des cellules souches hématopoïétiques (HSC) et des procédés d'utilisation de celles-ci pour l'édition de bases d'ADN génomique pour le traitement d'une maladie ou d'un trouble.
PCT/US2024/027460 2023-05-02 2024-05-02 Édition génique ciblée de cellules souches hématopoïétiques (hsc) et leurs procédés d'utilisation Ceased WO2024229251A2 (fr)

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US202363499580P 2023-05-02 2023-05-02
US63/499,580 2023-05-02

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WO2024229251A2 true WO2024229251A2 (fr) 2024-11-07
WO2024229251A3 WO2024229251A3 (fr) 2025-03-13

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PCT/US2024/027460 Ceased WO2024229251A2 (fr) 2023-05-02 2024-05-02 Édition génique ciblée de cellules souches hématopoïétiques (hsc) et leurs procédés d'utilisation

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Publication number Priority date Publication date Assignee Title
WO2021081244A1 (fr) * 2019-10-22 2021-04-29 Fred Hutchinson Cancer Research Center Réduction de l'expression de cd33 mediee par des editeurs de bases pour protéger sélectivement des cellules thérapeutiques
EP4225907A4 (fr) * 2020-10-12 2025-01-22 Duke University Composition d'édition de bases basée sur crispr/cas pour restaurer la fonction de la dystrophine
AU2022267356A1 (en) * 2021-04-30 2023-11-23 The Trustees Of The University Of Pennsylvania Compositions and methods for targeting lipid nanoparticle therapeutics to stem cells

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