WO2022165245A1 - Compositions et méthodes d'utilisation de variants leurres d'ace2 mutants - Google Patents

Compositions et méthodes d'utilisation de variants leurres d'ace2 mutants Download PDF

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WO2022165245A1
WO2022165245A1 PCT/US2022/014406 US2022014406W WO2022165245A1 WO 2022165245 A1 WO2022165245 A1 WO 2022165245A1 US 2022014406 W US2022014406 W US 2022014406W WO 2022165245 A1 WO2022165245 A1 WO 2022165245A1
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hace2
seq
protein
acid sequence
amino acid
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PCT/US2022/014406
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James M. Wilson
Joshua Joyner SIMS
Christian HINDERER
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The Trustees Of The University Of Pennsylvania
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Publication of WO2022165245A1 publication Critical patent/WO2022165245A1/fr
Priority to US18/304,143 priority Critical patent/US20230338478A1/en

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • SARS-CoV-2 coronavirus "SARS-CoV-2” outbreak that emerged in China and has rapidly spread across the world has been declared a public health emergency of international concern by the World Health Organization (WHO). Efforts are ongoing to discover effective therapeutic and prophylactic agents that will help to mitigate the spread of the disease.
  • AAVs are nonpathogenic parvoviruses that circulate broadly in humans and other species. Replacing all viral coding sequences with a gene of interest yields an AAV vector capable of efficient in vivo gene delivery without the insertional mutagenesis risks or robust inflammatory responses observed with lentiviral or adenoviral vectors.
  • AAV vectors have demonstrated an acceptable safety profile in thousands of human subjects, and two products are now approved in the US with dozens more in late-stage clinical development. The stability of AAV vectors make them practical for widespread distribution as prophylactic vaccines.
  • ACE2 angiotensin-converting enzyme 2
  • S 1 domain of the viral spike protein of SARS CoV-2 binds to host cells via the ACE2 receptor with low nanomolar affinity.
  • Soluble ACE2 has been administered intravenously to healthy volunteers and demonstrated an acceptable safety and immunogenicity profile.
  • a pilot efficacy study with soluble human recombinant ACE2 is ongoing in patients with COVID- 19 (Clinicaltrials.gov #NCT04287686).
  • Angiotensin-converting enzyme 2 (ACE2) is transmembrane glycoprotein with present increased expression in tissues of heart, kidneys, and testes (Kuba, K., et al., Pharmacol Ther, 2010, 128: 119-128).
  • Targeting ACE2 has been used as therapeutic approach for hypertension and heart failure, and described to provide a protective effect in cardiovascular systems.
  • Recent sequencing analysis studies have identified a large number of ACE2-expressing cells in the lung tissue, specifically the type I and II alveolar epithelial cells (Zhao,Y., et al., 2020, bioRxiv). This finding was consistent with the correlation of SARS -coronavirus (SARS-CoV) infection causing severe acute lung failure.
  • SARS-CoV SARS -coronavirus
  • SARS-CoV protein spikes contact ACE2 and utilize it to facilitate internalization, which thereby aids in infection (Kuba, K., et al., 2010, Pharmacol Ther, 128: 119-128).
  • ACE2 there is an HEXXH motif, containing two histidine in the active catalytic site, which is responsible for coordinating zinc ion, thereby facilitating the metabolism of circulating peptides.
  • compositions and methods for preventing one or more COVID-19 symptoms and/or infection including compositions and methods for reducing viral replication following infection.
  • a recombinant AAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises a 5 ’ inverted terminal repeat (ITR), a nucleic acid sequence encoding at least one mutant hAce2 soluble decoy protein under the control of regulatory control sequences which direct expression of the hAce2 soluble decoy protein, and a 3’ ITR, wherein the mutant hAce2 soluble decoy protein comprises an amino acid sequence of: (a) SEQ ID NO: 12 (hAce2- Variant2) or an amino acid sequence at least 95% identical thereto, optionally fused to an immunoglobulin Fc region; (b) SEQ ID NO: 10 (hAce2-Variantl) or an amino acid sequence at least 95% identical thereto, optionally fused to an immunoglobulin Fc region; (c) SEQ ID NO: 14 (hAce2-Variant3) or an amino acid sequence at least 95% identical
  • the rAAV comprises nucleic acid sequence encoding the mutant hAce2 soluble decoy protein selected from: (a) SEQ ID NO: 11 or a sequence at least about 90% identical thereto encoding SEQ ID NO: 12 (hAce2- Variant2); (b) SEQ ID NO: 9 or a sequence at least about 90% identical thereto encoding SEQ ID NO: 10 (hAce2-Variantl); (c) SEQ ID NO: 13 or a sequence at least about 90% identical thereto encoding SEQ ID NO: 14 (hAce2-Variant3); (d) SEQ ID NO: 15 or a sequence at least about 90% identical thereto encoding SEQ ID NO: 16 (hAce2-Variant4); (e) SEQ ID NO: 1 or a sequence at least about 90% identical thereto encoding SEQ ID NO: 2 (hAce2-Variantl- IgG4 fusion); (f) SEQ ID NO: 3 or a sequence at least about 90% identical thereto encoding
  • the mutant hAce2 soluble decoy is a hAce2 soluble decoy fusion protein further comprising an immunoglobulin Fc region, optionally wherein the Fc is a human Fc, a human IgGl Fc, a human IgG4 Fc or a human IgM Fc.
  • the mutant hAce soluble decoy protein is (a) a protein comprising SEQ ID NO: 4 (a hAce2-Variant2-IgG4 fusion) or an amino acid sequence at least 95% identical thereto; (b) a protein comprising SEQ ID NO: 109 (hAce2-Variant2-IgGl fusion) or an amino acid sequence at least 95% identical thereto; (c) a protein comprising SEQ ID NO: 113 (hAce2-Variant2-IgGl fusion with “GS” linker) or an amino acid sequence at least 95% identical thereto; (d) a protein comprising SEQ ID NO: 111 (hAce2-MR27-Variant-IgGl fusion) or an amino acid sequence at least 95% identical thereto; (e) a protein comprising SEQ ID NO: 115 (hAce2-MR27-Variant2-IgGl fusion with GS linker) or an amino acid sequence at least 95% identical thereto;
  • the rAAV comprises a vector genome comprising an expression cassette comprising regulatory control sequences comprising one or more of: a promoter, at least one enhancer, at least one intron, and a poly A signal, optionally wherein the regulatory control sequences comprise a CB7 hybrid promoter, a chicken beta actin intron, and a rabbit beta globin polyA.
  • the rAAV comprises the vector genome comprising an expression cassette having nucleotide sequence of SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 86; SEQ ID NO: 88; SEQ ID NO: 90, or SEQ ID NO: 92.
  • the rAAV comprises the vector genome having the nucleic acid sequence of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, or SEQ ID NO: 91.
  • a recombinant AAV comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises a 5 ’ inverted terminal repeat (ITR), a nucleic acid sequence encoding a fusion protein comprising a signal peptide, a mutant hAce2 soluble decoy protein, and an optional immunoglobulin Fc region, wherein the coding sequences for the fusion protein are under the control of regulatory control sequences which direct expression of the fusion protein, and a 3’ ITR, wherein the mutant hAce2 soluble decoy protein comprises a mutant amino acid in (a) R or M at residue 14 (K changed to R or M) and (b) V or K at residue (18) (E changed to V or K), and at least one further residue (c) P at residue (22) (L changed to P); (d) R at residue (25) (Q changes to R); (e) A at residue (30) (S changed to A); (f) A
  • a mutant hAce2 soluble decoy protein comprises an amino acid sequence selected from (i) the substitutions of (a), (b), and (j) or (ii) the substitutions of (a), (b), (g), and (j).
  • the signal peptide is a human signal peptide, optionally wherein the signal peptide is the native hAce2 signal peptide.
  • the AAV capsid is an AAV9 capsid, a AAVhu68 capsid, an AAV5 capsid, an AAV6 capsid, an AAV6.2 capsid, or an AAVrh91 capsid.
  • a pharmaceutical composition which comprises at least one recombinant AAV as described herein and one or more of any of: a pharmaceutically acceptable diluent, a suspending agent, a preservative, and/or a surfactant.
  • the pharmaceutical composition is formulated for intranasal administration.
  • the pharmaceutical composition is formulated for intrapulmonary administration.
  • the pharmaceutical composition is formulated for intravenous administration.
  • the pharmaceutical composition is formulated for intraperitoneal administration.
  • a method for treating and/or preventing one or more symptoms of SARS-CoV2 by administering or co-administering a pharmaceutically effective amount of synthetic or recombinant hAce2 soluble decoy protein, an rAAV or a pharmaceutical composition as described herein, and combinations thereof.
  • the symptoms are one or more of fever, cough, gastrointestinal distress, breathing difficulty, loss of taste, and/or loss of smell.
  • the method comprises administering or co-administering the hAce2 soluble decoy protein, the rAAV or a pharmaceutical composition intranasally.
  • the method comprises administering or co-administering the hAce2 soluble decoy protein, the rAAV or a pharmaceutical composition via inhalation. In other embodiments, the method comprises administering or co-administering the hAce2 soluble decoy protein, the rAAV or a pharmaceutical composition intravenously.
  • a mutant soluble human Ace2 (hAce2) protein useful in preventing infection with betacoronaviruses, including SARS-CoV2 is provided, as are compositions useful in treating disease associated with betacoronavirus-associated disease, including, e.g., COVID-19.
  • an rAAV in the manufacture (preparing) of a medicament for the prevention and/or treatment infection with a virus mediated by an Ace2 receptor, optionally wherein the virus is a SARS virus, or optionally wherein the virus is SARS-CoV2.
  • a packaging host cell in culture or suspension comprising: (a) a nucleic acid molecule encoding a vector genome comprising a 5’ inverted terminal repeat, an expression cassette comprising a mutant soluble hAce2 fusion protein as defined in any one of claims 1 to 16, and a 3’ inverted terminal repeat; (b) nucleic acid sequences encoding an rAAV capsid protein under control of sequences which regulate expression of the capsid protein in the packaging hose cell; and (c) helper sequences for replication and packaging of the vector genome into the rAAV capsid.
  • an rAAV stock is produced from the packaging host cell described herein.
  • FIGs. 1A and IB show a schematic representation of yeast-surface display technique.
  • FIG. 1A shows a schematic expression cassette for use in a mutational library.
  • FIG. IB shows a schematic representation of a yeast-surface display used in the high throughput screening (a yeast display (YD) system with an HA antigen, wherein the antigen is a coronavirus spike protein.
  • a large mutational library of Ace2 is generated using error prone PCR.
  • Aga2 fusion directs the Ace2 variant to be exported to and ligated onto the exterior yeast cell wall, thus coating the exterior of the yeast cell that contains that variant’s gene. This provides the link between genotype and phenotype allowing a high-throughput screening required for protein engineering technique.
  • FIGs. 2A and 2B show dot plots of results from florescence-activated cell sorting (FACS) following multiple consecutive rounds of selection for receptor binding domain (RBD)-targeted binder. Each dot represents two fluorescent signals of a separate yeast cell in the analyzed (sorted) sample, wherein X-axis is a measure of Ace2 expression, and Y-axis is measure of RBD-binding.
  • FIG. 2A shows results FACS run with goal to isolate the rare variants within the population with improved SARS-CoV-2 spike binding.
  • FIG. 2B shows first round of selection with FACS for improved SARS-CoV-2 spike binding.
  • FIG. 2C shows second round of selection with FACS for improved SARS-CoV-2 spike binding.
  • FIG. 2D shows third round of selection with FACS for improved SARS-CoV-2 spike binding.
  • FIG. 3 shows the neutralization data as measured across the most improved variants identified from screen. These variants neutralized SARS-CoC2 activity in the range of 3-10 ng/mL (Generation 2).
  • FIGs. 4A to 4G show selection and characterization of best variant from digital recombinant variants screened over wild-type Ace2-IgG (Generation 3).
  • FIG. 4A shows a confirmation of binding of the yeast clones from the final rounds of sorting to SARS-CoV2 RBD by flow cytometry.
  • FIG. 4B shows results of the neutralization assay with hAce2-Variant3-IgG4 purified decoy in full titration of SARS-CoV2 reporter virus.
  • FIG. 4C shows results of the neutralization assay with hAce2- Variant3-IgG4 purified decoy in full titration of SARS-CoVl reporter virus.
  • FIG. 4D shows results of the neutralization assay with hAce2-Variantl-IgG4 purified decoy in full titration of SARS-CoV2 reporter virus.
  • FIG. 4E shows results of the neutralization assay with hAce2- Variantl-IgG4 purified decoy in full titration of SARS-CoVl reporter virus.
  • FIG. 4F shows results of the neutralization assay with hAce2-Variant2-IgG4 purified decoy in full titration of SARS-CoV2 reporter virus.
  • FIG. 4G shows results of the neutralization assay with hAce2- Variant2-IgG4 purified decoy in full titration of SARS-CoVl reporter virus.
  • FIGs. 5 A to 5 C provide an alignment of wild-type (WT) human Ace2 (hAce2) soluble protein (SEQ ID NO: 25) and modified hAce2 soluble decoy proteins (SEQ ID NOs: 10 (hAce2-Variantl), 12 (hAce2-Variant2), 14 (hAce2-Variant 3), and 16(hAce2-Variant4)).
  • FIG. 5 A provides an alignment of amino acids 1 to 300 of wild-type and modified hAce2 soluble decoy protein.
  • FIG. 5B provides an alignment of amino acids 301 to 540 of wild-type and modified hAce2 soluble decoy proteins.
  • FIG. 5C provides an alignment of amino acids 541 to 600 of wild-type and modified hAce2 soluble decoy proteins.
  • FIGs. 6A and 6B show the measured binding affinity and fold change in binding affinity of modified hAce2-Variantl-IgG4 soluble decoy fusion protein and wild-type hAce2- IgG4 soluble decoy fusion protein for mutant RBD of SARS-CoV2.
  • FIG. 6A shows the measured binding affinity of modified hAce2-Variantl-IgG4 soluble decoy fusion protein and wild-type hAce2-IgG4 soluble decoy fusion protein for mutant RBD of SARS-CoV2.
  • FIG. 6A shows the measured binding affinity of modified hAce2-Variantl-IgG4 soluble decoy fusion protein and wild-type hAce2-IgG4 soluble decoy fusion protein for mutant RBD of SARS-CoV2.
  • 6B shows a fold change in binding affinity of modified hAce2-Variantl-IgG4 soluble decoy fusion protein and wild-type hAce2-IgG4 soluble decoy fusion protein for mutant RBD of SARS-CoV2 (affinity to mutant RBD of SARS-CoV2 over affinity to wild type RBD of SARS-CoV2).
  • FIG. 7 shows a graph comparison of mutant decoy affinity versus wild-type (WT) decoy affinity to various mutant RBD of SARS-CoV2.
  • FIGs. 8A and 8B show the alignment of wild-type (WT) CoV2 RBD, mutants of CoV2, and CoV 1 (SEQ ID NOs: 43-51).
  • FIG. 8A shows the alignment of amino acids 1 to 120.
  • FIG. 8B shows the alignment of amino acids 121 to 229.
  • FIG. 9 shows an example AAV vector genome with engineered hAce2 decoy (hAce2- Variantl-IgG4).
  • the construct contains Inverted Terminal Repeat (ITR) sequences for packaging, CMV IE enhancer with CB promoter (CB7 hybrid promoter), a Chicken Beta actin intron (chimeric intron), the engineered human Ace2 decoy gene fused to a human IgG4 Fc domain, and a Rabbit globin polyA terminator.
  • ITR Inverted Terminal Repeat
  • FIG. 10A to 10C show analytics from mouse study comprising intranasal delivery of IxlO 11 Genome Copies (GC) of rAAVs encoding hAce2-Variantl/2-IgG4-Fc fusions of engineered decoys in either AAVhu68 or AAVrh91 capsids.
  • BALF samples were collected at day 7 post-transduction.
  • FIG. 10A shows a mass spectrometry with hAce2-IgG4 standards to determine the concentration of decoy in BALF from 5 animals in each dosing group.
  • FIG. 10B shows results of CoV2 spike binding activity in the samples, wherein an immobilized recombinant CoV2 spike protein in an ELISA assay was with the human Fc tag on the decoy as the detection epitope.
  • FIG. 10C shows results of a commercial CoV2 reporter virus used to detect neutralizing activity in the BALF samples. The dotted line represents complete neutralization in the assay as established with saturating concentrations of a neutralizing monoclonal antibody.
  • FIGs. 11A to 1 IF show analytics from NHP study, intranasal delivery of 5xl0 12 GC of rAAVs encoding hAce2-Variantl/2-IgG4-Fc of mutant decoys.
  • the capsids used were AAVhu68 and AAVrh91. This data is on NLF samples collected at day 7 post-transduction.
  • FIG. 11A shows CoV2 spike binding activity in the lx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 11A shows CoV2 spike binding activity in the lx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 1 IB shows CoV2 spike binding activity in the lOx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 11C shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF IX samples (AAVrh91) from 2 animals in each doing group.
  • FIG. 1 ID shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF 10X samples (AAVrh91) from 2 animals in each doing group.
  • FIG. 1 IE shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF IX samples (AAVhu68) from 2 animals in each doing group.
  • FIG. 1 IF shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF 10X samples (AAVhu68) from 2 animals in each doing group.
  • FIGs. 12A to 12D show analytics from an NHP study with intranasal delivery of 5xl0 12 GC of rAAVs encoding hAce2-Variantl/2-IgG4-Fc of mutant decoys.
  • the capsids used were AAVhu68 and AAVrh91. This data is on NLF samples collected at day 14 posttransduction.
  • FIG. 12A shows CoV2 spike binding activity in the lx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 12A shows CoV2 spike binding activity in the lx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 12B shows CoV2 spike binding activity in the lOx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 12C shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF IX samples (AAVrh91) from 2 animals in each doing group.
  • FIG. 12D shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF 10X samples (AAVrh91) from 2 animals in each doing group.
  • FIGs. 13A to 13B shows neutralization data across various SARS-CoV-2 Variants as measured in an assay using a reporter virus.
  • FIG. 13A shows inhibition of SARS-CoV-2 RBD-hAce2 interactions in presence engineered hAce2 soluble decoy, hAce2-Variant2-IgG4.
  • FIG. 13B shows a plot of IC50 values for interaction inhibition between hAce2-Variant2-IgG4 and SARS-CoV2-RBD mutant variants, as measured in a neutralization assay.
  • FIG. 14 shows estimated IC50 of 6 “revertants” of hAce2-Variantl-IgG4 soluble decoy, wherein each “revertanf ’ comprised of one amino acid substitution reverted from engineered back to wild type at the indicated positions (based on numbering of amino acid sequence of SEQ ID NO: 25).
  • FIGs. 15A to 15B show the effect of varying linker length on IC50 values of interaction between hAce2-Variant2-IgG4 soluble decoy and SARS-CoV2-RBD.
  • FIG. 15A shows estimated IC50 values in comparison to varying “GSG” linker length, or varying decoy length (1-615 versus 1-740 amino acid of hAce2), which is linked at the amino terminus of IgG4 f the soluble decoy fusion protein.
  • FIG. 15B shows a schematic representation of protein interaction structure between hAce2 and RBD of SARS-CoV2, showing amino acids 1-615 and 1-740, as they are mapped onto the structure of protein interaction.
  • FIGs. 16A to 16D show analytics from a mouse challenge study with intranasal delivery of 1 X 10 11 GC of engineered decoy in the absence or presence of 280 PFU SARS- CoV-2. 7 days prior to the delivery of SARS-CoV-2 or PBS, all groups were given either the AAV decoy or PBS. At Day 0 all groups were given either SARS-CoV-2 or PBS. At day 4 and 7 mice were euthanized for histopathology and viral PCR.
  • FIG. 16A shows the change in body weight among the groups over a course of 5 days.
  • FIG. 16B shows the change in body weight among the groups over a course of 8 days.
  • FIG. 16C shows the concentration of AAV decoy in G4 and G5 at day 4 and 7, respectively.
  • FIG. 16D shows inflammation scores from lung sections derived from the 5 groups at day 4 and 7.
  • FIG. 17A to 17C shows analytics from a NHP study with intranasal delivery of 5x10 12 GC of rAAVs encoding hAce2-Variant2 (GTP14HL-IgG) of mutant decoys.
  • the capsids used were AAVhu68 and AAVrh91. This data is on NLF samples collected at days 7, 14 and 28 post-transduction.
  • FIG. 17A shows mass spectrometry to assess the change in concentration of the mutant decoys over a period of 28 days.
  • FIG. 17B shows mass spectrometry of the concentration of AAVrh91 at 5xl0 12 GC and 5xl0 n GC at day 14 post-transduction.
  • FIG. 17C shows CoV-2 spike binding activity in NLF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIGs. 18A to 18F show ACE2 Decoy Receptor Engineering.
  • FIG. 18A shows decoy affinity maturation and candidate selection process.
  • FIG. 18B shows flow cytometry analysis of the naive (dark gray) and sorted populations (light gray) from the secondary YD library.
  • FIG. 18C shows NGS analysis of plasmid populations recovered from rounds of YD.
  • FIG. 18D shows SPR binding analysis for CoV-2 RBD injected over surface-immobilized ACE2- wt.
  • FIG. 18E shows SPR binding analysis for CoV-2 RBD injected over surface-immobilized hAce2-Variant2.
  • 18F shows Wuhan CoV-2-Pseudotyped lentiviral reporter neutralization assay of ACE2-wt-Fc4 and hAce2-Variant2(CDY 14HL)-Fc4. Data for at least three independent measurements are presented as average ⁇ standard deviation.
  • FIGs. 19A to 19E show ACE2 Decoy Binding and Neutralization Across Diverse CoVs.
  • FIG. 19A shows structural models (7DF4.pdb [Xu C, Wang Y, Liu C, Zhang C, Han W, Hong X, et al. Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM. Sci Adv. 2021 ;7( 1). Epub 2020/12/06. doi: 10.1126/sciadv.abe5575. PubMed PMID: 33277323; PubMed Central PMCID: PMCPMC7775788.]) of Wuhan CoV-2 RBD (light) bound to human ACE2 (dark) RBD.
  • FIG. 19B shows SPR measurements: ACE2-wt-Fc4 or hAce2-Variant2-Fc4 binding to various purified recombinant RBD proteins.
  • FIG. 19C show Variant2 (CDY14HL)-Fc4 titrations using pseudotyped lentivirus reporters encoding CoV-2 spike proteins from four US-CDC Variants of Concern. RBD mutations are indicated in brackets, however in many cases other spike mutations exist that are not listed.
  • FIG. 19D shows.
  • FIG. 19E shows pseudotype titration for CoV-1 spike reporter virus. Reporter virus activity data are presented as mean ⁇ standard error of the mean for at least three replicate titrations.
  • FIGs. 20A to 20J show protection in the human ACE2 transgenic mouse model.
  • FIG. 20A shows BAL from vector-treated animals analyzed for decoy protein by MS.
  • FIG. 20B shows BAL from vector-treated animals analyzed for SARS-CoV-2 spike ELISA.
  • FIG. 20C shows BAL from vector-treated animals analyzed for neutralization of SARS-CoV-2 pseudotyped lentivirus.
  • FIG. 20D shows challenge study design.
  • FIG. 20E shows weight loss in the animals that were sustained for 7 days; one animal in the vehicle and vector treated groups required euthanasia.
  • FIG. 20F shows MS assay of expression in ASF (corrected for BAL dilution).
  • FIG. 20G shows pulmonary inflammation histopathology scores of tissues harvested at days 4 and 7.
  • FIG. 20H shows viral RNA in BAL.
  • FIG. 201 shows viral RNA in lung.
  • FIG. 20J shows sub-genomic RNA in lung. Outliers are indicated with
  • FIGs. 21A to 21E show AAV-Delivered Decoy Expression in the Airway of NHP.
  • FIG. 21 A shows pooled capsid comparison using mRNA barcoding.
  • mRNA barcode enrichment scores (average across 4 barcodes + SD) in (FIG. 21 A nasopharynx) and (FIG. 2 IB) septum.
  • FIG. 21C shows AAVrh91 transduction profile in airway barcode study.
  • FIG. 21E shows correlation between decoy protein by MS and spike binding by ELISA. Data includes d7 and dl4 samples from (FIG. 21D) plus NLF of
  • FIGs. 22A to 22E show characterization of initial ACE2 decoy construct.
  • FIG. 22A shows results for construct expressed in HEK293 cells and detected in supernatant using a sandwich ELISA to ACE2 for constructs without an Fc domain.
  • FIG. 22B shows result of an ELISA with SARS-CoV-2 spike protein as a capture antigen and an anti-human IgG polyclonal antibody used for detection of hAce2 with Fc fusion proteins expressed in HEK293 cells.
  • FIG. 22C shows the purified ACE2-NN-Fc4 protein was titrated against Wuhan CoV2 pseudotyped lentivirus bearing a luciferase reporter.
  • FIG. 22D shows the candidate construct (ACE2-NN-Fc4) was packaged in an AAV vector (hu68 capsid) and administered IN to WT mice. Seven days after administration, BAL was collected for measurement of fransgene expression using an ELISA with SARS- CoV-2 spike protein as a capture antigen to confirm that the decoy receptor expressed in vivo was functional. BAL from similar experiments was 6-fold diluted from the ASF as determined by comparison of BAL and serum urea. Thus, we determined that ASF concentrations of the decoy were likely below 2 ug/ml.
  • FIG. 22D shows the candidate construct (ACE2-NN-Fc4) was packaged in an AAV vector (hu68 capsid) and administered IN to WT mice. Seven days after administration, BAL was collected for measurement of fransgene expression using an ELISA with SARS- CoV-2 spike protein as a capture antigen to confirm that the decoy receptor expressed in vivo was functional. BAL from similar experiments was 6-fold diluted from the ASF
  • FIG. 22E shows two NHPs (IDs 258 and 396) received 9 x 10 12 GC of an AAVhu68 vector expressing a soluble ACE2-NN-Fc fusion protein via the MAD.
  • Nasal lavage samples were collected weekly after vector administration and concentrated 10-fold for analysis.
  • the concentration of the decoy receptor in NLF was measured by MS.
  • Urea measurements in similar experiments indicate that 10X nasal lavage is ⁇ 8-fold diluted from ASF. We therefore determined that ASF concentrations of the decoy were less than 100 ng/ml.
  • FIGs. 23A to 23D show design and selection of primary and secondary yeast display libraries.
  • FIG. 23A shows the designed two primary yeast display libraries: 1) the whole ACE2 gene fragment was mutagenized (Whole) and 2) the mutagenesis was limited to only the first 96 amino acids (NC) to concentrate the mutagenesis on the region most likely to impact RBD binding. The regions shaded gray were subjected to error prone PCR to introduce mutations.
  • FIG. 23B shows results of deep sequencing of yeast display plasmids extracted from the final round of sorting for the Whole and NC libraries. The fractional rate of mutation at each position in 18-615 of ACE2 is plotted. Improving mutations occurred mostly in the first 96 amino acids regardless of the input library.
  • FIG. 23C shows a detailed plot of the mutational frequencies in whole and NC library final round sorts for residues 18-100. The libraries yielded many of the same mutants this region with improved binding activity.
  • FIG. 23D Schematic representation of secondary library design.
  • FIG. 24A shows the parallel paths to the generation of affinity matured ACE2 decoy, selection and screening of primary display hits, and mutations from a secondary round of yeast display sorting.
  • FIG. 24B shows estimate IC50 (ng/mL) for Expression titer relative to Ace2- WT-Fc4 (for primary (1°), secondary (2°) digital, and secondary (2°) molecular).
  • FIG. 24C shows reporter virus activity for Ace-IgG variants GTP14-IgG (Variantl) and GTP14HL-IgG (Variant2)
  • FIGs. 25A and 25B show CoV2 challenge study in mice.
  • FIG. 25A shows a challenge study in mice plotted as average weight loss (percentage) in males.
  • FIG. 25B shows a challenge study in mice plotted as average weight loss (percentage) in females.
  • FIGs. 26A to 261 show AAV capsid selection for NHP IN delivery.
  • FIG. 26A shows mRNA barcode enrichment in airway tissues of Maxillary sinus for a mixture of 9 barcoded serotypes delivered IN at 2.7 x 10 11 GC each.
  • FIG. 26B shows mRNA barcode enrichment in airway tissues of Trachea prox. for a mixture of 9 barcoded serotypes delivered IN at 2.7 x 10 11 GC each.
  • FIG. 26C shows mRNA barcode enrichment in airway tissues of trachea mid. for a mixture of 9 barcoded serotypes delivered IN at 2.7 x 10 11 GC each.
  • FIG. 26D shows mRNA barcode enrichment in airway tissues of trachea dist.
  • FIG. 26E shows mRNA barcode enrichment in airway tissues of mainstem bronchi for a mixture of 9 barcoded serotypes delivered IN at 2.7 x 10 11 GC each.
  • FIG. 26F shows mRNA barcode enrichment in airway tissues of lung upper caudal for a mixture of 9 barcoded serotypes delivered IN at 2.7 x 10 11 GC each.
  • FIG. 26G shows mRNA barcode enrichment in airway tissues of “lung lower L” for a mixture of 9 barcoded serotypes delivered IN at 2.7 x 10 11 GC each.
  • FIG. 26H shows the biodistribution of vector genomes in airway tissues (ethmoid, maxilliary, cavity septum, nasopharynx) 28 days after dosing.
  • FIG. 261 shows the biodistribution of vector genomes in various airway tissues including lower airway tissues at 28 days after dosing.
  • Four NHP were IN dosed with AAVrh91 or AAVhu68 vectors encoding decoy transgenes at 5 x 10 12 GC.
  • AAVrh91 achieved higher gene transfer in upper airway tissues, particularly in the maxillary sinuses and cavity septum. Gene transfer in lower airway tissues was more variable.
  • FIG. 27 shows neutralization data as measured across different sampled pools of the purified engineered hAce2 decoy Fcl (IgG 1 Fc) fusion proteins, and compared with the engineered hAce2 decoy Fc4 (IgG4 Fc) fusion protein.
  • FIG. 28A shows a plot of neutralization data against Wuhan CoV2, as measured across different sampled pools of the purified engineered hAce2 decoy Fcl (IgG 1 Fc) fusion proteins, and compared with the engineered hAce2 decoy Fc4 (IgG4 Fc) fusion protein.
  • FIG. 28B shows a plot of neutralization data against Delta CoV2 (variant), as measured across different sampled pools of the purified engineered hAce2 decoy Fcl (IgG 1 Fc) fusion proteins, and compared with the engineered hAce2 decoy Fc4 (IgG4 Fc) fusion protein.
  • FIG. 29A shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected serum samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc decoy fusion at doses of 3 mg/kg, 10 mg/kg, and 30 mg/kg.
  • FIG. 29B shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected NJF samples, post intraperitoneal administration of hAceMR27-Variant-IgGl Fc decoy fusion at doses of 3 mg/kg, 10 mg/kg, and 30 mg/kg.
  • FIG. 29C shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected serum samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc or GTP14HL-Fcl decoy fusion from various pools of protein samples, when administered at doses of 3 mg/kg.
  • FIG. 29D shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected NLF samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc or GTP14HL-Fcl decoy fusion from various pools of protein samples, when administered at doses of 3 mg/kg.
  • FIG. 30A shows decoy protein levels as determined by mass spectrometry analysis (ng/mL) post administration of either AAVhu68.GTP14HL or AAVrh91.GTP14HL.
  • FIG. 3 OB shows decoy protein levels as determined by mass spectrometry analysis (ng/mL) post administration of either AAVhu68.GTP14HL or AAVrh91.GTP14HL, and plotted as urea-corrected decoy protein concentration (ng/mL).
  • FIG. 31 shows exemplary pool hAce2 decoy protein expression levels (mg/L) in a 10 day process in a 10L Stirred Tank Controlled Bioreactor. These results show about 1.2 g/L of protein purified using a one or two step process with higher percent monomer purity and good recovery (-90%).
  • FIG. 32A shows serum decoy levels in NHP following administration by IV infusion of hAce2 decoy protein (MR27HL-Fcl) at a dose of 30 mg/kg (broken x axis for showing early timepoints (Hours)).
  • FIG. 32B shows potency of decoy in serum samples of NHP following administration by IV infusion of hAce2 decoy protein (MR27HL-Fcl) at a dose of 30 mg/kg, plotted as a reporter virus activity over decoy MS concentration (ng/ml) at 1 hour, 7 hours, and 24 hours post administration.
  • hAce2 decoy protein MR27HL-Fcl
  • FIG. 33 shows viral neutralization assay using lentiviruses pseudotyped with the ancestral (Wuhan Hui) or Omicron variant spike protein.
  • FIG. 34 shows correlation of protein decoy expression in collected NLF following AAVrh91.hAce2GTP14HL-IgG4 administration at various doses (1.02 x 10 11 , 3.40 x 10 12 , and 1.02 x 10 12 GC).
  • FIG. 35 shows hAce2 decoy protein levels in NLF, plotted as ng/mL, measured in NHPs at day 30 and day 120 following AAVrh91.hAce2GTP14HL-IgG4 administration at various doses (1.02 x 10 11 , 3.40 x 10 12 , and 1.02 x 10 12 GC) in NHPs.
  • FIG. 36 shows Engineered ACE2 decoys bind diverse ACE2-dependent CoVs, plotted as decoy fluorescence for each specified CoV strain.
  • FIG. 37A shows a schematic overview of the fluorescence binding assay.
  • FIG. 37B shows binding of the CDY14HL-Fc4 with RBD, plotted as bound decoy fluorescence over decoy concentration in nM.
  • RBDs from SARS-CoV-2 variants were individually expressed in budding yeast as fusion proteins that are trafficked to the external cell wall. Decoy or unengineered ACE2 are incubated with the yeast, stained with antibodies, and the relative level of decoy binding is assessed by flow cytometry.
  • FIG. 38A shows phylogenetic tree of coronavirus RBDs tested in this study.
  • FIG. 38B shows a schematic overview of the fluorescence binding assay comprising budding yeast displaying RBD and ACE2 mFc.
  • FIG. 38C shows relative levels of decoy binding to diverse RBDs under several conditions as assessed by the yeast-display system. Similar to SARS-CoV2, all sarbecovirus RBDs retain >30% binding in the competition assay.
  • a mutant soluble Ace2 protein is provided, as are vector systems for expressing the protein in vitro or in vivo depending on whether a gene therapy or protein-based therapeutic approach is desired, or a combination approach.
  • the mutant soluble Ace2 proteins provided herein are believed to function as a decoy receptor for viral pathogens using the Ace2 receptor, including SARS CoV-2, the virus which causes COVID- 19 and SARS-CoVl.
  • a recombinant viral vector is replication-incompetent and comprises an exogenous viral genome which comprises coding sequences for at least one mutant hACE2 (or hAce2) soluble protein.
  • the compositions and methods provided herein have advantages over broadly neutralizing antibodies (bNAbs), as viral escape mutants that evade neutralization by the soluble receptor lose infectivity due to reduced affinity for the endogenous receptor.
  • the compositions and methods provide for use of a soluble decoy also avoids potential antibody -mediated enhancement of infection at nonneutralizing titers, which has been reported for SARS CoV-1 antibodies.
  • compositions provided herein are designed for intravenous delivery of a recombinant protein of an engineered soluble hACE2 decoy fusion protein.
  • the compositions provided herein are designed for mucosal expression in order to provide lower systemic exposure than occurs following intravenous or intramuscular delivery, thereby further enhancing the safety of an AAV -mediated approach.
  • a protein is provided herein (e.g., hAce2-Variant2-Fc4 (CDY14HL-Fc4; GTP14HL-IgG4 or GTP14HL-Fc4)), which has high binding and neutralizing activity against a full range of SARS-CoV-2 variants. Unexpectedly, this protein has also demonstrated equally potent binding and neutralization against other betacoronaviruses, including SARS-CoV-1, which was responsible for the 2003 SARS pandemic. This may be formulated for delivery in protein form or for expression in vivo from a suitable nucleic acid molecule (e.g., a viral vector such as AAV).
  • a suitable nucleic acid molecule e.g., a viral vector such as AAV.
  • a mutant human Ace2 (hAce2) soluble decoy protein which comprises a mutated amino acid sequence of SEQ ID NO: 81 or a hAce2 decoy protein at least 95% identical to SEQ ID NO: 81, wherein the mutant hAce2 soluble protein has one or more of the following amino acid residues, based on numbering of SEQ ID NO: 81, (a) R or M at residue 14 (K changed to R or M); (b) V or K at residue (18) (E changed to V or K); (c) P at residue (22) (L changed to P); (d) R at residue (25) (Q changes to R); (e) A at residue (30) (S changed to A); (f) A at residue (42) (V changed to A); (g) I or F at residue (62) (L changes to I or F); (h) D at residue (73) (N changed to D); (i) P at residue (74) (L changed to P); (j) Y at residue (313) (N changed
  • a mutant human Ace2 (hAce2) soluble decoy protein wherein the mutant hAce2 soluble protein has one or more of the following amino acid residues, based on numbering of SEQ ID NO: 81, (i) A at residue 10 (T changed to A), (ii) G at residue 26 (S changed to G), (ii) G or D at residue 32 (N changed to O or D), (iv) D at residue 44 (N changed to D), (v) K at residue 58 (E changed to K), (vi) R at residue 59 (Q changed to R), (vii) F at residue 62 (L changed to F), (vii) R at residue 64 (Q changed to R), (ix) D or S at residue 73 (N changed to D or S), (x) P at residue 74 (L changed to P), (xi) A at residue 75 (T changed to A), (xii) Y at residue 313 (N changed to Y), (xiii) H at residue (328) (H changed to
  • the mutant hAce2 soluble decoy protein comprises two or more of the substitutions of (a), (b), (g), (i) and (j). In certain embodiments, the mutant hAce2 soluble decoy protein comprises three or more of the substitutions of (a), (b), (g), (i) and (j), wherein (a) is a Met and/or (g) is a Phe.
  • the mutant hAce2 soluble decoy protein comprises an amino acid sequence selected from: (a) all of the substitutions of (a), (b), and (j); (b) all of the substitutions of (a), (b), (g), and (j); (c) an amino acid sequence having the residues of (a), (b), (g), (i) and/or (j); (Variant 4); (d) an amino acid sequence having the residues of (a), (b), (e), (g), (i) and/or (j); (Variant 1); (e) an amino acid sequence having the residues of (a), (b), (e), (g), (j), (i) and/or (k); (Variant 2); (f) an amino acid sequence having the residues of (a), (b), (c), (f), (g), (h), and/or (j) (Variant 3); (g) amino acid sequence having the residues of (a), (b), (d), (i), and/or (j)
  • a mutant hAce2 soluble decoy protein may be a fusion protein which comprises a mutated amino acid sequence of SEQ ID NO: 81 or a hAce2 decoy protein at least 95% identical to SEQ ID NO: 81 fused, directly or via a linker, to an immunoglobulin Fc region.
  • the Fc region is a human IgG Fc or a human IgM Fc.
  • the Fc region is a human IgG2 Fc or a human IgG4 Fc.
  • a mutant hAce2 soluble decoy fusion protein is (a) hAce2- Variantl-IgG4 fusion (SEQ ID NO: 35), (b) hAce2-Variant2-IgG4 fusion (SEQ ID NO: 37), (c) hAce2-Variant3-IgG4 fusion (SEQ ID NO: 39), (d) hAce2-Variant4-IgG4 fusion (SEQ ID NO: 41), (e) hAce2-Variant5-IgG4 fusion (SEQ ID NO: 74 linked to SEQ ID NO: 77), (f) hAce2-Variant6-IgG4 fusion (SEQ ID NO: 75 linked to SEQ ID NO: 77), or (g) or an amino acid sequence at least 95% identical to any of claims (a) to (f).
  • a mutant hAce2 soluble decoy fusion protein is (a) hAce2-Variant2-IgGl fusion (SEQ ID NO: 109), (b) hAce2-Variant2-IgGl fusion with “GS” linker (SEQ ID NO: 113), (c) hAce2-MR27- Variant-IgGl fusion (SEQ ID NO: 111), (d) hAce2-MR27-Variant2-IgGl fusion with “GS” linker (SEQ ID NO: 115), or (e) an amino acid sequence at least 95% identical to any one of (a) to (d).
  • Compositions and therapeutic regimens may contain a single mutant hAce2 soluble protein, fusion protein, or a mixture (cocktail) of these, optionally further in combination with other components.
  • a nucleic acid molecule encoding a soluble Ace2 protein operably linked to regulatory sequences which direct expression of the protein thereof in a cell wherein the soluble Ace2 protein comprises: (a) a fusion protein comprising a signal peptide and the mutant Ace2 soluble protein; or a fusion protein comprising a signal peptide, the mutant Ace2 soluble decoy and an Fc tail.
  • the vector e.g., a plasmid
  • the signal peptide may be a non-human or non-mammalian signal peptide suited for the production cell type.
  • the vector is selected for delivery to a patient.
  • the signal peptide is suitably a human signal peptide.
  • a vector having an expression cassette comprising a nucleic acid molecule is provided.
  • the hAce2 soluble decoy transgene encoding the hAce2 soluble decoy protein is: (a) hAce2-Variantl (SEQ ID NO: 27), (b) hAce2- Variant2 (SEQ ID NO: 29), (c) hAce2-Variant3 (SEQ ID NO: 31), (d) hAce2-Variant4 (SEQ ID NO: 33), (e) hAce2-Variant5 (SEQ ID NO: 74), (f) hAce2-Variant6 (SEQ ID NO: 75), (g) hAce2-Variantl-IgG4 fusion (SEQ ID NO: 35), (h) hAce2-Variant
  • the hAce2 soluble decoy transgene encoding the hAce2 soluble decoy protein is selected from SEQ ID NO: 93, 95, 97, and 99. In certain embodiments, the hAce2 soluble decoy transgene encoding the hAce2 soluble decoy protein is selected from SEQ ID NO: 108, 110, 112, and 114. In certain embodiments, the hAce2 soluble decoy transgene encoding the hAce2 soluble decoy protein has an amino acid sequence selected from SEQ ID NOs: 62 to 71.
  • the vector is used for protein expression in a producer host cell (e.g., a plasmid), which may be a mammalian cell, a bacterial cell, a yeast cell, or an insect cell.
  • a producer host cell e.g., a plasmid
  • the cells may be cell lines in cell suspension or another type of cell culture.
  • the vector is a viral vector for delivery of the transgene and expression of the hAce2 in vivo.
  • a viral vector is used for delivery of the mutant soluble Ace2- protein to the patient.
  • Suitable viral vectors may include, e.g., adenoviruses, vaccinia, lentivirus, or parvoviruses.
  • the vector genome comprises a nucleic acid sequence encoding a mutant hAce2 soluble decoy protein under the control of regulatory control sequences which direct expression of the hAce2 soluble decoy protein, wherein the mutant hAce2 soluble decoy protein comprises a signal peptide and a mutated amino acid sequence of SEQ ID NO: 81 or a hAce2 decoy protein at least 95% identical to SEQ ID NO: 81, wherein the mutant hAce2 soluble protein has one or more of the following amino acid residues, based on numbering of SEQ ID NO: 81, (a) R or M at residue 14 (K changed to R or M); (b) V or K at residue (18) (E changed to V or K); (c) P at residue (22) (L changed to P); (d) R at residue (25) (Q changes to R); (e) A at residue (30) (S changed to A); (f) A at residue (42) (V changed to A); (g) I or F at
  • the mutant hAce2 protein comprises two or more of the substitutions of (a), (b), (f), (h) and (i). In other embodiments, the mutant hAce2 protein comprises three or more of the substitutions of (a), (b), (f), (h) and (i), wherein (a) is a Met and/or (f) is a Phe.
  • the mutant hAce2 protein comprises an amino acid sequence selected from: (a) all of the substitutions of (a), (b), and (j); (b) all of the substitutions of (a), (b), (g), and (j); (c) an amino acid sequence having the residues of (a), (b), (g), (i) and/or (j); (Variant 4); (d) an amino acid sequence having the residues of (a), (b), (e), (g), (i) and/or (j); (Variant 1); (e) an amino acid sequence having the residues of (a), (b), (e), (g), (j), (i) and/or (k); (Variant 2); (f) an amino acid sequence having the residues of (a), (b), (c), (f), (g), (h), and/or (j) (Variant 3); (g) amino acid sequence having the residues of (a), (b), (d), (i), and/or (j) (Variant 5
  • the hAce2 soluble decoy is a hAce2 soluble decoy fusion protein further comprising an immunoglobulin Fc region, which may be a human IgG Fc or an IgM Fc.
  • the signal peptide is a human signal peptide. In certain embodiments, the signal peptide is the native Ace2 signal.
  • the transgene encodes a mutant soluble Ace2 protein which comprises an amino acid of: (a) hAce2-Variantl (SEQ ID NO: 10), (b) hAce2-Variant2 (SEQ ID NO: 12), (c) hAce2-Variant3 (SEQ ID NO: 14), (d) hAce2-Variant4 (SEQ ID NO: 16), (e) hAce2-Variant5 (SEQ ID NO: 72), (f) hAce2- Variant6 (SEQ ID NO: 73), (g) hAce2-Variantl-IgG4 fusion (SEQ ID NO: 2), (h) hAce2- Variant2-IgG4 fusion (SEQ ID NO: 4), (i) hAce2-Variant3-IgG4 fusion (SEQ ID NO: 6), (j) hAce2-Variant4-IgG4 fusion (SEQ ID NO: 8), (k)
  • the nucleic acid sequence encoding the hAce2 soluble decoy protein is: (a) SEQ ID NO: 9 or a sequence at least about 90% identical thereto encoding hAce2- Variantl (SEQ ID NO: 10), (b) SEQ ID NO: 11 or a sequence at least about 90% identical thereto encoding hAce2-Variant2 (SEQ ID NO: 12), (c) SEQ ID NO: 13 or a sequence at least about 90% identical thereto encoding hAce2-Variant3 (SEQ ID NO: 14), (d) SEQ ID NO: 15 or a sequence at least about 90% identical thereto encoding hAce2-Variant4 (SEQ ID NO: 16), (e) SEQ ID NO: 1 or a sequence at least about 90% identical thereto encoding hAce2- Variantl-IgG4 fusion (SEQ ID NO: 2), (f) SEQ ID NO: 3 or a sequence at least about 90% identical thereto encoding hA
  • the capsid is an AAV capsid which transduce lung and/or epithelial cells.
  • the AAV capsid is an AAV9 capsid, a AAVhu68 capsid, an AAV5 capsid, an AAV6 capsid, an AAV6.2 capsid, or an AAVrh91 capsid.
  • a composition comprises one or more of a mutant hAce2 soluble decoy protein, a nucleic acid molecule, a vector, or an rAAV as described herein, optionally in combination with each other.
  • the composition is formulated for aerosol administration.
  • the composition is formulated for intravenous administration.
  • a producer host cell comprising a nucleic acid molecule encoding the protein for expression in vitro and for purification for delivery of a mutant soluble Ace2-protein-based therapeutic.
  • a composition comprising a population of mutant hAce2 soluble protein decoy produced using a producer host cell.
  • a composition comprising a population of mutant soluble Ace2-proteins may include up to 5% variation from the sequences provided herein in view of post-translational modifications such as, e.g., glycosylation, oxidation and deamidation. In certain embodiments, there is 0.5% to 5% variation, in other embodiments, there is about 1%, about 2%, about 3%, or about 4% variation.
  • no detectable variation is observed.
  • post-translation modification may be detected by assessed by any suitable technique including, e.g., chromatographic and/or mass spectrometric analysis, or peptide mapping. These detection methods are not a limitation on the present invention.
  • a pharmaceutical composition which comprises at least one mutant hAce2 soluble decoy protein as provided herein and one or more of any of: a pharmaceutically acceptable diluent, a suspending agent, a preservative, and/or a surfactant.
  • a pharmaceutical composition which comprises the nucleic acid molecule and one or more of any of: a pharmaceutically acceptable diluent, a suspending agent, a preservative, and/or a surfactant.
  • the pharmaceutical composition is formulated for intranasal administration.
  • the pharmaceutical composition is formulated for intrapulmonary administration.
  • the pharmaceutical composition is formulated for intravenous administration.
  • the novel soluble Ace2 proteins provided herein are believed to function as decoys for viruses which utilize an Ace 2 receptor as a mechanism for cellular update.
  • the soluble Ace2 proteins significantly reduce and/or prevent viral attachment to the native Ace2 receptor in cells.
  • Human respiratory coronaviruses have been associated with sudden acute respiratory syndrome (SARS), including SARS CoV-2 and CoVl, and are putatively associated with the common cold, non-A, B or C hepatitis.
  • SARS sudden acute respiratory syndrome
  • pre-emergent coronaviruses such as WIV- CoV, are present in reservoir animal populations, use Ace2 as a receptor, and have a recognized potential to one day cross into humans and cause disease.
  • the coronavirus family also includes a number of non-human viruses such as infectious bronchitis virus (poultry), porcine transmissible gastroenteric virus (pig), porcine hemagglutinatin encephalomyelitis virus (pig), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), canine coronavirus (dog). Still other infectious viruses utilizing the Ace2 receptor may be treated using the protein and viral vectors described herein.
  • these proteins and vector constructs are useful in humans. Additionally or in alternative embodiments, these proteins and vector constructs are useful in non-human mammals, including, e.g., members of the dog and cat families.
  • novel sequences encoding soluble hACE2 proteins and fusion proteins thereof which comprise an Fc and/or a hinge region designed to increase half-life of the soluble proteins.
  • the soluble hACE2 proteins comprise of a leader sequence, (with reference to the residue numbering of NCBI Reference ID: NP_068576; SEQ ID NO: 25) which comprises the extracellular/zinc binding domain (HEMGH), and a truncation in the C- terminus of the hACE2 protein, including the transmembrane domain (aa 741 to 760) and a C- terminal fragment (amino acids 761 to 805).
  • the protein comprises at least amino acid 18 to 615 of the hACE2 protein of SEQ ID NO: 25 (also referenced to as amino acids 1 to 588 of SEQ ID NO: 42, or amino acids of SEQ ID NO: 81).
  • the protein comprises at least amino acid sequence of SEQ ID NO: 107.
  • the native leader signal (aa 1-17) may be present to provide a construct comprising amino acids 1 to 615 of the native sequence.
  • the protein comprises at least amino acid 18 to 740 of the hACE2 protein of SEQ ID NO: 25 (also referenced to as amino acids 1 to 725 of SEQ ID NO: 42, or amino acids of SEQ ID NO: 83).
  • the protein comprises at least amino acid 1 to 615 of the hACE2 protein of SEQ ID NO: 25 (also referenced to amino acids of SEQ ID NO: 80). In certain embodiments, the protein comprises at least amino acid 1 to 740 of the hACE2 protein of SEQ ID NO: 25 (also referenced to amino acids of SEQ ID NO: 82). In certain embodiments, the protein comprises at least amino acid sequence of SEQ ID NO: 105.
  • an internal deletion mutant may be selected which deletes the transmembrane domain entirely or functionally (positions 741 to 760). In certain embodiments, the native zinc binding domain is retained. In other embodiments, this domain is rendered non-catalytic by mutating the histidine (H) residues.
  • Such mutations may be at one or both of the H residues.
  • a suitable mutation may be from H to asparagine (N).
  • other suitable amino acids may be substituted in order to render this domain non-functional.
  • a suitable alternative mutation in a separate site may be H345L, which renders Ace2 inactive.
  • modified hACE2 soluble decoy proteins and/or mutant hACE2 soluble decoy fusion proteins contain a signal sequence encoding a signal peptide which directs the expressed protein within the host cell, wherein the signal peptide (also may be referred to as a leader peptide) is at the amino terminus of the protein.
  • the signal sequence may encode a native hACE2 leader or may be from a source exogenous to the hACE2.
  • a sequence encoding a human interleukin-2 signal peptide, or a thrombin signal peptide may be selected.
  • another suitable signal peptide e.g., human or a viral, may be selected.
  • the signal peptide may be a nonhuman or non-mammalian signal peptide suited for the production cell type. In certain other embodiments, the signal peptide is suitably a human signal peptide. In certain embodiments, modified hACE2 soluble decoy proteins and/or modified hACE2 soluble decoy fusion proteins do not contain a signal sequence.
  • a linking sequence may be present between a hACE2 soluble decoy proteins and the Fc region and/or hinge region.
  • the linking sequence may be immediately adjacent to the C-terminus of a hACE2 protein and N-terminus of a second domain, being used to separate two, three or four of the domains or regions. Any suitable sequence of about 1 to 20 amino acids in length may be selected, but it is preferably 3 to 18 amino acids, or about 5 to about 12 amino acids in length. Multiple linking sequences may be present in a single encoded protein sequence, and these may be independently selected.
  • the vector genome may be designed to contain an F2A or IRES between the coding sequences for the two proteins.
  • the proteins further comprise an Fc tail, i.e., hACE2 soluble decoy fusion proteins.
  • the hACE2 soluble decoy proteins are linked to an Fc fragment of an antibody, preferably a human antibody, such as the Fc fragment of a human IgG antibody, e.g., an IgGl (Fcl), IgG2 (Fc2), IgG3 (Fc3), or IgG4 (Fc4), IgA or a human IgM antibody.
  • a human antibody e.g., an IgGl (Fcl), IgG2 (Fc2), IgG3 (Fc3), or IgG4 (Fc4), IgA or a human IgM antibody.
  • the hACE2 soluble decoy protein may be genetically fused to an Fc fragment, either directly or using a linker. See also, US Patent Provisional Application No. 63/136,497, filed January 12, 2021, and US Provisional Patent Application No. 63/137,519, filed January 14, 2021, which are incorporated herein by reference.
  • the binding molecules are linked to the Fc fragment by a linking sequence comprising from 1 to 100 amino acids, preferably from 1 to 60 amino acids, or from 10 to 60 amino acids. Examples of linkers include, but are not limited to, the linking sequences listed above.
  • an immunoglobulin domain is genetically fused to the C-terminus of an Fc fragment.
  • an immunoglobulin domain or fusion protein is fused to both the N- and the C-terminus of an Fc fragment. See, e.g., WO 2016/124768, which is incorporated by reference herein.
  • the mutant hAce2 soluble decoy is connected to IgG4 Fc (also referred to as Fc4) domain via a flexible “GSG” linker, wherein the flexible “GSG” linker is selected from at least one or more repeats of “GSG”, yielding to total length of the linker of about 3 to about 125 amino acids.
  • the mutant hAce2 soluble decoy is directly connected to IgG4 FC domain.
  • the mutant hAce2 soluble decoy is connected to IgGl Fc (also referred to as Fcl or IgGl) domain via a flexible “GSG” linker, wherein the flexible “GSG” linker is selected from at least one or more repeats of “GSG”, yielding to total length of the linker of about 3 to about 125 amino acids.
  • the mutant hAce2 soluble decoy is directly connected to IgGl FC domain.
  • the mutant hAce2 soluble decoy is connected to IgGl Fc (also referred to as Fcl or IgGl) domain via a “GS” linker.
  • the mutant hAce2 soluble decoy is connected to IgGl Fc (also referred to as Fcl or IgGl) domain via a “EPKSC” linker (SEQ ID NO: 101).
  • the mutant hAce2 soluble compromises amino acid sequence with length of 615 amino acids, based on the numbering of amino acids 1 to 615 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 80). In some embodiments, the mutant hAce2 soluble compromises amino acid sequence with length of 588 amino acids, based on the numbering of amino acids 18 to 615 SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 81). In some embodiments, the mutant hAce2 soluble decoy comprises amino acid sequence with length 740 amino acids, based on amino acids 1 to 740 of SEQ ID NO SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 82).
  • the mutant hAce2 soluble decoy comprises amino acid sequence with length of 723 amino acids, based in the numbering or amino acids 18 to 740 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 83).
  • the mutant hAce2 soluble decoy with length of 615 amino acids is directly connected to IgG4 Fc domain.
  • the mutant hAce2 soluble decoy with length of 615 amino acids is connected to IgG4 Fc domain via a flexible linker, as described herein.
  • the mutant hAce2 soluble decoy with length of 588 amino acids is directly connected to IgG4 Fc domain.
  • the mutant hAce2 soluble decoy with length of 588 amino acids is connected to IgG4 Fc domain via a flexible linker as described herein.
  • the mutant hAce2 soluble decoy with length of 740 amino acids is directly connected to IgG4 Fc domain.
  • the mutant hAce2 soluble decoy with length of 723 amino acids is directly connected to IgG4 Fc domain.
  • the mutant hAce2 soluble decoy with length of 615 amino acids is directly connected to IgGl Fc domain. In certain embodiments, the mutant hAce2 soluble decoy with length of 615 amino acids is connected to IgGl Fc domain via a flexible linker, as described herein. In certain embodiments, the mutant hAce2 soluble decoy with length of 588 amino acids is directly connected to IgGl Fc domain. In certain embodiments, the mutant hAce2 soluble decoy with length of 588 amino acids is connected to IgGl Fc domain via a flexible linker as described herein.
  • the mutant hAce2 soluble decoy with length of 740 amino acids is directly connected to IgGl Fc domain. In certain embodiments, the mutant hAce2 soluble decoy with length of 723 amino acids is directly connected to IgGl Fc domain.
  • compositions which are useful for intravenous delivery of a mutant hACE2 soluble decoy and/or hAce2 soluble decoy fusion proteins for therapeutic or prophylactic purpose.
  • the mutant hAce2 may be linked via a hinge region to an Fcl or Fc4 domain encoding an hAce2_Fcl or hAce2_Fc4 (hAce2-IgGl, hAce2- IgG4 or hAce2-Variant 1/2/3 /4-IgG) soluble decoy fusion protein (hAce2 fusion).
  • the mutant hAce2 may be linked to an IgM domain.
  • the mutant hAce2 may be linked to an IgA domain. In some embodiments, the mutant hAce2 contains two inactivating mutations H374N and H378N. In other embodiments, the mutant hAce2 may comprise at least one inactivating mutation of H345L.
  • the mutant hAce2 soluble decoy comprise one or more of the mutations: R or M at residue 14 (K changed to R or M), V or K at residue (18) (E changed to V or K), P at residue (22) (L changed to P), R at residue 25 (Q changed to A), A at residue (30) (S changed to A), A at residue (42) (V changed to A), I or F at residue (62) (L changes to I or F), D at residue (73) (N changed to D), P at residue (74) (L changed to P), Y at residue (313) (N changed to Y), and/or H at residue (328) (H changed to L), wherein the numbering is based on SEQ ID NO: 81.
  • the mutant hAce2 soluble decoy comprise one or more of the mutations: A at residue 10 (T changed to A), G at residue 26 (S changed to G), G or D at residue 32 (N changed to O or d), D at residue 44 (N changed to D), K at residue 58 (E changed to K), R at residue 59 (Q changed to R), F at residue 62 (L changed to F), R at residue 64 (Q changed to R), D or S at residue 73 (N changed to D or S), P at residue 74 (L changed to P), A at residue 75 (T changed to A), Y at residue 313 (N changed to Y), and/or H at residue (328) (H changed to L), wherein the numbering is based on SEQ ID NO: 81.
  • the mutated hAce2 soluble decoy comprise at least one or more of above-mentioned mutations and is linked to an IgGl, an IgG4, IgA or an IgM Fc domain encoding mutant hAce2 soluble decoy fusion protein.
  • compositions which are useful for intracellular delivery and expression of a soluble form of the mutated soluble ACE2 viral constructs (hAce2) for therapeutic or prophylactic purpose.
  • the modified hAce2 may be linked via a hinge region to an Fcl or Fc4 domain encoding an hAce2_Fcl or hAce2_Fc4 fusion protein (hAce2 fusion).
  • hAce2 contains two inactivating mutations H374N and H378N encoding hAce2_NN.
  • the mutated hAce2_NN is linked to a IgGl, IgG4, IgA or IgM Fc domain.
  • the mutated hAce2 soluble decoy used for intracellular delivery comprises at least one or more mutations as described herein.
  • the mutant hAce2 soluble decoy and/or the mutant hAce2 soluble decoy fusion protein can be engineered into a suitable expression cassette in which the coding sequence for the amino acid sequence is operably linked to a regulatory sequence which direct expression thereof.
  • the expression cassette may optionally contain varied leader sequences preceding a mature protein or protein fragment.
  • hAce2 DNA sequence contains a native leader sequence encoding a native signal peptide.
  • hAce2 DNA sequence contains an interleukin-2 (IL-2) encoding a IL-2 signal peptide.
  • hAce2 DNA sequence contains a thrombin leader sequence (Trb) encoding a Trb signal peptide.
  • the mutant hAce2 soluble decoy fusion protein comprises a native signal peptide, wherein the native signal peptide comprises amino acid residues of 1 to 17 of SEQ ID NO: 25 (or amino acid sequence of SEQ ID NO: SEQ ID NO: 76), the hAce2 soluble decoy comprises amino acid residues of 18 to 615 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 81), comprising one or more of mutations as described herein, and an Fc domain (i.e., IgG, IgA, or IgM).
  • native signal peptide comprises amino acid residues of 1 to 17 of SEQ ID NO: 25 (or amino acid sequence of SEQ ID NO: SEQ ID NO: 76)
  • the hAce2 soluble decoy comprises amino acid residues of 18 to 615 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 81), comprising one or more of mutations as described herein, and an Fc domain (i
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 1 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 2 (hAce2-Variantl-IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 3 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 4 (hAce2-Variant2-IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 5 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 6 (hAce2-Variant3-IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 7 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 8 (hAce2-Variant4- IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 93 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 94 (hAce2-Variant2-IgGl).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 97 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 98 (hAce2-Variant2-IgGl with “GS” linker).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 95 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 96 (hAce2-MR27HL-Variant-IgGl).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein comprising a native signal peptide is SEQ ID NO: 99 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 100 (hAce2-MR27HL-Variant-IgGl with “GS” linker).
  • the mutant hAce2 soluble decoy protein comprises a native signal peptide, wherein the native signal peptide comprises amino acid residues of 1 to 17 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 76), and the hAce2 soluble decoy comprises amino acid residues of 18 to 615 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 81), comprising one or more of listed mutations as described herein.
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 9 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 10 (hAce2-Variantl).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 11 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 12 (hAce2-Variant2).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 13 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 14 (hAce2-Variant3).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 15 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 16 (hAce2-Variant4).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 104 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 105 (hAce2- MR27HL-Variant).
  • the mutant hAce2 soluble decoy fusion protein comprises amino acid residues of 18 to 615 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 81), comprising one or more of mutations as described herein, and an Fc domain (i.e., IgG, IgA or IgM).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 34 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 35 (hAce2-Variantl- IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 36 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 37 (hAce2-Variant2-IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 38 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 39 (hAce2-Variant3-IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 40 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 41 (hAce2-Variant4-IgG4).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 108 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 109 (hAce2-Variant2-IgGl).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 110 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 111 (hAce2- MR27HL-Variant-IgGl).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 112 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 113 (hAce2- Variant2-IgGl comprising “GS” linker).
  • the nucleic acid sequence encoding the mutant hAce2 soluble decoy fusion protein is SEQ ID NO: 114 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 115 (hAce2-MR27HL-Variant-IgGl comprising “GS” linker).
  • the mutant hAce2 soluble decoy protein comprises amino acid residues of 18 to 615 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 81), comprising one or more of listed mutations as described herein.
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 27 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 28 (hAce2-Variantl).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 29 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 30 (hAce2-Variant2).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 31 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 32 (hAce2-Variant3).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 33 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 34 (hAce2-Variant4).
  • the nucleic acid sequence encoding mutant hAce2 soluble decoy protein comprising a native signal peptide is SEQ ID NO: 106 or a sequence at least about 85% identical thereto and encoding an amino acid sequence of SEQ ID NO: 107 (hAce2- MR27-Variant).
  • the mutant hAce2 soluble decoy fusion protein comprises an engineered hAce2 decoy protein connected via a linker to a IgGl Fc domain.
  • the hAce2 soluble decoy fusion protein is selected from: (a) hAce2-Variant2- IgGl fusion (aa 18 to 847 of SEQ ID NO: 94 or an amino acid sequence of SEQ ID NO: 109), (b) hAce2-Variant2-IgGl fusion with “GS” linker (aa 18 to 844 of SEQ ID NO: 98 or an amino acid sequence of SEQ ID NO: 113), (c) hAce2-MR27-Variant-IgGl fusion (aa 18 to 847 of SEQ ID NO: 96 or an amino acid sequence of SEQ ID NO: 111), (d) hAce2-MR27- Variant2-IgGl fusion with “GS” linker (aa 18 to 8
  • the mutant hAce2 soluble decoy protein comprises amino acid residues of 18 to 615 of SEQ ID NO: 25 (or SEQ ID NO: 81), comprising one or more of listed mutations as described in Table 1 below. In some embodiments, the mutant hAce2 soluble decoy protein comprises amino acid residues of 1 to 615 of SEQ ID NO: 25 (or an amino acid sequence of SEQ ID NO: 80), comprising one or more of listed mutations as described in Table 1 below.
  • an amino acid sequence for the mutant hAce2 soluble decoy is selected from SEQ ID NO: 52 to 61. In certain embodiments, an amino acid sequence for the mutant hAce2 soluble decoy (without signal or leader peptide) is selected from SEQ ID NO: 62 to 71. In certain embodiments, an amino acid sequence for the mutant hAce2 soluble decoy (with signal or leader peptide) is selected from SEQ ID NO: 10, 12, 14, 16, 72 or 73. In certain embodiments, an amino acid sequence for the mutant hAce2 soluble decoy (without signal or leader peptide) is selected from SEQ ID NO: 27, 29, 31, 33, 74, or 75.
  • the mutant hAce2 soluble decoy (with signal or leader peptide) is linked to the IgGl, IgG4, IgA, or IgM Fc domain. In certain embodiments, the mutant hAce2 soluble decoy (without signal or leader peptide) is linked to the IgGl, IgG4, IgA or IgM Fc domain.
  • hAce2-Variantl soluble decoy protein has an amino acid sequence of SEQ ID NO: 10, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 10 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 10 or an amino acid sequence of SEQ ID NO: 27), or a sequence at least about 95% identical thereto.
  • hAce2-Variant2 soluble decoy protein has an amino acid sequence of SEQ ID NO: 12, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 12 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 12 or an amino acid sequence of SEQ ID NO: 29), or a sequence at least about 95% identical thereto.
  • hAce2- Variants soluble decoy protein has an amino acid sequence of SEQ ID NO: 14, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 14 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 14 or an amino acid sequence of SEQ ID NO: 31), or a sequence at least about 95% identical thereto.
  • hAce2-Variant4 soluble decoy protein has an amino acid sequence of SEQ ID NO: 16, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 16 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 16 or an amino acid sequence of SEQ ID NO: 33), or a sequence at least about 95% identical thereto.
  • hAce2- Variants soluble decoy protein has an amino acid sequence of SEQ ID NO: 72, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 72 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 72 or an amino acid sequence of SEQ ID NO: 74), or a sequence at least about 95% identical thereto.
  • hAce2-Variant6 soluble decoy protein has an amino acid sequence of SEQ ID NO: 73, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 73 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 73 or an amino acid sequence of SEQ ID NO: 75), or a sequence at least about 95% identical thereto.
  • hAce2-MR27-Variant soluble decoy protein has an amino acid sequence of SEQ ID NO: 105, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 105 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 105 or an amino acid sequence of SEQ ID NO: 107), or a sequence at least about 95% identical thereto.
  • a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 105 or an amino acid sequence of SEQ ID NO: 76), and a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 105 or an amino acid sequence of SEQ ID NO: 107), or a sequence at least
  • hAce2-Variantl-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 2, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO:
  • soluble hAce2 protein fragment i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 35), and a human Fc immunoglobulin fragment IgG4 (aa 616 to 844 of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • hAce2-Variant2-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 4, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 76), a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 37), and a human Fc immunoglobulin fragment IgG4 (aa 616 to 844 of SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • a native signal peptide aa 1 to 17 of SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 76
  • a soluble hAce2 protein fragment i.e., soluble decoy (aa 18 to 615
  • hAce2-Variant3-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 6, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 76), a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 39), and a human Fc immunoglobulin fragment IgG4 (aa 616 to 844 of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • a native signal peptide aa 1 to 17 of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 76
  • a soluble hAce2 protein fragment i.e., soluble decoy (aa 18 to 6
  • hAce2-Variant4-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 8, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 8 or an amino acid sequence of SEQ ID NO: 76), a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 8 or an amino acid sequence of SEQ ID NO: 41), and a human Fc immunoglobulin fragment IgG4 (aa 616 to 844 of SEQ ID NO: 8 or an amino acid sequence of SEQ ID NO:
  • hAce2- Variant5-IgG4 soluble decoy fusion protein has an amino acid sequence, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy is an amino acid sequence of SEQ ID NO: 72), and is linked to a human Fc immunoglobulin fragment IgG4 (SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • hAce2-Variant6-IgG4 soluble decoy fusion protein has an amino acid sequence, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy is an amino acid sequence of SEQ ID NO: 73), and is linked to a human Fc immunoglobulin fragment IgG4 (SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • a soluble decoy fusion protein comprises a soluble hAce2 protein fragment selected from hAce2-Variantl, 2, 3, 4, 5, 6, or MR27HL, wherein the soluble decoy is further linked to a human Fc immunoglobulin fragment IgGl (SEQ ID NO: 103).
  • a hAce2-Variant2-IgGl soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 94, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 94 or an amino acid sequence of SEQ ID NO: 76), a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 94 or an amino acid sequence of SEQ ID NO: 29), a linker “EPKSC” (aa 616 to 620 of SEQ ID NO: 94 or an amino acid sequence if SEQ ID NO: 101), and a human Fc immunoglobulin fragment IgGl (aa 621 to 847 of SEQ ID NO: 94 or an amino acid sequence of SEQ ID NO: 103), or a sequence at least about 95% identical thereto.
  • a native signal peptide aa 1 to 17 of SEQ ID NO
  • a hAce2-Variant2-IgGl soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 98, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 98 or an amino acid sequence of SEQ ID NO: 76), a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 98 or an amino acid sequence of SEQ ID NO: 29), a linker “GS” (aa 616 to 617 of SEQ ID NO: 98), and a human Fc immunoglobulin fragment IgGl (aa 618 to 844 of SEQ ID NO: 98 or an amino acid sequence of SEQ ID NO: 103), or a sequence at least about 95% identical thereto.
  • a native signal peptide aa 1 to 17 of SEQ ID NO: 98 or an amino acid sequence of SEQ ID NO:
  • a hAce2-MR27HL-Variant-IgGl soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 96, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 96 or an amino acid sequence of SEQ ID NO: 76), a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 96 or an amino acid sequence of SEQ ID NO: 107), a linker “EPKSC” (aa 616 to 620 of SEQ ID NO: 96 or an amino acid sequence of SEQ ID NO: 101), and a human Fc immunoglobulin fragment IgGl (aa 619 to 844 of SEQ ID NO: 96 or an amino acid sequence of SEQ ID NO: 103), or a sequence at least about 95% identical thereto.
  • a native signal peptide aa 1 to 17 of
  • a hAce2-MR27HL-Variant-IgGl soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 100, wherein a soluble decoy fusion protein comprising a native signal peptide (aa 1 to 17 of SEQ ID NO: 100 or an amino acid sequence of SEQ ID NO: 76), a soluble hAce2 protein fragment, i.e., soluble decoy (aa 18 to 615 of SEQ ID NO: 100 or an amino acid sequence of SEQ ID NO: 107), a linker “GS” (aa 616 to 617 of SEQ ID NO: 100), and a human Fc immunoglobulin fragment IgGl (aa 619 to 844 of SEQ ID NO: 100 or an amino acid sequence of SEQ ID NO: 103), or a sequence at least about 95% identical thereto.
  • a native signal peptide aa 1 to 17 of SEQ ID NO: 100 or an amino acid sequence of SEQ ID NO: 76
  • a hAce2-Variantl soluble decoy protein has an amino acid sequence of SEQ ID NO: 27, or a sequence at least about 95% identical thereto.
  • hAce2-Variant2 soluble decoy protein has an amino acid sequence of SEQ ID NO: 29, or a sequence at least about 95% identical thereto.
  • hAce2- Variant3 soluble decoy protein has an amino acid sequence of SEQ ID NO:31, or a sequence at least about 95% identical thereto.
  • hAce2-Variant4 soluble decoy protein has an amino acid sequence of SEQ ID NO: 33, or a sequence at least about 95% identical thereto.
  • a hAce2-MR27HL-Variant soluble decoy protein has an amino acid sequence of SEQ ID NO: 107, or a sequence at least about 95% identical thereto.
  • a hAce2-Variantl-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 35, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 35 or an amino acid sequence of SEQ ID NO: 27), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 35 or an amino acid sequence of SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 35 or an amino acid sequence of SEQ ID NO: 27), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 35 or an amino acid sequence of SEQ
  • a hAce2-Variant2-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 37, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 37 or an amino acid sequence of SEQ ID NO: 29), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 37 or an amino acid sequence of SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 37 or an amino acid sequence of SEQ ID NO: 29), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 37 or an amino acid sequence of SEQ ID
  • a hAce2-Variant3-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 39, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 39 or an amino acid sequence of SEQ ID NO: 31), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 39 or an amino acid sequence of SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 39 or an amino acid sequence of SEQ ID NO: 31), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 39 or an amino acid sequence of SEQ
  • a hAce2- Variant4-IgG4 soluble decoy fusion protein has an amino acid sequence of SEQ ID NO: 41, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 41 or an amino acid sequence of SEQ ID NO: 33), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 41 or an amino acid sequence of SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy (aa 1 to 598 of SEQ ID NO: 41 or an amino acid sequence of SEQ ID NO: 33), and a human Fc immunoglobulin fragment IgG4 (aa 599 to 827 of SEQ ID NO: 41 or an amino acid sequence of SEQ ID
  • a hAce2-Variant5-IgG4 soluble decoy fusion protein has an amino acid sequence, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy is an amino acid sequence of SEQ ID NO: 74), and is linked to a human Fc immunoglobulin fragment IgG4 (SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • a hAce2-Variant6-IgG4 soluble decoy fusion protein has an amino acid sequence, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy comprises an amino acid sequence of SEQ ID NO: 75), and is linked to a human Fc immunoglobulin fragment IgG4 (SEQ ID NO: 77), or a sequence at least about 95% identical thereto.
  • a hAce2-Variant2-IgGl soluble decoy fusion protein has an amino acid sequence, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy comprises an amino acid sequence of SEQ ID NO: 29), and is linked to a human Fc immunoglobulin fragment IgGl (SEQ ID NO: 103), or a sequence at least about 95% identical thereto.
  • a hAce2-MR27HL-Variant-IgGl soluble decoy fusion protein has an amino acid sequence, wherein a soluble decoy fusion protein a soluble hAce2 protein fragment, i.e., soluble decoy comprises an amino acid sequence of SEQ ID NO: 107), and is linked to a human Fc immunoglobulin fragment IgGl (SEQ ID NO: 103), or a sequence at least about 95% identical thereto.
  • an expression cassette or a vector genome comprises at least one mutated soluble hAce2 decoy protein (mutated hAce2 soluble decoy and/or mutated hAce2 soluble decoy fusion proteins) is provided herein.
  • an expression cassette or a vector genome comprises a single mutated soluble hAce2 decoy (mutated hAce2 soluble decoy and/or mutated hAce2 soluble decoy fusion proteins) protein as described herein and a second coding sequence encoding a different gene product.
  • suitable expression vectors include, without limitation, plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses, parvovirues (e.g., bocavirus or adeno-associated viruses, lentiviruses and herpes viruses, among others.
  • the vectors are selected for delivery of an expression cassette encoding the protein to a patient and in vivo expression of the protein.
  • a selected protein(s) e.g., MR27HL fusion proteins
  • MR27HL fusion proteins is suited for expression in vitro and delivery to a patient as a protein-based therapeutic.
  • a nucleic acid molecule (e.g., a plasmid) is used for in vitro expression of a recombinant protein to produce a recombinant fusion protein therapeutic.
  • the signal peptide may be a non-human or non-mammalian signal peptide suited for the production cell type.
  • the vector is selected for expression in a human cell.
  • the signal peptide is suitably a human signal peptide.
  • the vector comprises CMV (cytomegalovirus), EFl (elongation factor- 1 alpha), or EEF2 (Eukaryotic Translation Elongation Factor 2) promoter.
  • the protein production is with co-transfection of glycosylation enzymes to promote sialyation of decoy proteins produced.
  • the transgene and other nucleic acids may be contained within a production vector.
  • a vector may include, but is not limited to, any plasmid, phagemid, F-factor, virus, cosmid, or phage in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable, or non-therapeutic because it is designed for in vitro protein production and may contain elements not suitable for delivery to a patient.
  • the production vector can also transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extra-chromosomally (e.g., autonomous replicating plasmid with an origin of replication).
  • polypeptides or peptide fragments of the invention include, but are not limited to, cells or microorganisms that are transformed with a recombinant nucleic acid construct that contains a nucleic acid segment of the invention.
  • recombinant nucleic acid constructs may include bacteriophage DNA, plasmid DNA, cosmid DNA, or viral expression vectors.
  • cells and microorganisms that may be transformed include bacteria (for example, E. coli or B. subtilis),' yeast (for example, Saccharomyces and Pich ay.
  • insect cell systems for example, baculovirus
  • plant cell systems for example, COS, Chinese Hamster Ovary (CHO), BHK, 293, VERO, HeLa, MDCK, W138, and NIH 3T3 cells.
  • mammalian cell systems for example, COS, Chinese Hamster Ovary (CHO), BHK, 293, VERO, HeLa, MDCK, W138, and NIH 3T3 cells.
  • Also useful as host cells are primary or secondary cells obtained directly from a mammal that are transfected with a plasmid vector or infected with a viral vector. Synthetic methods may also be used to produce polypeptides and peptide fragments of the invention. Such methods are known and have been reported. Merrifield, Science, 85:2149 (1963).
  • the production cell lune is banked, thawed, and cultured in chemically defined media without any components of animal origin.
  • the production cell line is a suspension cell line, wherein the cells are grown in suspension mode.
  • the protein production comprises process in a single use stir tank-controlled bioreactor in fed batch mode, wherein the protein is secreted into the cell culture and separated from cell components as a part of harvest.
  • the harvest is performed using continuous centrifugation followed by depth filtration.
  • the harvest is performed with depth filtration.
  • the filtered product is further treated with stability enhancing components.
  • the filtered product is further concentrated and buffer-exchanged using tangential flow filtration.
  • the recombinant protein is produced using a production cell line as described herein, wherein the production process comprises a chromatography step comprising binding and elution steps; a neutralization step; a filtration step using 0.2-micron depth filter; a further filtration step using virus removal nanofilter; and a further filtration step (e.g., using a 0.2 micron filter).
  • This may optionally further comprise at least one or more further chromatography steps; and/or solvent detergent viral inactivation step before or after chromatography steps.
  • the at least one or more chromatography steps are selected from ion exchange membrane chromatography, an anion exchange resin chromatography, a cation exchange resin chromatography, a hydrophobic interaction chromatography (HIC) resin chromatography, a mixed mode of anion and HIC chromatography, or a mixed mode of cation and HIC resin column chromatography.
  • the protein product is then formulated into the final formulation buffer by initial concentration to a target concentration, a lOx buffer exchange, followed by a final concentration and filter flush.
  • the protein product is examined for bioburden, endotoxins and related contaminants (i.e., host cell proteins), residual DNA, and/or residual affinity leached ligand throughout protein production process and in the final formulation.
  • the protein product comprises a final spike of formulation component and filtration step to produce the bulk drug (protein) substance.
  • the formulation buffer for the protein drug product is a buffered saline.
  • One suitable buffered saline is a phosphate buffer saline with 0.001% poloxamer 188 (137 mM Sodium Chloride, 2.7 mM Potassium Chloride, 10 mM Sodium Phosphate Dibasic, 1.8 mM Potassium Phosphate Monobasic, 0.001% Poloxamer 188, pH 7.3- 7.5).
  • Other suitable buffers may be selected.
  • the final protein product is stored in -80°.
  • the final protein product is formulated in a suitable buffer for stability and stored, e.g., at about 2 °C to about 8°C. In certain embodiments, the final protein product is formulated in a buffer suitable for lyophilization, which is further suitable for storage at room temperature.
  • modified hAce2 soluble decoy protein and/or hAce2 soluble decoy fusion protein as described herein may comprise any suitable number of additional amino acid residues, i.e., at least one additional amino acid residue.
  • the additional amino acid residues may be added in order to improve or simplify production, purification and/or detection of a protein.
  • additional amino acid residue may include an addition of a cysteine residue at either the amino or carboxy terminus of a protein, which may provide a "tag" for purification or detection of the protein, i.e., a His6 tag, a c-myc tag, a FLAG tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of the hexa-histidine tag.
  • a cysteine residue at either the amino or carboxy terminus of a protein, which may provide a "tag” for purification or detection of the protein, i.e., a His6 tag, a c-myc tag, a FLAG tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of the hexa-histidine tag.
  • IMAC immobilized metal affinity chromatography
  • the nucleic acid molecule selected for in vitro production comprises a transgene encoding a modified hAce2 soluble decoy protein and/or hAce2 soluble decoy fusion protein as described herein under regulatory control of sequences designed to optimize in vitro production levels.
  • the transgene comprises a nucleic acid sequence of SEQ ID NO: 1 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 2.
  • the transgene comprises a nucleic acid sequence of SEQ ID NO: 3 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 4.
  • the transgene comprises a nucleic acid sequence of SEQ ID NO: 95 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 6
  • the transgene comprises a nucleic acid sequence of SEQ ID NO: 7 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 8.
  • the transgene comprises a nucleic acid sequence of SEQ ID NO: 93 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 94.
  • the transgene comprises a nucleic acid sequence of SEQ ID NO: 95 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 96. In certain embodiments, the transgene comprises a nucleic acid sequence of SEQ ID NO: 97 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 98. In certain embodiments, the transgene comprises a nucleic acid sequence of SEQ ID NO: 99 or a sequence at least about 95% identical thereto, encoding an amino acid sequence of SEQ ID NO: 100.
  • hAce2-MR27HL-Variant-IgGl amino acid sequence of SEQ ID NOs: 96 and 100 (comprising leader sequence); SEQ ID NOs: 111 and 115
  • the hAce2- MR27HL fusion proteins are believed to be particularly well suited for in vitro production and delivery as a protein-based therapeutic.
  • vectors are particularly well suited for prophylactic expression of a soluble protein(s) provided herein.
  • such vectors may be used in combination with protein therapeutics which confer therapeutic effect or passive immunization.
  • suitable expression vectors include, without limitation, plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses, parvoviruses (e.g., bocavirus or adeno-associated viruses, lentiviruses and herpes viruses, among others.
  • a parvovirus capsid is used to generate a recombinant vector suitable for delivery to a patient.
  • the parvovirus capsid is selected from adeno-associated viruses which target nasal epithelial cells, nasopharynx cells, lung cells, or another target tissue which expresses the soluble protein.
  • capsids from Clade F AAV such as AAVhu68 or AAV9 may be selected.
  • Methods of generating vectors having the AAV9 capsid or AAVhu68 capsid, and/or chimeric capsids derived from AAV9 have been described. See, e.g., US 7,906, 111, which is incorporated by reference herein.
  • AAV serotypes which transduce nasal cells or another suitable target may be selected as sources for capsids of AAV viral vectors including, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVhu68, AAVrh91, rhlO, AAVrh64Rl, AAVrh64R2, rh8 (See, e.g., US Published Patent Application No. 2007- 0036760-Al; US Published Patent Application No. 2009-0197338-Al; and EP 1310571). See, e.g., US 7,906,111, which is incorporated by reference herein.
  • WO 2003/042397 AAV7 and other simian AAV
  • US Patent 7790449 and US Patent 7282199 AAV8
  • WO 2005/033321 AAV9
  • WO 2006/110689 or yet to be discovered, or a recombinant AAV based thereon, may be used as a source for the AAV capsid.
  • WO 2020/223232 Al AAV rh90
  • WO 2020/223231 Al AAV rh91
  • WO 2020/223236 Al AAV rh92, AAV rh93, AAVrh91.93
  • a modified rAAV capsid is selected, wherein the capsid comprises at least one exogenous peptide which is a targeting motif (i.e., nasal epithelial cells, nasopharynx cells, and/or lung cells).
  • a targeting motif i.e., nasal epithelial cells, nasopharynx cells, and/or lung cells.
  • an AAV capsid (cap) for use in the viral vector can be generated by mutagenesis (i.e., by insertions, deletions, or substitutions) of one of the aforementioned AAV caps or its encoding nucleic acid.
  • the AAV capsid is chimeric, comprising domains from two or three or four or more of the aforementioned AAV capsid proteins.
  • the AAV capsid is a mosaic of Vpl, Vp2, and Vp3 monomers from two or three different AAVs or recombinant AAVs.
  • an rAAV composition comprises more than one of the aforementioned caps.
  • the term “clade” as it relates to groups of AAV refers to a group of AAV which are phylogenetically related to one another as determined using a Neighbor- Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence.
  • the Neighbor-Joining algorithm has been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.
  • AAV vp 1 capsid protein implements the modified Nei-Gojobori method.
  • G Gao et al, J Virol, 2004 Jun; 78(12): 6381-6388, which identifies Clades A, B, C, D, E and F, and provides nucleic acid sequences of novel AAV, GenBank Accession Numbers AY530553 to AY530629. See, also, WO 2005/033321.
  • an “AAV9 capsid” is a self-assembled AAV capsid composed of multiple AAV9 vp proteins.
  • the AAV9 vp proteins are typically expressed as alternative splice variants encoded by a nucleic acid sequence which encodes the vpl amino acid sequence of GenBank accession: AAS99264. These splice variants result in proteins of different length.
  • “AAV9 capsid” includes an AAV having an amino acid sequence which is 99% identical to AAS99264 or 99% identical thereto. See, also US 7,906,111 and WO 2005/033321.
  • AAV9 variants include those described in, e.g., WO2016/049230, US 8,927,514, US 2015/0344911, and US 8,734,809.
  • a rAAVhu68 is composed of an AAVhu68 capsid and a vector genome.
  • An AAVhu68 capsid is an assembly of a heterogenous population of vpl, a heterogenous population of vp2, and a heterogenous population of vp3 proteins.
  • the term “heterogenous” or any grammatical variation thereof refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.
  • the AAVhu68 capsid coding sequence is a coding sequence which is useful in manufacturing of recombinant AAV (rAAV) for generating higher yields of recombinant AAV having AAVhu68 capsids. See also, US Provisional Patent Application No. 63/093,275, filed October 18, 2020, and International Patent Application No. PCTPCT/US2021/055436, filed October 18, 2021, which are incorporated herein by reference in their entireties.
  • the expression cassettes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a packaging host cell.
  • a suitable vector e.g., a plasmid
  • the plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art.
  • AAV -based vectors having an AAV9 or another AAV capsid
  • methods of preparing AAV -based vectors are known. See, e.g., US Published Patent Application No. 2007/0036760 (February 15, 2007), which is incorporated by reference herein.
  • the invention is not limited to the use of AAV9 or other clade F AAV amino acid sequences, but encompasses peptides and/or proteins containing the terminal P-galactose binding generated by other methods known in the art, including, e.g., by chemical synthesis, by other synthetic techniques, or by other methods.
  • the sequences of any of the AAV capsids provided herein can be readily generated using a variety of techniques. Suitable production techniques are well known to those of skill in the art.
  • peptides can also be synthesized by the well-known solid phase peptide synthesis methods (Merrifield, (1962) J. Am. Chem. Soc., 85:2149; Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62).
  • These methods may involve, e.g., culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; a minigene composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the minigene into the AAV capsid protein.
  • ITRs AAV inverted terminal repeats
  • the components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans.
  • any one or more of the required components e.g., minigene, rep sequences, cap sequences, and/or helper functions
  • a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
  • a stable host cell will contain the required component(s) under the control of an inducible promoter.
  • the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene.
  • a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
  • a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contains the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
  • compositions of the invention may also be used for production of a desired gene product in vitro.
  • a desired product e.g., a protein
  • a desired culture following transfection of host cells with a rAAV containing the molecule encoding the desired product and culturing the cell culture under conditions which permit expression.
  • the expressed product may then be purified and isolated, as desired. Suitable techniques for transfection, cell culturing, purification, and isolation are known to those of skill in the art. Methods for generating and isolating AAVs suitable for use as vectors are known in the art.
  • the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassettes.
  • the cap and rep genes can be supplied in trans.
  • the expression cassettes described herein are engineered into a genetic element (e.g., a shuttle plasmid) which transfers the immunoglobulin construct sequences carried thereon into a packaging host cell for production a viral vector.
  • a genetic element e.g., a shuttle plasmid
  • the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Stable AAV packaging cells can also be made.
  • the expression cassettes may be used to generate a viral vector other than AAV, or for production of mixtures of antibodies in vitro.
  • AAV intermediate or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non- functional to transfer the gene of interest to a host cell.
  • the recombinant AAV described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2.
  • Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein.
  • ITRs AAV inverted terminal repeats
  • Methods of generating the capsid, coding sequences therefore, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186A1.
  • cells are manufactured in a suitable cell culture (e.g., HEK 293 cells).
  • Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors.
  • the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid.
  • the vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media.
  • the harvested vector-containing cells and culture media are referred to herein as crude cell harvest.
  • the gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors.
  • Zhang et al., 2009 "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, which is incorporated herein by reference in its entirety.
  • the crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulation and filtration to prepare bulk vector.
  • a two-step affinity chromatography purification at high salt concentration followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT/US2016/065970, filed December 9, 2016, entitled “Scalable Purification Method for AAV9”, which is incorporated by reference. Purification methods for AAV8, International Patent Application No. PCT/US2016/065976, filed December 9, 2016, and rhlO, International Patent Application No. PCT/US 16/66013, filed December 9, 2016, entitled “Scalable Purification Method for AAVrhlO”, also filed December 11, 2015, and for AAV1, International Patent Application No. PCT/US2016/065974, filed December 9, 2016 for “Scalable Purification Method for AAV1”, filed December 11, 2015, are all incorporated by reference herein.
  • the number of particles (pt) per 20 pL loaded is then multiplied by 50 to give particles (pt) /mL.
  • Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC).
  • Pt/mL-GC/mL gives empty pt/mL.
  • Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.
  • methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6: 1322-1330; and Sommer et al., Molec. Ther. (2003) 7: 122-128.
  • the methods include subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon.
  • Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281- 9293).
  • a secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep antimouse IgG antibody covalently linked to horseradish peroxidase.
  • a method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex).
  • Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains.
  • the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA.
  • the samples are further diluted and amplified using primers and a TaqManTM Anorogenic probe specific for the DNA sequence between the primers.
  • the number of cycles required to reach a defined level of Auorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System.
  • Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction.
  • the cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.
  • Sedimentation velocity as measured in an analytical ultracentrifuge (AUC) can detect aggregates, other minor components as well as providing good quantitation of relative amounts of different particle species based upon their different sedimentation coefficients.
  • AUC analytical ultracentrifuge
  • This is an absolute method based on fundamental units of length and time, requiring no standard molecules as references.
  • Vector samples are loaded into cells with 2-channel charcoal-epon centerpieces with 12mm optical path length.
  • the supplied dilution buffer is loaded into the reference channel of each cell.
  • the loaded cells are then placed into an AN- 60Ti analytical rotor and loaded into a Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge equipped with both absorbance and RI detectors.
  • the rotor After full temperature equilibration at 20 °C the rotor is brought to the final run speed of 12,000 rpm. A280 scans are recorded approximately every 3 minutes for ⁇ 5.5 hours (110 total scans for each sample). The raw data is analyzed using the c(s) method and implemented in the analysis program SEDFIT. The resultant size distributions are graphed and the peaks integrated. The percentage values associated with each peak represent the peak area fraction of the total area under all peaks and are based upon the raw data generated at 280nm; many labs use these values to calculate empty: full particle ratios. However, because empty and full particles have different extinction coefficients at this wavelength, the raw data can be adjusted accordingly. The ratio of the empty particle and full monomer peak values both before and after extinction coefficient- adjustment is used to determine the empty-full particle ratio.
  • an optimized q-PCR method which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size.
  • the proteinase K buffer may be concentrated to 2 fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but may be varied from 0. 1 mg/mL to about 1 mg/mL.
  • the treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes).
  • heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes).
  • Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay. Quantification also can be done using ViroCyt or flow cytometry.
  • droplet digital PCR may be used.
  • ddPCR droplet digital PCR
  • methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10. 1089/hgtb.2013. 131. Epub 2014 Feb 14.
  • compositions containing at least one mutant hAce2 soluble decoy protein and an optional carrier, excipient and/or preservative may contain at least one mutant hAce2 soluble decoy fusion protein and an optional carrier, excipient and/or preservative.
  • the composition may comprise at least a second, different hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein.
  • the composition may comprise of hAce2- Variant 1-IgG soluble decoy fusion protein and hAce2-Variant4-IgG soluble decoy fusion protein.
  • compositions containing at least one rAAV stock e.g., an rAAV9.hACE2 stock
  • an optional carrier, excipient and/or preservative e.g., an rAAV9.hACE2 stock
  • An rAAV stock refers to a plurality of rAAV vectors which are the same, e.g., such as in the amounts described below in the discussion of concentrations and dosage units.
  • AAV.hAce2 refers to an rAAV having a coding sequence for either wild-type or mutated hAce2, where indicated and as defined herein, which has packaged therein a vector genome encoding the hACE2 soluble protein.
  • AAV.hACE2-VariantX-IgG refers to a rAAV having a coding sequence for mutated hACE2 soluble decoy fusion as defined herein, wherein X stands for 1, 2, 3, or 4, which has packaged therein a vector genome encoding the hACE2 soluble protein and an IgG Fc portion, designed to expression and assemble as a fusion protein in a target cell.
  • AAV9.hACE2-VariantX-IgG refers to a rAAV having an AAV9 capsid as defined herein which has packaged therein the vector genome, wherein X stands for 1, 2, 3, 4, 5 or 6.
  • the rAAV stock comprise a vector genome selected from: SEQ ID NO: 17 (hAce2-Variantl-IgG4), SEQ ID NO: 19 (hAce2-Variant2-IgG4), SEQ ID NO: 21 (hAce2-Variant3-IgG4), SEQ ID NO: 23 (hAce2-Variant4-IgG4), SEQ ID NO: 85 (hAce2-Variant2-IgGl), SEQ ID NO: 87 (hAce2-MR27-Variant-IgGl), SEQ ID NO: 89 (hAce2-Variant2-IgGl comprising “GS” linker between hAce2 and IgGl Fc domain), and/or SEQ ID NO: 91 (hAce2-MR27-Variant-IgGl comprising “GS” linker).
  • the rAAV stock comprise an expression cassette selected from: nt 198 to 4666 of SEQ ID NO: 17 or SEQ ID NO: 116 (hAce2-Variantl-IgG4), nt 198 to 4666 of SEQ ID NO: 19 or SEQ ID NO: 117 (hAce2-Variant2-IgG4), nt 198 to 4666 of SEQ ID NO: 21 or SEQ ID NO: 118(hAce2-Variant3-IgG4), nt 198 to 4666 of SEQ ID NO: 23 or SEQ ID NO: 119 (hAce2-Variant4-IgG4), SEQ ID NO: 86 (hAce2-Variant2-IgGl), SEQ ID NO: 88 (hAce2-MR27-Variant-IgGl), SEQ ID NO: 90 (hAce2-Variant2-IgGl comprising “GS” linker between hAce2 and IgGl Fc domain),
  • the rAAV stock comprise a vector genome comprising a nucleic acid sequence encoding for an hAce2 soluble decoy with an amino acid sequence selected from SEQ ID NOs: 10, 12, 14, 16, 72 or 73, wherein the hAce2 soluble decoy is further linked to an Fc domain selected from IgGl, IgG4, IgA or IgM.
  • the rAAV stock comprise a vector genome comprising a nucleic acid sequence encoding for an hAce2 soluble decoy with an amino acid sequence selected from SEQ ID NOs: 52 to 61, wherein the hAce2 soluble decoy is further linked to an Fc domain selected from IgGl, IgG4, IgA or IgM.
  • a composition may contain at least a second, different rAAV stock. This second vector stock may vary from the first by having a different AAV capsid and/or a different vector genome.
  • a composition as described herein may contain a different vector expressing an expression cassette as described herein, or another active component (e.g., an antibody construct, another biologic, and/or a small molecule drug).
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically - acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells.
  • the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • soluble refers to the ability of a polypeptide to be solvated in an aqueous solution.
  • a soluble peptide can be mixed with an aqueous medium such that at least a detectable portion of the peptide is present in the aqueous medium.
  • the peptide may be detected through use of common techniques, such as absorbance of light, fluorescence, the ability to bind dyes, the ability to reduce silver ions, and the like.
  • a pharmaceutical composition comprising the mutant hACE2 protein(s) or a mutant hACE2-encoding nucleic acid may be made.
  • Such compositions may include pharmaceutically suitable salts thereof, plus buffers, tonicity components or pharmaceutically suitable vehicles.
  • Pharmaceutical vehicle substances are used to improve the tolerability of the composition and allow a better solubility as well as better bioavailability of the active substances. Examples here include emulsifiers, thickeners, redox components, starch, alcohol solutions, polyethylene glycol, or lipids.
  • Other carriers may include additives used in tablets, granules and capsules, etc.
  • Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients.
  • excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients.
  • Such carriers may also include flavor and color additives or other ingredients.
  • Compositions comprising such carriers are formulated by well-known conventional methods. The selection of a suitable pharmaceutical vehicle depends to a great extent on how the substance is administered. Liquid or solid vehicles may be used for oral administration, but for injections the final composition must be a liquid.
  • the medication to be used may comprises buffer substances or tonic substances.
  • buffers By means of buffers, the pH of the medication can be adjusted to physiological conditions and furthermore fluctuations in pH can be diminished and/or buffered.
  • a phosphate buffer One example of this is a phosphate buffer.
  • Tonic substances are used to adjust the osmolarity and may include ionic substances, for example, inorganic salts such as NaCl or nonionic salts such as glycerol or carbohydrates.
  • the composition to be used according to the present invention is preferably prepared to be suitable for systemic, topical, oral, or intranasal administration. These forms of administration of the medication according to the present invention allow a rapid and uncomplicated uptake.
  • solid and/or liquid medications may be taken directly or dissolved and/or diluted.
  • the medication to be used according to the invention is preferably prepared for intravenous, intra-arterial, intramuscular, intravascular, intraperitoneal or subcutaneous administration.
  • injections or transfusions are suitable for this purpose.
  • Administration directly into the bloodstream has the advantage that the active ingredients of the medication are distributed throughout the entire body and rapidly reach the target tissue.
  • the dosage of the compositions and/or the pharmaceutical composition comprising modified hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein of the invention depends on factors including the route of administration, and physical characteristics of subject in need, e.g., age, weight, general health, of the subject.
  • the amount of an hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein in a single dose may be in an amount that effectively prevents, ameliorate symptoms, or treats the SARS-CoV (or other virus mediated by binding the Ace2 receptor) without inducing significant toxicity.
  • a pharmaceutical composition of the invention may include a dosage of an hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein, as described herein, ranging from 0.01 to 500 mg/kg (e.g., 0.01 , 0 .1, 0.2, 0.3, 0.4, 0.5, 1, 2 , 3 , 4 , 5 , 10 , 15 , 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, at least about 1 to at least about 100 mg/kg and, at least about 1 to at least about 50 mg/kg.
  • 0.01 to 500 mg/kg e.g., 0.01 , 0 .1, 0.2, 0.3, 0.4, 0.5, 1, 2 , 3 , 4 , 5 , 10 , 15 , 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg
  • the dosage may be adapted in accordance to the extent of the SARS-CoV (or other virus mediated by binding the Ace2 receptor) infection and according to different parameters of the subject.
  • the pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an amelioration of the symptoms, treatment and/or prevention of a viral infection mediated by the Ace2 receptor, e.g., SARS- CoV 1 or SARS-CoV2, among others identified herein.
  • the pharmaceutical compositions are administered in a variety of dosages, e.g., intravenous dosage, intranasal dosage, intramuscular dosage and oral dosage.
  • one or more mutant soluble human Ace2 (hAce2) proteins provided herein are useful in treating and, in certain embodiments preventing, infection with betacoronaviruses, including SARS-CoV2 is provided, as are compositions useful in treating disease associated with betacoronavirus-associated disease, including, e.g., SARS-associated infection and COVID- 19.
  • compositions comprising one or more hAce2 soluble decoy proteins and/or hAce2 soluble decoy fusion proteins may be dosed at 1 mg/kg to 100 mg/kg, or doses therebetween, e.g., 1 mg/kg to 50 mg/kg.
  • the composition may be administered to a subject in a need thereof, at least one or more times, daily for a predetermined time period, weekly, monthly, biannually, annually, or as necessary.
  • the compositions are delivered intravenously.
  • effectiveness of hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein may be used in determining the effective dose.
  • IC50 is used to measure the effectiveness of hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein. “IC50” is the half maximal inhibitory concentration, which is a measure of the effectiveness of a substance for inhibiting a specific quantifiable biological or biochemical function. This quantitative measure indicates how much of a particular substance is needed to inhibit a specific biological function by 50% and is commonly used in the art.
  • the IC50 of hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein is measured as capable of blocking binding ACE2 binding to RBD. In certain embodiments, the IC50 is at least about 3 ng/mL to at least about 10 ng/mL. In certain embodiments, the IC50 for mutant hAce2 soluble decoy fusion protein is at least about 10 times lower, at least about 100 times lower, and/or at least about 1000 times lower as compared wild-type hAce2 soluble decoy fusion protein.
  • Methods suitable for assessing antibodies that bind to ACE2 extracellular domain include 20 those of Enzyme Linked Immunosorbent Assay (ELISA). Specifically, NOVUS NBP2 human ACE-2 ELISA Chemiluminescent Kit, which can specifically detect human ACE2 in various samples such as serum, plasma and other biological fluids (available from the following website: novusbio.eom/products/ace-2-elisa-kit_nbp2-66387#datasheet). The kit is utilized according to manufacturing instruction. In some embodiments of the claimed, the kit is used to immobilize the 25 hAce2, and hAce2_NN for SARS-CoV-2 S-protein binding assay. For hAce2-fuson and hAce2_NN-fuison protein binding with S-protein of SARS-CoV- 2, anti-human IgG-HRP linked detection antibody was used.
  • ELISA Enzyme Linked Immunosorbent Assay
  • assessing levels of SARS-CoV neutralization activity is performed using the adopted SARS-CoV-2 S-protein binding assay, as indicated above.
  • a pseudotyped Human Immunodeficiency Viral (HIV) vector with the S-protein of SARSCoV-2 and expressing eGFP was used in evaluating in vitro neutralization activity of the constructs (Kobinger, G.P., et al., 2007, Hum Gene Ther, 18(5): 413-422).
  • Vero E 6 cells (ATCC CRL1586) is used for viral replication of S-protein pseudotyped HIV vector.
  • Ace2 is expressed in stably transduced HEK293 cells (selected for expression with Zeocin).
  • hACE2 soluble decoy and/or hAce2 soluble decoy fusion proteins are tested for neutralizing activity using a pseudotyped retroviral transduction inhibition assay.
  • a lentiviral vector is generated carrying a GFP-reporter gene and pseudotyped with a SARS-CoV-2 spike protein with a heterologous C-terminus for optimal incorporation into a lentiviral vector.
  • the pseudotyped vector is used to transduce target HEK 293 cells that carry a human ACE2 transgene.
  • Neutralizing activity of candidate soluble ACE2 proteins and ACE2-Fc fusion proteins are evaluated by pre-incubating the purified protein with the lentiviral vector before it is applied to the ACE2-expressing target cells. Neutralization are quantified based on the concentration of the protein that yields a 50% reduction in GFP expression in target cells relative to cells treated with the lentiviral vector alone.
  • KD value is used to measure the effectiveness of hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein, wherein KD value reflects the interaction between the hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein, and RBD of SARS-CoV. In certain embodiments, KD value is at least about 40 pM to at least about 300 pM.
  • a composition in one embodiment, includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
  • a surfactant are present in the formulation.
  • the composition may be transported as a concentrate which is diluted for administration to a subject.
  • the composition may be lyophilized and reconstituted at the time of administration.
  • a suitable surfactant, or combination of surfactants may be selected from among nonionic surfactants that are nontoxic.
  • a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400.
  • Pluronic® F68 also known as Poloxamer 188
  • Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide)), SOLUTOL® HS 15 (Macrogol-15 Hydroxystearate), LABRASOL® (Polyoxy capryllic glyceride), poly oxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
  • the formulation contains a poloxamer.
  • copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content.
  • Poloxamer 188 is selected.
  • the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.
  • the formulation buffer is phosphate-buffered saline (PBS) with total salt concentration of 200 mM, 0.001% (w/v) pluronic F68 (Final Formulation Buffer, FFB).
  • the formulation buffer is phosphate buffer saline with 0.001% poloxamer 188 (137 mM Sodium Chloride, 2.7 mM Potassium Chloride, 10 mM Sodium Phosphate Dibasic, 1.8 mM Potassium Phosphate Monobasic, 0.001% Poloxamer 188, pH 7.3- 7.5).
  • the vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts.
  • the vectors are formulated for delivery via intranasal delivery devices for targeted delivery to nasal and/or nasopharynx epithelial cells.
  • vectors are formulated for aerosol delivery devices, e.g., via a nebulizer or through other suitable devices.
  • routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., lung), oral inhalation, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration.
  • the vector is administered intranasally using intranasal mucosal atomization device (LMA® MAD NasalTM- MAD110).
  • the vector is administered intrapulmonary in nebulized form using Vibrating Mesh Nebulizer (Aerogen® Solo) or MADgicTM Laryngeal Mucosal Atomizer. Routes of administration may be combined, if desired.
  • Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients.
  • a therapeutically effective human dosage of the viral vector is generally in the range of from about 25 to about 1000 microliters to about 5 mL of aqueous suspending liquid containing doses of from about 10 9 to 4x10 14 GC of AAV vector.
  • the dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed.
  • the levels of expression of the transgene can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the minigene.
  • dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention.
  • the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 10 9 GC to about 10 16 GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 10 12 GC to 10 14 GC for a human patient.
  • the compositions are formulated to contain at least 10 9 , 2xl0 9 , 3xl0 9 , 4xl0 9 , 5xl0 9 , 6xl0 9 , 7xl0 9 , 8xl0 9 , or 9xl0 9 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 10 , 2xlO 10 , 3xl0 10 , 4xlO 10 , 5xl0 10 , 6xlO 10 , 7xlO 10 , 8xl0 10 , or 9xlO 10 GC per dose including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least 10 11 , 2xlO n , 3xl0 n , 4xlO n , 5xl0 n , 6xlO n , 7xlO n , 8xl0 n , or 9xlO n GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 12 , 2xl0 12 , 3xl0 12 , 4xl0 12 , 5xl0 12 , 6xl0 12 , 7xl0 12 , 8xl0 12 , or 9xl0 12 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 13 , 2xl0 13 , 3xl0 13 , 4xl0 13 , 5xl0 13 , 6xl0 13 , 7xl0 13 , 8xl0 13 , or 9x10 13 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 14 , 2xl0 14 , 3xl0 14 , 4xl0 14 , 5xl0 14 , 6xl0 14 , 7xl0 14 , 8xl0 14 , or 9xl0 14 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 10 15 , 2xl0 15 , 3xl0 15 , 4xl0 15 , 5xl0 15 , 6xl0 15 , 7xl0 15 , 8xl0 15 , or 9xl0 15 GC per dose including all integers or fractional amounts within the range.
  • the dose can range from 10 10 to about 10 12 GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from 10 9 to about 7xl0 13 GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose ranges from 6.25xl0 12 GC to 5.00xl0 13 GC. In a further embodiment, the dose is about 6.25xl0 12 GC, about 1.25xl0 13 GC, about 2.50xl0 13 GC, or about 5.00xl0 13 GC.
  • the dose is about 1.02xl0 n GC, about 3.40xl0 n GC, or about 1.02xl0 12 GC. In certain embodiments, the dose is about 1.5 x 10 11 GC, about 1.5 x 10 12 GC, or about 1.5 x 10 13 GC. In certain embodiments, the dose is about 5 x 10 11 GC, about 5 x 10 12 GC, or about 5 x 10 13 GC. In certain embodiment, the dose is divided into one half thereof equally and administered to each nostril. In certain embodiments, for human application the dose ranges from 6.25x10 12 GC to 5.00xl0 13 GC administered as two aliquots of 0.2 ml per nostril for a total volume delivered in each subject of 0.8 ml.
  • the volume of carrier, excipient or buffer is at least about 25 pL. In one embodiment, the volume is about 50 pL. In another embodiment, the volume is about 75 pL. In another embodiment, the volume is about 100 pL. In another embodiment, the volume is about 125 pL. In another embodiment, the volume is about 150 pL. In another embodiment, the volume is about 175 pL.
  • the volume is about 200 pL. In another embodiment, the volume is about 225 pL. In yet another embodiment, the volume is about 250 pL. In yet another embodiment, the volume is about 275 pL. In yet another embodiment, the volume is about 300 pL. In yet another embodiment, the volume is about 325 pL. In another embodiment, the volume is about 350 pL. In another embodiment, the volume is about 375 pL. In another embodiment, the volume is about 400 pL. In another embodiment, the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 550 pL. In another embodiment, the volume is about 600 )1L. In another embodiment, the volume is about 650 pL. In another embodiment, the volume is about 700 pL. In another embodiment, the volume is between about 700 and 1000 pL.
  • the recombinant vectors may be dosed intranasally by using two sprays to each nostril.
  • the two sprays are administered by alternating to each nostril, e.g., left nostril spray, right nostril spray, then left nostril spray, right nostril spray.
  • each nostril may receive multiple sprays which are separated by an interval of about 10 to 60 seconds, or 20 to 40 seconds, or about 30 seconds, to a few minutes, or longer.
  • Such sprays may deliver, e.g., about 150 pL to 300 pL, or about 250 pL in each spray, to achieve a total volume dosed of about 200 pL to about 600 pL, 400 pL to 700 pL, or 450 pL to 1000 pL.
  • the recombinant AAV vector may be dosed intranasally to achieve a concentration of 5-20 ng/ml of the expression product of the transgene as measured in a nasal wash solution post-dosing, e.g., one week to four weeks, or about two weeks after administration of the vector.
  • a nasal wash solution post-dosing e.g., one week to four weeks, or about two weeks after administration of the vector.
  • such suspensions may be volumes doses of about 1 mL to about 25 mL, with doses of up to about 2.5 x 10 15 GC.
  • the intranasal delivery device provides a spay atomizer which delivers a mist of particles having an average size range of about 30 microns to about 100 microns in size. In certain embodiments, the average size range is about 10 microns to about 50 microns.
  • Suitable devices have been described in the literature and some are commercially available, e.g., the LMA MAD NASALTM (Teleflex Medical; Ireland); Teleflex VaxINatorTM (Teleflex Medical; Ireland); Controlled Particle Dispersion® (CPD) from Kurve Technologies. See, also, PG Djupesland, Drug Deliv and Transl. Res (2013) 3: 42-62.
  • the particle size and volume of delivery is controlled in order to preferentially target nasal epithelial cells and minimize targeting to the lung.
  • the mist of particles is about 0. 1 micron to about 20 microns, or less, in order to deliver to lung cells. Such smaller particle sizes may minimize retention in the nasal epithelium.
  • One device mists particles at an average diameter of about 16 microns to about 22 microns.
  • the mist may be delivered directly to the tracheobronchial tree inserted through the suction channel of a 3.5-mm flexible fiberoptic bronchoscope (Olympus, Melville, NY).
  • Other suitable delivery devices may include a laryngo-tracheal mucosal atomizer, which provides for administration across the upper airway past the vocal cords. It fits through vocal cords and down a laryngeal mask or into nasal cavity.
  • the droplets are atomized at an average diameter of about 30 microns to aboutlOO microns.
  • a standard device has a tip diameter of about 0.
  • Doses may be administered is 10 aliquots (approximately 150 pl each) of 99 control with saline or 99 rAAV.hACE2 sprayed into right and left main stem bronchi.
  • a frozen composition which contains an rAAV in a buffer solution as described herein, in frozen form.
  • one or more surfactants e.g., Pluronic F68
  • stabilizers or preservatives is present in this composition.
  • a composition is thawed and titrated to the desired dose with a suitable diluent, e.g., sterile saline or a buffered saline.
  • a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of the reference group, unless otherwise specified.
  • a “subpopulation” of vpl proteins is at least one (1) vpl protein and less than all vpl proteins in an assembled AAV capsid, unless otherwise specified.
  • a “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified.
  • vpl proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid.
  • vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g., at least one, two, three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs.
  • highly deamidated refers to at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidated at a referenced amino acid position, as compared to the predicted amino acid sequence at the reference amino acid position.
  • Such percentages may be determined using 2D-gel, mass spectrometry techniques, or other suitable techniques.
  • composition neutralizes more than one subtype of the target airborne pathogen.
  • the target airborne pathogen is a virus which binds to the Ace2 receptor.
  • the virus is selected from SARS-CoV2, SARS- CoVl, or mutated SARS-CoV2.
  • the composition treats infection with mutated SARS-CoV2 variants, e.g., EU, UK, South Africa), mink, and further variants including, e.g., Delta, Delta plus, Gamma, Epsioon, Kappa, loata, Zeta, omnicron, or related betacoronaviruses, e.g., CoVl.
  • the composition neutralizes mutated SARS-CoV2, wherein SARS- CoV2 comprises one or more variants, such as listed above.
  • the composition is useful for binding RBD of human ACE2 of betacoronaviruses, including both SARS-COV1 and SARS-CoV2 and having neutralizing activity for both.
  • compositions are provided herein which comprise the hAce2 fusion proteins in a non-viral delivery system and one or more suspending agents, carriers, excipients, preservatives, or the like.
  • Excipients and carriers that can be used to prepare parenteral formulations comprising the proteins include, without limitation, aqueous solvents such as water, saline, physiological saline, buffered saline (e.g., phosphate-buffered saline), balanced salt solutions [e.g., Ringer's BSS] and aqueous dextrose solutions), isotonic/iso- osmotic agents (e.g., salts [e.g., NaCl, KCI and CaC12] and sugars [e.g., sucrose]), buffering agents and pH adjusters (e.g., sodium dihydrogen phosphate [monobasic sodium phosphate]/disodium hydrogen phosphate [dibasic sodium phosphate],
  • Suitable doses may include, e.g., 1 pg up to 100 mg/dose, but may vary based on the delivery system and route of administration.
  • intranasal delivery systems may delivery about 10 pg/ml to about 1 mg/ml, or about 100 pg/ml to about 1 to 10 mg/ml per dose.
  • Higher doses may be delivered intravenously, or via other delivery routes. Dosing may be over period of 1 to 3 days, or daily over a week, two weeks, three weeks, 4 weeks, 6 weeks, 2 months, 3 months, or longer. The doses may be higher in the beginning and taper.
  • doses may be delivered multiple times a day and/or days may be skipped between doses.
  • the rAAV encoding mutated hAce2 soluble decoy and/or mutated hAce2 soluble decoy fusion protein may be administered intravenously to achieve expression levels sufficient for treatment of coronaviruses which utilize and/or rely on ACE2 receptor for intracellular entry and infection.
  • the coronavirus is SARS-CoVl.
  • the coronavirus is SARS-CoV2.
  • the coronavirus is a naturally mutated SARS-CoV2.
  • the hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein may be administered intravenously at an effective dose for treatment of betacoronaviruses which utilize and/or rely on ACE2 receptor for intracellular entry and infection.
  • the coronavirus is SARS-CoVl .
  • the coronavirus is SARS-CoV2.
  • the coronavirus is mutated SARS- CoV2.
  • the hAce2 soluble decoy and/or hAce2 soluble decoy fusion protein may be administered intravenously at an effective dose for treatment in a subject in need thereof, wherein the subject had been diagnosed with COVID- 19 (SARS-CoV2, including SARS-CoV2 mutants).
  • the mutant hAce2 soluble decoy fusion protein is administered in a composition comprising a mixture of the mutant hAce2 soluble decoy proteins, e.g.,hAce2-Variant2-Fc and hAce2-MMR27-Fc.
  • rAAV encoding mutated hAce2 soluble decoy fusion protein may be administered intranasally to achieve expression levels sufficient for COVID- 19 prophylaxis and prevention in un-infected subjects in the need thereof.
  • rAAV encoding mutated hAce2 soluble decoy fusion protein may be administered intrapulmonary to achieve expression levels sufficient for COVID- 19 treatment in infected subjects in need thereof.
  • rAAV encoding mutated hAce2 soluble decoy fusion protein may be administered intravenously.
  • rAAV encoding mutated hAce2 soluble decoy fusion protein may be administered intramuscularly.
  • a co-therapy may be selected either prior to, simultaneously with, or following administration of a rAAV provided herein.
  • a co-therapy may involve co-administration of a combination of two or more different populations of rAAV expressing an hACE2 soluble protein as described herein.
  • a co-therapy may involve co-administration of a combination of different populations of rAAV expressing two or more of mutated hAce2 soluble decoy fusion proteins, i.e., hAce2-Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2- Variant4-IgG, hAce2-MR27HL-Variant-IgG, wherein the IgG is IgGl Fc or IgG4 Fc.
  • the co-therapy may involve co-administration of a combination of rAAV expressing an hAce2 soluble protein as described herein and an rAAV expressing an hAce2 soluble protein as described in US Provisional Patent Application No. 63/002,100, filed March 3, 2020; US Provisional Patent Application No. 63/027,731, filed May 20, 2020; and US Provisional Patent Application No. 63/069,651, filed August 24, 2020, which are incorporated herein by reference.
  • a co-therapy may involve co-administration of a combination of two or more of mutated hAce2 soluble decoy fusion proteins, i.e., hAce2- Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG, hAce2- Variant5-IgG, hAce2-Variant6-IgG, hAce2-MR27HL-Variant-IgG.
  • the co-therapy may involve co-administration of a combination of rAAV expressing an hAce2 soluble protein as described herein a mutated hAce2 soluble decoy fusion proteins as described herein.
  • a different drug or biological may be co-administered to a subject for treatment of one or more symptoms of COVID- 19.
  • a co-therapeutic may be, e.g., remdesivir (GS-5734), Sarilumab, chloroquine or hydroxychloroquine, hyperimmune plasma, DAS181 (e.g., Nebulized DAS181 9mg/day (4.5 mg bid/day) for 10 days), recombinant human angiotensin-converting enzyme 2 (rhACE2), Sildenafil, Tocilizumab, tetrandrine 60 m QD for one week (take 6 days, stop using for 1 day), methylprednisolone, camostat mesylate (a Serine protease inhibitor that blocks TMPRSS-2 mediated cell entry of SARS-CoV-2), bevacizumab, danoprevirr combined with ritonavir, baricitinib, hydroxy
  • a co-therapy may involve co-administration of a combination of rAAV-hAce2 and at least one or more of mutated hAce2 soluble decoy fusion proteins, i.e., hAce2-Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG, hAce2-Variant5-IgG, hAce2-Variant6-IgG, hAce2- MR27HL-Variant-IgG intravenously.
  • mutated hAce2 soluble decoy fusion proteins i.e., hAce2-Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG, hAce2-Variant5-IgG, hA
  • a co-therapy may involve coadministration via a combination of routes intravenously, intramuscularly, intrapulmonary and/or intranasally.
  • the rAAV-hAce2 may encode for wild type hAce2 or comprise “NN” mutation as described herein, and co-administered with and at least one or more of mutated hAce2 soluble decoy fusion proteins, i.e., hAce2-Variantl-IgG, hAce2- Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG hAce2-Variant5-IgG, hAce2- Variant6-IgG, hAce2-MR27HL-Variant-IgG intravenously.
  • the rAAV- hAce2 may encode for mutated hAce2 as described herein, and co-administered with and at least one or more of mutated hAce2 soluble decoy fusion proteins, i.e., hAce2-Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG, hAce2-Variant5-IgG, hAce2- Variant6-IgG, hAce2-MR27HL-Variant-IgG intravenously.
  • mutated hAce2 soluble decoy fusion proteins i.e., hAce2-Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG, hAce2-Varian
  • the rAAV- Ace2 may be administered intranasally, wherein a co-therapy of and at least one or more of mutated hAce2 soluble decoy fusion proteins, i.e., hAce2-Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG, hAce2-Variant5-IgG, hAce2-Variant6-IgG, hAce2- MR27HL-Variant-IgG may be administered intravenously.
  • a co-therapy of and at least one or more of mutated hAce2 soluble decoy fusion proteins i.e., hAce2-Variantl-IgG, hAce2-Variant2-IgG, hAce2-Variant3-IgG, hAce2-Variant4-IgG,
  • a “synthetic protein” or “recombinant protein” refers to protein, which has been expressed and purified from a producer host cell. Wherein the producer host cell, comprising a vector encoding for soluble Ace2 proteins as described herein, is cultured under conditions suitable for leading to expression of protein.
  • a mutant soluble Ace2 proteins (hAce2 soluble decoy or hAce2 soluble decoy fusion proteins) may comprise a population of mutant soluble Ace2-proteins, which may include up to 5% variation from the sequences provided herein in view of post-translational modifications such as, e.g., glycosylation, oxidation and deamidation.
  • there is 0.5% to 5% variation in other embodiments, there is about 1%, about 2%, about 3%, or about 4% variation. In other embodiments, no detectable variation is observed.
  • Such post-translation modification may be detected by assessed by any suitable technique including, e.g., chromatographic and/or mass spectrometric analysis, or peptide mapping. These detection methods are not a limitation on the present invention.
  • a “post-translational modification” may encompass any one of or combination of modification (s), including covalent modification, which a protein undergoes after translation is complete and after being released from the ribosome or on the nascent polypeptide co- translationally.
  • Post-translational modification includes but is not limited to phosphorylation, myristylation, ubiquitination, glycosylation, coenzyme attachment, methylation and acetylation.
  • Post-translational modification can modulate or influence the activity of a protein, its intracellular or extracellular destination, its stability or half-life, and/or its recognition by ligands, receptors or other proteins. Post-translational modification can occur in cell organelles, in the nucleus or cytoplasm or extracellularly.
  • an antibody “Fc region” and or “Fc domain” refers to the crystallizable fragment which is the region of an antibody which interacts with the cell surface receptors (Fc receptors).
  • the Fc region is a human IgG4 Fc (amino acid sequence of SEQ ID NO: 77).
  • the Fc region is a human IgGl Fc (amino acid sequence of SEQ ID NO: 103).
  • the Fc region is a human IgAl Fc (UniProt amino acid sequence of SEQ ID NO: 125).
  • the Fc region is a human IgA2 Fc (amino acid sequence of SEQ ID NO: 126).
  • the Fc region is a human IgM Fc (amino acid sequence of SEQ ID NO: 78). In one embodiment, the Fc region is a modified human IgM Fc without N-terminal “I” (amino acid sequence of SEQ ID NO: 79). See, also, US Provisional Patent Application No. 63/087,053, filed October 2, 2020. In one embodiment, the Fc region is an engineered Fc fragment.
  • An antibody “hinge region” is a flexible amino acid portion of the heavy chains of IgG and IgA immunoglobulin classes, which links these two chains by disulfide bonds.
  • immunoglobulin molecule is a protein containing the immunologically-active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen.
  • the immunoglobulin molecules described herein as IgM may be used in regimens including immunoglobulins of other types, e.g., IgG, IgE, IgM, IgD, IgA and IgY, classes (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclasses.
  • immunoglobulins of other types e.g., IgG, IgE, IgM, IgD, IgA and IgY, classes (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclasses.
  • antibodies and “immunoglobulin” may be used interchangeably herein.
  • a “polypeptide J chain” is referred to a joining chain, which is a polypeptide comprising in IgM antibodies, wherein the polypeptide J chain regulates the antibody formation for isotype IgA or IgM.
  • the J chain is of sequence identified by sequence with NCBI accession Gene ID: 3512 (SEQ ID NO: 127).
  • this chain may not be expressed when the antibody is delivered via a viral vector and expressed in vivo.
  • a “hexavalent structure” is referred to an Fc region of the fusion protein which comprises six monomers of IgM Fc region.
  • a “pentavalent structure” is referred to an Fc region of the fusion protein which comprises five monomers of IgM Fc region and a joining polypeptide J chain.
  • immunoglobulin heavy chain is a polypeptide that contains at least a portion of the antigen binding domain of an immunoglobulin and at least a portion of a variable region of an immunoglobulin heavy chain or at least a portion of a constant region of an immunoglobulin heavy chain.
  • the immunoglobulin derived heavy chain has significant regions of amino acid sequence homology with a member of the immunoglobulin gene superfamily.
  • the heavy chain in a Fab fragment is an immunoglobulin-derived heavy chain.
  • immunoglobulin light chain is a polypeptide that contains at least a portion of the antigen binding domain of an immunoglobulin and at least a portion of the variable region or at least a portion of a constant region of an immunoglobulin light chain.
  • the immunoglobulin-derived light chain has significant regions of amino acid homology with a member of the immunoglobulin gene superfamily.
  • NAb titer a measurement of how much neutralizing antibody (e.g., anti-AAV NAb) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV).
  • Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009, 199 (3): p. 381-390, which is incorporated by reference herein.
  • Methods suitable for assessing antibodies that bind to ACE2 extracellular domain include those of Enzyme Linked Immunosorbent Assay (ELISA). Specifically, NOVUS NBP2 human ACE-2 ELISA Chemiluminescent Kit, which can specifically detect human ACE2 in various samples such as serum, plasma and other biological fluids (novusbio.eom/products/ace-2-elisa-kit_nbp2-66387#datasheet). The kit is utilized according to manufacturing instruction.
  • ELISA Enzyme Linked Immunosorbent Assay
  • sc refers to self-complementary.
  • Self-complementary AAV refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template.
  • dsDNA double stranded DNA
  • operably linked refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • heterologous when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid.
  • the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene.
  • the promoter is heterologous.
  • a “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.
  • a “recombinant AAV” or “rAAV” is a nuclease-resistant viral particle containing two elements, an AAV capsid and a vector genome containing at least non-AAV coding sequences packaged within the AAV capsid.
  • the capsid contains about 60 proteins composed of vpl proteins, vp2 proteins, and vp3 proteins, which self-assemble to form the capsid.
  • “recombinant AAV” or “rAAV” may be used interchangeably with the phrase “rAAV vector”.
  • the rAAV is a “replication-defective virus” or "viral vector”, as it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny.
  • the only AAV sequences are the AAV inverted terminal repeat sequences (ITRs), typically located at the extreme 5’ and 3’ ends of the vector genome in order to allow the gene and regulatory sequences located between the ITRs to be packaged within the AAV capsid.
  • ITRs AAV inverted terminal repeat sequences
  • nuclease-resistant indicates that the AAV capsid has assembled around the expression cassette which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.
  • a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome.
  • a stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected.
  • a “vector genome” refers to the nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid which forms a viral particle.
  • a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ ITR, coding sequence(s), and an AAV 3’ ITR.
  • ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected.
  • the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV.
  • the vector genome contains regulatory sequences which direct expression of the gene products. Suitable components of a vector genome are discussed in more detail herein.
  • the vector genome is an expression cassette having inverted terminal repeat (ITR) sequences necessary for packaging the vector genome into the AAV capsid at the extreme 5’ and 3’ end and containing therebetween an hAce2 decoy as described herein operably linked to sequences which direct expression thereof.
  • ITR inverted terminal repeat
  • non-viral particles used in manufacture of a rAAV will be referred to as vectors (e.g., production vectors).
  • these production vectors are plasmids, but the use of other suitable genetic elements is contemplated.
  • Such production plasmids may encode sequences expressed during rAAV production, e.g., AAV capsid or rep proteins required for production of a rAAV, which are not packaged into the rAAV.
  • such a production plasmid may carry the vector genome which is packaged into the rAAV.
  • an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
  • a biologically useful nucleic acid sequence e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.
  • regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
  • “operably linked” sequences include both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis nucleic acid sequence.
  • Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal.
  • the expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region (3’ UTR) comprising a polyadenylation site, among other elements.
  • the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, wherein the regulatory sequences are separated from nucleic acid sequence of a gene product by an intervening nucleic acid sequences, i.e., 5 ’-untranslated regions (5’UTR).
  • the expression cassette comprises nucleic acid sequence of one or more of gene products.
  • the expression cassette can be a monocistronic or a bicistronic expression cassette.
  • the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell.
  • such an expression cassette can be used for generating a viral vector and contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
  • a vector genome may contain two or more expression cassettes.
  • translation in the context of the present invention relates to a process at the ribosome, wherein an mRNA strand controls the assembly of an amino acid sequence to generate a protein or a peptide.
  • RNA or of RNA and protein are used herein in its broadest meaning and comprises the production of RNA or of RNA and protein. Expression may be transient or may be stable.
  • nucleic acid indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences.
  • the homology is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.
  • sequence identity “percent sequence identity” or “percent identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
  • percent sequence identity may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof.
  • a fragment is at least about 8 amino acids in length and may be up to about 700 amino acids. Examples of suitable fragments are described herein.
  • the term “substantial homology” or “substantial similarity,” when referring to amino acids or fragments thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions with another amino acid (or its complementary strand), there is amino acid sequence identity in at least about 95 to 99% of the aligned sequences.
  • the homology is over full-length sequence, or a protein thereof, e.g., an immunoglobulin region or domain, an AAV cap protein, or a fragment thereof which is at least 8 amino acids, or more desirably, at least 15 amino acids in length. Examples of suitable fragments are described herein.
  • highly conserved is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art.
  • aligned sequences or alignments refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Examples of such programs include, “Clustal Omega”, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet.
  • nucleotide sequence identity can be measured using FastaTM, a program in GCG Version 6. 1.
  • FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).
  • an “effective amount” may refer to the amount of the rAAV composition which delivers and expresses in the target cells an amount of decoy receptor sufficient to reduce or prevent SARS-CoV2 infection and/or one or more symptoms thereof. Additionally, an “effective amount” may refer to the amount of recombinant protein composition which delivers an amount of decoy receptor sufficient to reduce or prevent SARS-CoV2 infection and/or one or more symptoms thereof. An effective amount may be determined based on an animal model, rather than a human patient. Examples of a suitable murine model are described herein.
  • target tissue refers to a tissue, an organ or a cell type, that an embodiment, a regimen or a composition as described herein targets.
  • the target tissue is a respiratory organ or a respiratory tissue.
  • the target tissue is lung.
  • the target tissue is nose, e.g., nasal epithelial.
  • the target tissue is nasopharynx.
  • the target tissue is respiratory epithelium.
  • the target tissue is nasal airway epithelium.
  • the target tissue is nasal cells.
  • the target tissue is nasopharynx cells.
  • the target tissue is nasal epithelial cells, which may be ciliated nasal epithelial cells, columnar epithelial cells, goblet cells (which secrete mucous onto the surface of the nasal cavity which is composed of the ciliated epithelial cells) and stratified squamous nasal epithelial cells which line the surface of the nasopharynx.
  • the target tissue is lung epithelial cells.
  • the target tissue is muscle, e.g., skeletal muscle.
  • the term “about” when used to modify a numerical value means a variation of ⁇ 10%, ( ⁇ 10%, e.g., ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ 10, or values therebetween) from the reference given, unless otherwise specified.
  • the term “E+#” or the term “e+#” is used to reference an exponent.
  • “5E10” or “5el0” is 5 x IO 10 . These terms may be used interchangeably.
  • a refers to one or more, for example, “an enhancer”, is understood to represent one or more enhancer(s).
  • the terms “a” (or “an”), “one or more,” and “at least one” is used interchangeably herein.
  • variants of SARS-CoV-2 emerged and were characterized by increased binding to the angiotensin-converting enzyme 2 (ACE2) receptor as well as enhanced pathogenicity and transmissibility. These variants can circumvent preexisting immunity to COVID- 19 infections, which suggests that first- generation vaccines and monoclonal antibodies may now be less effective.
  • ACE2 angiotensin-converting enzyme 2
  • AAV adeno- associated viral
  • ACE2 decoy is not only highly active against all relevant CoV-2 variants, but also against other CoV members of the Clade 1 betacoronavirus family.
  • Intranasal delivery of an AAV expressing the affinity -matured ACE2 decoy significantly diminished clinical and pathologic consequences of a SARS-CoV-2 challenge in a mouse model.
  • ACE2 decoys soluble form of angiotensin-converting enzyme 2 -are considered a protein therapeutic in the treatment of COVID- 19 patients [Chan KK, et al, An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants. Sci Adv. 2021;7(8). Epub 2021/02/19. doi: 10.1126/sciadv.abfl738.
  • AAV adeno-associated virus
  • Example 1 Engineering an ACE2 decoy variant with enhanced activity for intranasal gene therapy to prevent infection by SARS-CoV-2 variants
  • ACE2 decoy affinity maturation of ACE2 decoy enhances neutralization 100-fold of
  • FIG. 22A to 22 E shows characterization of initial ACE2 decoy construct.
  • FIG. 22A shows results for construct expressed in HEK293 cells and detected in supernatant using a sandwich ELISA to ACE2 for constructs without an Fc domain.
  • FIG. 22B shows result of an ELISA with SARS-CoV-2 spike protein as a capture antigen and an anti -human IgG polyclonal antibody used for detection of hAce2 with Fc fusion proteins expressed in HEK293 cells.
  • FIG. 22C shows the purified ACE2-NN-Fc4 protein was titrated against Wuhan CoV2 pseudotyped lentivirus bearing a luciferase reporter. The IC50 was obtained from a fit of these data (15 pg/ml).
  • 22D shows the candidate construct (ACE2-NN-Fc4) was packaged in an AAV vector (hu68 capsid) and administered IN to WT mice.
  • BAL was collected for measurement of transgene expression using an ELISA with SARS-CoV-2 spike protein as a capture antigen to confirm that the decoy receptor expressed in vivo was functional.
  • BAL from similar experiments was 6-fold diluted from the ASF as determined by comparison of BAL and serum urea. Thus, we determined that ASF concentrations of the decoy were likely below 2 pg/ml.
  • FIG. 22E shows two NHPs (IDs 258 and 396) received 9 x 10 12 GC of an AAVhu68 vector expressing a soluble ACE2-NN-Fc fusion protein via the MAD .
  • Nasal lavage samples were collected weekly after vector administration and concentrated 10-fold for analysis.
  • the concentration of the decoy receptor in NLF was measured by MS.
  • Urea measurements in similar experiments indicate that 10X nasal lavage is ⁇ 8-fold diluted from ASF. We therefore determined that ASF concentrations of the decoy were less than 100 ng/ml. We therefore set out to affinity -mature the ACE2 protein sequence.
  • PubMed PMID: 22806834] which we screened using stringent off-rate sorting [Boder ET, Midelfort KS, Wittrup KD. Directed evolution of antibody fragments with monovalent femtomolar antigenbinding affinity. Proc Natl Acad Sci U S A. 2000;97(20): 10701-5. Epub 2000/03/14. doi: 10. 1073/pnas. 170297297. PubMed PMID: 10984501; PubMed Central PMCID: PMCPMC27086.] (FIG. 18B).
  • ACE2 variants from several stages of the yeast-display screening as soluble IgG4 Fc fusions, evaluated expression titers, and predicted ICso for SARS-CoV-2 neutralization using reporter virus (FIGs. 24A to 24C).
  • the most potent neutralizing variants converged upon similar substitutions at five positions: 31, 35, 79, 330, and N90 glycan disruption (top 5 accumulated mutations in hAce2 decoy (from molecular library) are shown in a table immediately below).
  • hAce2-Variantl-Fc4 CDY14-FC4
  • H345L we ablated ACE2 enzyme activity by introducing H345L at no cost to potency
  • hAce2-Variant2-Fc4 bound SARS-CoV-2 RBD with 1,000-fold improved affinity (29 nM for wtACE2 vs. 31 pM for hAce2-Variant2-Fc4; see FIG. 18D and FIG. 18E).
  • hAce2-Variant2-Fc4 neutralized Wuhan-Hu- 1 SARS-CoV-2 reporter nearly 300-fold better than the un-engineered ACE2 decoy (ICso 127 ng/ml for hAce2- hAce2-Variant2-Fc4 (CDY14HL- Fc4) vs. 11 pg/ml for ACE2-wt-Fc4; see FIG. 18F).
  • ACE2 decoy is effective against SARS-CoV-2 variants and SARS-CoV-1
  • decoy inhibitors may achieve broad neutralization and escape mutant resistance; changes that reduce decoy binding would also decrease ACE2 receptor binding, thus reducing viral fitness.
  • Escape mutations at the immunodominant ACE2 binding site of the RBD is of major concern for emerging SARS-CoV-2 variants [Starr, T. N. et al. Prospective mapping of viral mutations that escape antibodies used to treat COVID-19. Science 371, 850-854, doi: 10. 1126/science.abf9302 (2021)].
  • RBDs from more distant ACE2- dependent CoVs also differ substantially from the original SARS-CoV-2 at the ACE2 interface (FIG. 19A).
  • decoys should maintain neutralizing potency even as viruses evolve tighter binding to their receptor to achieve more efficient infection. Mutations at six sites at the RBD:ACE2 interface have arisen in multiple independent SARS-CoV-2 lineages ( Figure 19A). To varying degrees, mutations at these sites have been reported to confer enhanced ACE2 binding, transmissibility, virulence, and escape from first-generation monoclonal antibody therapeutics or even active vaccines. To assess the potential of our decoy for broad neutralization, we measured ACE2-Fc4 (a surrogate for the native receptor) and hAce2-Variant2-Fc4 (CDY14HL- Fc4) (the therapeutic decoy) affinities across a diverse panel of CoV RBDs using SPR.
  • ACE2-Fc4 a surrogate for the native receptor
  • hAce2-Variant2-Fc4 CDY14HL- Fc4
  • hACE2 TG mouse model We therefore selected the hACE2 TG mouse model for three reasons: 1) we can characterize disease by measuring viral loads, clinical sequalae, and histopathology; 2) we can use an IN route of administration as we would in humans, realizing this deposits vector in the proximal and distal airways of the mouse, while IN delivery in humans is restricted to the proximal airway; and 3) we can leverage the extensive experience of murine models in de-risking human studies of AAV gene transfer.
  • AAVhu68-hAce2-Variant2-Fc4 AAVhu68- CDY14HL- Fc4
  • vehicle hACE2-TG mice
  • SARS-CoV-2 280 pfu
  • observation and daily weights mice were challenged with SARS-CoV-2 (280 pfu), followed clinically (observation and daily weights), and necropsied on days 4 and 7 after challenge for tissue and BAL analysis (FIG. 20D).
  • SARS-CoV-2 280 pfu
  • FIG. 20D Expression of hAce2-Variant2-Fc4 (CDY14HL-Fc4) in BAL normalized for dilution was in the range of the IC50 measured in vitro and in the pilot studies (FIG 20F).
  • Sham-treated SARS-CoV-2 challenged animals demonstrated statistically significant weight loss as has been described by others [Winkler, E. S. et al. SARS-CoV-2 infection of human ACE2- transgenic mice causes severe lung inflammation and impaired function. Nature Immunology 21, 1327-1335, doi: 10.1038/s41590- 020-0778-2 (2020); Zheng, J. et al. COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice. Nature 589, 603-607, doi: 10. 1038/s41586-020-2943-z (2021); Yinda, C. K. et al. K18-hACE2 mice develop respiratory disease resembling severe COVID-19.
  • FIG. 20G Compared to vehicle-treated animals, viral RNA in BAL and lung homogenate was diminished at day 4 and 7 in AAVhu68-hAce2-Variant2-Fc4 (AAVhu68.CDY14HL-Fc4) treated animals (FIGs. 20H and 201). The greatest reductions were at day 7 for both BAL (26-fold) and lung tissue (35- fold).
  • AAV delivery yields therapeutic ACE2 decoy levels in nonhuman primates
  • AAV capsid is most efficient at transducing cells of the nonhuman primate (NHP) proximal airways — the desired cellular targets for COVID-19 prophylaxis following nasal delivery of vector.
  • NHP nonhuman primate
  • MAD NasalTM intranasal mucosal atomization device
  • a mixture of 9 AAV serotypes with uniquely barcoded transgenes were administered via the MAD NasalTM to an NHP. Tissues were harvested 14 days later for evaluation of relative transgene expression using the mRNA bar-coding technique [Zheng J, Wong, et al.
  • AAVrh91 The novel Clade A capsid (AAVrh91) we isolated from macaque liver performed best in the nasopharynx and septum (FIGs. 21A and 2 IB) with low but detectable expression levels in large airways and distal lung (FIGs.26A to 261). Clade E and F capsids performed better than AAVrh91 in some non-target tissues such as distal lung (FIGs.26A to 261).
  • the profile of expression from AAVrh91 illustrates relative distribution of transgene expression with proximal airway structures>intra-pulmonary conducting airway >distal lung (FIG. 21C).
  • NHP study To determine the candidate for clinical evaluation, we conducted a final NHP study where groups of 2 animals were administered 5x10 12 GC of vectors that differed with respect to capsid (AAVhu68 vs. AAVrh91) and transgene cassettes (hAce2-Variantl-Fc4 (CDY14- Fc4) vs. hAce2-Variant2-Fc4 (CDY14HL-Fc4)). NLFs were harvested on days 7, 14, and 28, and animals were necropsied on day 28 for biodistribution.
  • hAce2-Variant2-Fc4 (CDY14HL-Fc4).
  • a subset of samples evaluated for binding to the spike protein of SARS-CoV-2 showed a good correlation with decoy protein as measured by MS. This indicates that the decoy protein produced in vivo in proximal airways is indeed functional (FIG. 21E).
  • the density of ACE2 in the nose and airway has been linked to pathogenicity and transmissibility of SARS-CoV-2 [Zhou D, Dejnirattisai W, Supasa P, Liu C, Mentzer AJ, Ginn HM, et al. Evidence of escape of SARS-CoV-2 variant B. 1.351 from natural and vaccine-induced sera. Cell. 2021. Epub 2021/03/18. doi:
  • SARS-CoV-2 variants achieve greater infection and transmission through increased affinity [Adachi, et al, cited above; Bunyavanich S, et al, Nasal Gene Expression of Angiotensin-Converting Enzyme 2 in Children and Adults. JAMA. 2020;323(23):2427-9. Epub 2020/201721. doi: 10.100 l/jama.2020.8707. PubMed PMID: 32432657; PubMed Central PMCID: PMCPMC7240631]; here we confirm increased affinity for ACE2 in SARS-CoV-2 strains under positive selection during 2020.
  • the engineered decoy may be the Achilles’ heel of any ACE2-dependent CoV whose primary driver of fitness - higher binding to its receptor - should further enhance the potency of the ACE2 decoy.
  • PubMed PMID 33093202
  • PubMed Central PMCID PMCPMC7668070
  • Adam VS et al., Adeno-associated virus 9-mediated airway expression of antibody protects old and immunodeficient mice against influenza virus.
  • hAce2-Variant2-Fc4 (CDY14HL-Fc4) is in the prevention and treatment of future outbreaks caused by new CoVs that utilize ACE2 as a receptor.
  • AAVrh91-hAce2- Variant2-Fc4 could be rapidly deployed from stockpiles to contain the initial outbreak and the hAce2-Variant2-Fc4 (CDY14HL-Fc4) protein can be leveraged to improve outcomes in those who are infected.
  • the hAce2-Variant2-Fc4 (CDY14HL-Fc4) products may be useful in the current COVID-19 pandemic if SARS-CoV-2 variants confound current treatment and prevention strategies.
  • An immediate application could be in immune-suppressed individuals who do not respond to traditional vaccines, develop chronic infection with SARS-CoV-2, and may be reservoirs for new variants [Kemp SA, et al. SARS-CoV-2 evolution during treatment of chronic infection. Nature. 2021. Epub 2021/02/06. doi: 10.1038/s41586-021-03291-y. PubMed PMID: 33545711],
  • the advantage of vector-expressed decoy in preventing COVID- 19 infections in immune-suppressed individuals is that this therapy does not rely on the recipient’s adaptive immune system to be effective.
  • Yeast display We generated mutagenized ACE2 gene fragments by error prone PCR using the Diversify PCR Random Mutagenesis Kit (TakaraBio) at multiple mutation levels, mixing the PCR products.
  • the plasmid contained an upstream Aga2 gene fragment, a downstream HA epitope tag with flexible GSG linkers, and was driven by an inducible GAL 1 promoter, and contained a low-copy centromeric origin, similar to pTCON2 [Chao G, Lan WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD. Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006;l(2):755-68. Epub 2007/04/05. doi: 10. 1038/nprot.2006.94.
  • hAce2- Variantl-Fc4 CDY14-Fc4
  • hAce2-Variant2-Fc4 CDY14HL-Fc4
  • We determined the concentration using the predicted extinction coefficient at 280nm (1 g/1 1.995).
  • Some neutralization measurements (figure not shown) used crude decoy protein tittered using and human IgG4 ELISA kit; this typically yielded higher IC50s.
  • Soluble hACE2Fc (produced in-house) was spiked at different levels (0.5-500 ng/mL) into PBS or NLF acquired from a naive rhesus macaque. Samples were denatured and reduced at 90°C for 10 minutes in the presence of lOmM dithiothreitol (DTT) and 2M Guanadinium- HC1 (Gnd-HCl). We cooled the samples to room temperature, then alkylated samples with 30mM iodoacetamide (IAM) at room temperature for 30 minutes in the dark. The alkylation reaction was quenched by adding IpL DTT.
  • DTT dithiothreitol
  • Gnd-HCl 2M Guanadinium- HC1
  • the column size was 75 cm x 15 urn I.D. and was packed with 2-micron C18 media (Acclaim PepMap). Due to the loading, lead-in, and washing steps, the total time for an LC-MS/MS run was about 2 hours.
  • BioPharma Finder 1.0 software (Thermo Fischer Scientific) to analyze all data.
  • BioPharma Finder 1.0 software (Thermo Fischer Scientific) to analyze all data.
  • peptide mapping we used a single-entry protein FASTA database to perform searches. The mass area of the target peptide was plotted against the spike concentration to complete a standard curve.
  • ANHYEDYGDYWR SEQ ID NO: 84
  • test articles lx or lOxNLF and/or bronchoalveolar lavage fluid (BAL) is treated as previously described without any dilution or protein precipitation.
  • BAL bronchoalveolar lavage fluid
  • SARS-CoV-2 Spike Protein RBD (Sinobio #40592-V08H) was immobilized on a 96- well plate (0.25ug/mL in PBS, lOOul/well) at 4°C overnight. Plates were then washed 5x with PBS/0.05% Tween and blocked with PBS/1.0% BSA for 1 hour with shaking. Samples (2x dilution in PBS/2.0% BSA) and standards (soluble hACE2-Fc at starting concentration lOOng/ml, 12-point, 1:2 serial dilution, plus a O.Ong/ml blank, in PBS/1.0% BSA) were added at lOOul/well in duplicate and incubated for 2 hours at room temperature with shaking.
  • Soluble hACE2Fc was spiked into NLF (0.0, 0.5, 2.0, and 10.0 ng/ml) acquired from a naive rhesus macaque on the same plate with a standard curve (soluble hACE2Fc starting concentration lOOng/ml, 12-point, 1:2 serial dilution, plus a O.Ong/ml blank) in PBS/1.0% BSA.
  • NLF 0.0, 0.5, 2.0, and 10.0 ng/ml
  • a standard curve soluble hACE2Fc starting concentration lOOng/ml, 12-point, 1:2 serial dilution, plus a O.Ong/ml blank
  • mice were purchased from The Jackson Laboratory. Anesthetized mice received an IN (intranasal) administration of 10 11 GC of AAVhu68-hAce2-Variantl-Fc4 (AAVhu68.CDY14- Fc4), AAVrh91-hAce2-Variantl-Fc4 (AAVrh91.CDY14-Fc4), AAVhu68-hAce2-Variant2- Fc4 (AAVhu68.CDY14HL-Fc4), or AAVrh91-hAce2-Variant2-Fc4 (AAVrh91.CDY14HL- Fc) in a volume of 50 pL or the same volume of vehicle control (PBS) on day 0. On day 7, mice were euthanized, and BAL was collected (1 ml of PBS administered intratracheally).
  • PBS vehicle control
  • mice were administered with either vehicle or 10 11 GC of AAVhu68-hAce2-Variant2-Fc4 (AAVhu68.CDY14HL-Fc4) IN on day -7 as described above.
  • mice were administered with mock or the SARS- CoV-2 challenge (50 pl of 2.8xl0 2 pfu of SARS-CoV-2, USA_WAl/2020 isolate [NR-52281, BEI Resources]).
  • mice were euthanized on either day 4 or 7 via cervical dislocation.
  • BAL was collected as described above and aliquoted for viral load assays into Trizol LS (Thermo Fisher Scientific, Waltham, MA) or heat inactivated (60°C for 30 minutes) for decoy protein expression.
  • the lung was collected and split for histopathology into 10% neutral buffered formalin or snap frozen for viral load analysis.
  • RNA extraction for RT-qPCR, the quantitative RT-PCR assay for SARS-CoV-2 RNA, and subgenomic RNA were performed as described [Baum A, Ajithdoss D, Copin R, Zhou A, Lanza K, Negron N, et al.
  • REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters. Science. 2020;370(6520): 1110-5. Epub 2020/10/11. doi: 10.1126/science.abe2402. PubMed PMID: 33037066; PubMed Central PMCID: PMCPMC7857396],
  • Rhesus and cynomolgus macaques were obtained from Primgen (PreLabs). NHP studies were conducted at the University of Pennsylvania or Children’s Hospital of Philadelphia within facilities that are United States Department of Agriculture-registered, Association for Assessment and Accreditation of Laboratory Animal Care-accredited, and Public Health Service- assured.
  • 4xl0 12 GC of the pool AAV preps was delivered IN in a total volume of 0.28 ml to an adult male rhesus macaque using the MAD NasalTM device. After 14 days, we collected airway tissues at necropsy, and extracted total RNA using Trizol Reagent (Thermo Fisher).
  • cynomolgus macaques were administered IN with 5xl0 12 GC of AAVhu68-hAce2-Variantl-Fc4 (AAVhu68.CDY14), AAVrh91-hAce2-Variantl-Fc4 (AAVrh91.CDY14), AAVhu68-hAce2-Variant2-Fc4 (AAVhu68.CDY14HL), or AAVrh91-hAce2-Variant 2-Fc4 (AAVrh91.CDY14HL) as described above.
  • NHPs were administered with 5xl0 n GC of AAVrh91- hAce2-Variant 2-Fc4 (AAVrh91.CDY14HL). All NHPs were negative for pre-existing neutralizing antibody titers to the administered AAV capsid prior to study initiation (Immunology Core at the Gene Therapy Program). Animals were monitored throughout the in-life phase for complete blood counts, clinical chemistries, and coagulation panels by Antech Diagnostics (Lake Success, NY). On days 7, 14, and 28 NLF was collected (animals placed in ventral recumbency with head tilted to the right, up to 5 mL of PBS delivered in ImL aliquots, and fluid collected via gravity). Animals were necropsied on day 28 and a full histopathological evaluation was performed.
  • BIOQUAL Institute Institutional Animal Care and Use Committee IACUC
  • BIOQUAL Institutional Animal Care and Use Committee
  • BIOQUAL is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council. Animals were monitored twice daily for clinical signs (specifically ruffled fur, heavy breathing, lethargy) and weighed daily.
  • This example describes identifying ACE2 variants of Example 1 with increased affinity for the SARS-CoV-2 spike protein, as well as increased stability and expression yield.
  • yeast display is especially appropriate for ACE2 decoy engineering for several reasons.
  • yeast display uses conserved eukaryotic protein secretion pathways in the budding yeast Saccharomyces cerevisiae to fold and export protein variants to the yeast cell surface.
  • large, disulfide-linked extracellular proteins such as ACE2
  • This system also applies selection pressure on the folding and secretion of protein variants that is relevant to the ultimate application in human gene therapy.
  • large libraries (>10 9 variants) of decoy variants can be generated and selected rapidly, allowing the discovery of a small number of rare decoy mutations that improve both target-binding affinity and decoy expression.
  • the improving mutations at each hotspot were allowed to vary in relationship to their observed frequency in the sequencing data set.
  • the frequency for wild type (WT) was set at 10% at each position. Yields included 4, 5, and 6 mutation recombinants with highest likelihood of super-decoy activity.
  • We cloned 50 of these digital recombinants with and without a mutation at a 7 th spot (amino acid 330, based on hAce2 amino acid numbering, SEQ ID NO: 25). About 100 digital recombinants are expressed and screened.
  • yeast clones from the final rounds of sorting, confirmed their binding to SARS-CoV2 RBD by flow cytometry (FIG. 4A), and sequenced the best performing yeast clones.
  • AA position numbering is based on SEQ ID NO: 25, which reproduces the human Ace2 isoform 1 protein of NCBI Reference NP 068576.1; AA position numbering as indicated in parenthesis is based on SEQ ID NO: 81 (without signal/leader peptide); SEQ ID NO in parenthesis is without signal/leader peptide.
  • FIGs. 14 shows estimated IC50 of 6 “revertants” of hAce2- Variantl-IgG4 soluble decoy, wherein each “revertant” comprised of one amino acid substitution reverted from engineered back to wild type at the indicated positions (based on numbering of amino acid sequence of SEQ ID NO: 25).
  • F is the fractional activity of the luciferase reporter for the sample, that is, the level of luciferase activity as compared to a negative control (fully active) and a positive control (fully inhibited), ranging from 0- 1.
  • the [decoy concentration] is the concentration of the decoy in the measurement well. This estimation is only performed with the specific rang of F value, optimally wherein F is between 0.2 and 0.8.
  • FIG. 15A shows estimated IC50 values in comparison to varying “GSG” linker length, or varying decoy length (1-615 versus 1-740 amino acid of hAce2), which is linked at the amino terminus of IgG4 f the soluble decoy fusion protein.
  • FIG. 15B shows a schematic representation of protein interaction structure between hAce2 and RBD of SARS-CoV2, showing amino acids 1-615 and 1-740, as they are mapped onto the structure of protein interaction.
  • Example 4 Therapeutic affinity of hAce2 soluble decoy fusion protein to receptor binding domain (RBD) variant of SARS-CoV2 of Example 1
  • the SARS-CoV2 RBD variants comprise of amino acid mutations at: 439K, 417V (EU), 501Y (UK), 417N, 484K, 501Y (South Africa), 501T (mink), 486L (mink), 453F (mink), and 439R, 455Y, 486L, 493N, 498Y, 501T (CoVl).
  • WT wild-type
  • CoV2 RBD mutants of CoV2 RBD
  • CoV 1 SEQ ID NOs: 43-51
  • FIG. 8A show the measured fold change in binding affinity of modified hAce2- Variantl-IgG4 soluble decoy fusion protein and wild-type hAce2-IgG4 soluble decoy fusion protein for mutant RBD of SARS-CoV2 (affinity to mutant RBD of SARS-CoV2 over affinity to wild type RBD of SARS-CoV2).
  • FIG. 7 shows a graph comparison of mutant decoy affinity (hAce2-Variant2-IgG4) versus wild-type (WT) decoy affinity to various mutant RBD of SARS-CoV2.
  • This binding analysis was performed using SPR binding analysis using a Biacore T200 instrument (GE Healthcare) at room temperature in HBS-EP(+) buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20 surfactant, Cat# BR100669, Cytiva) using a protein A/G derivatized sensor chip (Cat# SCBS PAGHC30M, XanTec Bioanalytics).
  • FIG. 13A shows inhibition of SARS-CoV-2 RBD-hAce2 interactions in presence engineered hAce2 soluble decoy, hAce2-Variant2-IgG4.
  • FIG. 13B shows a plot of IC50 values for interaction inhibition between hAce2-Variant2-IgG4 and SARS-CoV2-RBD mutant variants, as measured in neutralization assay. Additional results for hAce2-Variant-2-IgG4 are provided in the following table.
  • Example 5. rAAV-mediated expression of engineered Ace2 decoys of Example 1
  • hAce2 decoys hAce2-Variantl-IgG4 (Variantl), hAce2- Variant2-IgG4 (Variant2)
  • AAV packaging plasmids comprising a vector genome (FIG. 9) and manufactured rAAVhu68 or AAVrh91 using conventional triple transduction techniques, i.e., co-expression of the plasmid encoding the vector genome to be packaged, a plasmid expressing the rep and the AAV capsid coding sequences (AAVhu68 or AAVrh91, respectively) and a plasmid having helper functions.
  • FIG 10A to 10C is on BALF samples collected at day 7 post-transduction. Analysis of the BALF samples indicates that rAAVs encoding the engineered decoy-Fc fusions when dosed intranasally are capable of generating concentrations of active decoy protein in the air-surface liquid (ASL) of mice (FIG 10A). Decoy recovered from the mouse ASL is capable of binding the CoV2 spike protein and neutralizing CoV2 reporter virus (FIGs 10B and 10C). It is important to note that the BALF fluid is collected by introducing a volume of PBS to the lungs of the mice, and then recovering the buffer solution with a syringe. Thus, the BALF represents a significant dilution of the ASL with the PBS buffer. We expect concentrations of the active decoy in the actual ASL to be significantly higher than in the BALF.
  • the NLF fluid is collected by introducing a volume of PBS to the nasal passages of the sedated NHP, and then recovering the buffer solution.
  • the NLF represents a significant dilution of the nose ASL with the PBS buffer.
  • concentrations of the active decoy in the actual NHP nose ASL to be significantly higher than in the NLF.
  • FIGs. 11A to 1 IB show analytics from NHP study, intranasal delivery of 5xl0 12 GC of rAAVs encoding hAce2-Variantl/2-IgG4-Fc of mutant decoys.
  • the capsids used were AAVhu68 and AAVrh91.
  • the engineered decoys were hAce2-Variantl-IgG4 (Variantl) and hAce2-Variant2-IgG4 (Variant2), the latter of which is a variant with the H345L mutation which inactivates the Ace2 enzymatic activity.
  • FIG. 11A to 1 IB show analytics from NHP study, intranasal delivery of 5xl0 12 GC of rAAVs encoding hAce2-Variantl/2-IgG4-Fc of mutant decoys.
  • the capsids used were AAVhu68 and AAVrh91.
  • FIG. 11A shows CoV2 spike binding activity in the lx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 1 IB shows CoV2 spike binding activity in the lOx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 11C shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF IX samples (AAVrh91) from 2 animals in each doing group.
  • FIG. 1 ID shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF 10X samples (AAVrh91) from 2 animals in each doing group.
  • FIG. 1 IE shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF IX samples (AAVhu68) from 2 animals in each doing group.
  • FIG. 1 IF shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF 10X samples (AAVhu68) from 2 animals in each doing group.
  • FIGs. 12A to 12D show analytics from NHP study, intranasal delivery of 5xl0 12 GC of rAAVs encoding hAce2-Variantl/2-IgG4-Fc of mutant decoys.
  • the capsids used were AAVhu68 and AAVrh91. This data is on NLF samples collected at day 14 post-transduction.
  • FIG. 12A shows CoV2 spike binding activity in the lx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 12A shows CoV2 spike binding activity in the lx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 12B shows CoV2 spike binding activity in the lOx BALF samples (AAVrh91), wherein an immobilized recombinant CoV2 spike protein in an ELISA assay, with the human Fc tag on the decoy as the detection epitope.
  • FIG. 12C shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF IX samples (AAVrh91) from 2 animals in each doing group.
  • FIG. 12D shows mass spectrometry with Ace2-IgG4 standards to determine the concentration of decoy in NLF 10X samples (AAVrh91) from 2 animals in each doing group.
  • AAVrh91.CB7.CI.hAce2GTP14HL-IgG.rBG (AAVrh91.CDY14HL-Fc4; comprising hAce2-Variant2-IgG4 (Variant2))) is a final drug product (FDP) that consists of a nonreplicating recombinant adeno-associated viral (rAAV) vector active ingredient and a formulation buffer.
  • FDP final drug product
  • ITR Inverted Terminal Repeat
  • AAV2 130 base pairs [bp], GenBank: NC_001401
  • the ITRs function as both the origin of vector DNA replication and the packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans.
  • the ITR sequences represent the only cis sequences required for vector genome replication and packaging.
  • CB7 promoter This promoter is composed of a hybrid between a CMV IE enhancer and a chicken P-actin promoter (SEQ ID NO: 122).
  • CMV IE Human Cytomegalovirus Immediate-Early Enhancer
  • CB Chicken -Actin Promoter
  • the hybrid intron consists of a chicken P-actin splice donor (973 bp, GenBank: X00182.1) and a rabbit P-globin splice acceptor element.
  • the intron is transcribed, but removed from the mature messenger ribonucleic acid (mRNA) by splicing, bringing together the sequences on either side of it.
  • mRNA messenger ribonucleic acid
  • the presence of an intron in an expression cassette has been shown to facilitate the transport of mRNA from the nucleus to the cytoplasm, thus enhancing the accumulation of a steady level of mRNA for translation. This is a common feature in gene vectors intended for increased levels of gene expression (SEQ ID NO: 123).
  • Coding sequence The coding sequence consists of a portion of the complementary deoxyribonucleic acid (cDNA) of the human ACE2 gene encoding a secreted version of the human ACE2 protein (1845 bp; 615 amino acids [aa], GenBank: AB 193259.1), which is the receptor protein used by SARS-CoV-2 for cell entry.
  • the secreted version of human ACE2 protein has been mutated to eliminate peptidase activity and improve antiviral potency.
  • the Fc from IgG4 was chosen to have reduced or no effector function.
  • rBG PolyA Rabbit P-Globin Poly adenylation Signal
  • the rBG polyadenylation (PolyA) signal (127 bp, GenBank: V00882.1) facilitates efficient polyadenylation of the transgene mRNA in cis. This element functions as a signal for transcriptional termination, a specific cleavage event at the 3' end of the nascent transcript and the addition of a long polyadenylation tail (SEQ ID NO: 124).
  • FDP is delivered via IN administration using a MAD NasalTM Device into each nostril (referred to as nare hereafter).
  • nare a MAD NasalTM Device into each nostril
  • Two aliquots of 0.2 mL each are administered into each nare for a total administered volume of 0.4 mL per nare, leading to a total administered volume of 0.8 mL per subject.
  • the three dose cohorts include: Cohort 1 (low dose - 5.0 x 10 10 GC or 3.00 x 10 11 GC), Cohort 2 (mid dose - 5.0 x 10 11 GC or 1.00 x 10 12 GC), and Cohort 3 (high dose - 5.0 x lO 12 GC or 3.00 x 10 12 GC).
  • the product for the Phase 1 FIH clinical trial is manufactured by transient transfection of HEK293 cells using plasmid DNA followed by downstream purification.
  • FIGs. 22A to 22E show design and characterization of initial ACE2 decoy construct.
  • ACE2 decoy constructs contained the extracellular domain of ACE2 (amino acids 18-615) with one of three candidate signal peptides (IL-2, native, or thrombin).
  • IL-2 candidate signal peptides
  • two catalytic histidine residues were mutated to asparagine to abrogate enzymatic activity (designated NN).
  • Constructs were designed with no Fc, or the Fc domain of IgGl or IgG4.
  • Constructs were expressed in HEK293 cells and detected in supernatant using a sandwich ELISA to ACE2 (for constructs without an Fc domain) or an ELISA with SARS-CoV-2 spike protein as a capture antigen and an anti-human IgG polyclonal antibody for detection (for Fc fusion proteins).
  • the candidate ACE2-NN-Fc4 fusion protein was expressed in HEK293 cells, purified by protein A chromatography, and analyzed by SDS PAGE under reducing and nonreducing conditions. The affinity of the purified ACE2-NN-Fc4 decoy protein for monomeric spike protein in solution was quantified by Biacore SPR.
  • the purified ACE2-NN-Fc4 protein was titrated against Wuhan CoV2 pseudotyped lentivirus bearing a luciferase reporter. The ICso was obtained from a fit of these data (15 ug/ml).
  • the candidate construct (ACE2-NN-Fc4) was packaged in an AAV vector (hu68 capsid) and administered IN to WT mice.
  • BAL was collected for measurement of transgene expression using an ELISA with SARS-CoV-2 spike protein as a capture antigen to confirm that the decoy receptor expressed in vivo was functional.
  • BAL from similar experiments was 6-fold diluted from the ASF as determined by comparison of BAL and serum urea. Thus, we determined that ASF concentrations of the decoy were likely below 2 ug/ml.
  • Two NHPs IDs 258 and 396) received 9 x 10 12 GC of an AAVhu68 vector expressing a soluble ACE2-NN-Fc fusion protein via the MAD.
  • Nasal lavage samples were collected weekly after vector administration and concentrated 10-fold for analysis. The concentration of the decoy receptor in NLF was measured by MS. Urea measurements in similar experiments indicate that 10X nasal lavage is ⁇ 8-fold diluted from ASF. We therefore determined that ASF concentrations of the decoy were less than 100 ng/ml.
  • FIGs. 23A to 23D show design and selection of primary and secondary yeast display libraries. Structure of CoV-2 RBD (blue spheres) bound to human ACE2 (green ribbons, red and yellow spheres) (6M17.pdb [Yan, R. et al. Structural basis for the recognition of SARS- CoV-2 by ful llength human ACE2. Science 367, 1444-1448, doi: 10. 1126/science.abb2762 (2020)]). Most ACE2 contacts with RBD are limited to the amino acids 18-88 (red spheres) and a patch of amino acids that are more C terminal (yellow spheres). The gene sequence for ACE2 is shown below in the same coloring.
  • FIGs. 24A and 24C show a schematic of parallel paths to the generation of affinity matured ACE2 decoy.
  • Primary library hits, digital recombinants of those hits and isolated clones from the second, more stringent round of yeast display sorting were all cloned as Fc4-fusion proteins and screened in a CoV2 pseudotype neutralization assay.
  • a pool of plasmid DNA from those 90 isolates was subjected to deep sequencing for mutational analysis, and the rates of all possible amino acid substitutions are presented in this heat map by amino acid position. Black squares represent the wt amino acid at each position.
  • the goal was to generate a collection of synthetic (digital) recombinants of the observed mutations in this data set.
  • FIGs. 25A and 25B show challenge study in mice. Average weight loss (percentage) in males and females hACE2-TG mice that received Challenge Placebo and Challenge Decoy.
  • FIGs. 26A to 261 show AAV capsid selection for NHP IN delivery.
  • Data show the enrichment score (tissue abundance in RT-PCR-NGS/ injection mixture abundance in PCR- NGS) for all 4 barcodes per capsid with mean and SD.
  • Four NHP were IN dosed with rh91 or hu68 vectors encoding decoy transgenes at 5xl0 12 GC.
  • Data show the biodistribution of vector genomes in airway tissues 28 days after dosing.
  • AAVrh91 achieved higher gene transfer in upper airway tissues, particularly in the maxillary sinuses and cavity septum. Gene transfer in lower airway tissues was more variable.
  • the purpose of the 120-day study is to evaluate the safety, tolerability, biodistribution, and excretion of AAVrh91.CB7.CI.hAce2GTP14HL-IgG.rBG following inranasal (IN) administration using the MAD110 Nasal Device in an anatomically relevant model of adult cynomolgus macaque NHPs.
  • Safety pharmacology CNS, cardio/respiratory and renal
  • PK pharmacokinetics
  • immunological readouts immunological readouts
  • expression data neutralization
  • BAL nasal lavages
  • angiotensin related assessments at 30 days and 120 days post administration is examined.
  • Table below shows a summarized layout of the IND-enabling NHP Pharm/Tox study.
  • Intranasal (IN) administration is performed using the MAD Nasal device. A total volume of 0.28 mL will be administered, which will be delivered in four aliquots of 0.070 mL (two aliquots per nare).
  • F female; FFB, final formulation buffer; GC, genome copies; FDP (final drug product), AAVrh91.CB7.CI.hAce2GTP14HL-IgG4.rBG; IN, intranasal; M, male; N, number of animals; N/A, not applicable, ROA, route of administration.
  • AAVrh91.CB7.CI.hAce2GTP14HL-IgG4.rBG to interact with the renin angiotensin system, angiotensin II, angiotensin-(l-7), and angiotensin-(l-9) is assessed as part of the clinical pathology panel.
  • the expression of the transgene product (i.e., hAce2GTP14HL-IgG4) is measured in serum, NLF, and BALF using a SARS-CoV-2 spike binding assay and MS analysis.
  • NAb responses against the AAVrh91 capsid are measured at baseline to assess the impact on transduction (biodistribution) and throughout the study to assess the kinetics of the NAb response.
  • PBMCs are collected at baseline and every 30 days thereafter to evaluate T cell responses to the capsid and/or transgene product using an interferon gamma (IFN-y) enzyme-linked immunospot (ELISpot) assay.
  • IFN-y interferon gamma
  • ELISpot enzyme-linked immunospot
  • Blood is collected to assess vector distribution, and urine and feces are collected to assess vector excretion (shedding). These samples are collected at frequent time points and quantified by qPCR to enable assessment of the kinetics of vector distribution and excretion post treatment.
  • tissues are selected based on the biodistribution data from above mentioned studies, and include possible target tissues of the airways, along with additional tissues of the nervous system (brain, spinal cord, peripheral nerves, DRG) and highly perfused peripheral organs (such as the liver and kidneys).
  • the dose examined are: low dose - 5.0 x 10 10 GC, mid-dose - 5.0 x 10 11 GC, high dose - 5.0 x 10 12 GC. These examined doses are used for dose scaling from animal subject to human subject, which is based on the surface area of nasal cavity. More specifically, the surface area of the human nasal cavity (0.0181 m 2 ) is approximately 2.94 times greater than the surface area of the NHP nasal cavity (0.00616 m 2 ) [Hoffman G.M., Cracknell S., Damiano J.M., Macri N.P., & Moore S. (2014)]. "Inhalation Toxicology.” Handbook of Toxicology: CRC Press: 233-300).
  • the vector doses are similarly scaled between NHPs and humans based on the surface area of the nasal cavity such that the low dose, mid-dose, and high dose for the planned Phase 1 FIH clinical trial are equivalent to the low dose, mid-dose, and high dose evaluated in the NHP toxicology study, respectively, and summarized in the table below.
  • aClinical doses have been selected to be equivalent to the doses in the planned NHP toxicology study.
  • the clinical doses were extrapolated from NHPs to humans by scaling based on the surface area of the nasal cavity for adult humans (0.0181 m 2 ) versus adult NHPs (0.00616 m 2 ) (Hoffman et al., 2014).
  • the administration volume selected for use in humans is the recommended administration volume for the MAD NasalTM device based on the manufacturer’s procedure guide.
  • the administration volume selected for NHPs was therefore extrapolated from humans to NHPs by scaling based on the surface area of the nasal cavity for adult humans (0.0181 m 2 ) versus adult NHPs (0.00616 m 2 ) (Hoffman et al., 2014).
  • 0.80 mL the volume in which vector will be delivered in the human nose
  • 2.94 0.272 mL.
  • FIG. 34 shows correlation of protein decoy expression in collected NLF following AAVrh91.hAce2GTP14HL-IgG4 administration at various doses (1.02 x 10 11 , 3.40 x 10 12 , and 1.02 x 10 12 GC).
  • FIG. 35 shows hAce2 decoy protein levels in NLF, plotted as ng/mL, measured in NHPs at day 30 and day 120 following AAVrh91.hAce2GTP14HL-IgG4 administration at various doses (1.02 x 10 11 , 3.40 x 1012, and 1.02 x 10 12 GC) in NHPs.
  • FIG. 2 ID shows a comparison of hAce2 decoy protein level expression in NHPs, plotted as ng/mL, following administration with either AAVrh91.CDY14HL-Fc4, AAVrh91.CDY14HL-Fc4, AAVhu68.CDY14HL-Fc4, or AAVhu68.CDY14-Fc4 administered at doses of 5 x 10 12 GC or 5 x 10 11 GC.
  • FIG. 30A shows decoy protein levels as determined by mass spectrometry analysis
  • FIG. 30B shows decoy protein levels as determined by mass spectrometry analysis (ng/mL) post administration of either AAVhu68.GTP14HL or AAVrh91.GTP14HL, and plotted as urea-corrected decoy protein concentration (ng/mL). Furthermore, we performed an additional ferret transduction study to confirm the previously obtained results of the pilot study at a dose of a dose of 2.5xI0 12 GC. The layout of the study is summarized in the table below.
  • Example 10 In Vitro Neutralization of CoV2 variants by engineered hAce2 protein decoy fusion.
  • hACe2-Variant2-IgG4Fc and for the Protein Decoy MR27HL-Fcl (hAce2-MR27HL- Variant-IgGl Fc), against various SARS-CoV2 (CoV2 variants) and presented in the table immediately below as a ICso (IC50) values in ng/mL.
  • FIG. 27 shows a plot of neutralization data as measured across different sampled pools of the purified engineered hAce2 decoy Fcl (IgGl Fc) fusion proteins, and compared with the engineered hAce2 decoy Fc4 (IgG4 Fc) fusion protein.
  • FIG. 28A shows a plot of neutralization data against Wuhan CoV2, as measured across different sampled pools of the purified engineered hAce2 decoy Fcl (IgGl Fc) fusion proteins, and compared with the engineered hAce2 decoy Fc4 (IgG4 Fc) fusion protein.
  • FIG. 28A shows a plot of neutralization data against Wuhan CoV2, as measured across different sampled pools of the purified engineered hAce2 decoy Fcl (IgGl Fc) fusion proteins, and compared with the engineered hAce2 decoy Fc4 (IgG4 Fc) fusion protein.
  • EXAMPLE 11 High Activity of an Affinity Matured ACE2 Decoy against Omicron SARS-CoV-2 and Pre-emergent Coronaviruses
  • Viral sequences can change dramatically during pandemics lasting multiple years. Likewise, evolution over centuries has generated genetically diverse virus families posing similar threats to humans. This variation presents a challenge to drug development, in both the breadth of achievable protection against related groups of viruses and the durability of therapeutic agents or vaccines during extended outbreaks. This phenomenon has played out dramatically during the coronavirus disease 2019 (COVID- 19) pandemic.
  • the highly divergent Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has upended previous gains won by vaccine and monoclonal antibody development.
  • ecological surveys have increasingly revealed a broad class of SARS-CoV-2-like viruses in animals, each poised to cause a future human pandemic.
  • Monoclonal antibody therapeutics with the ability to bind SARS-CoV-2 spike protein and prevent cell entry have been critical tools in managing the COVID- 19 pandemic [Wee AZ, Wrapp D, Herbert AS, Maurer DP, Haslwanter D, Sakharkar M, et al. Broad neutralization of SARS-related viruses by human monoclonal antibodies. Science. 2020;369(6504):731-6; Weinreich DM, Sivapalasingam S, Norton T, Ali S, Gao H, Bhore R, et al. REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19. N Engl J Med.
  • Receptor decoys may provide a mode of viral neutralization that is more resistant to continued viral evolution and escape-mutant generation [Higuchi Y, Suzuki T, Arimori T, Ikemura N, Mihara E, Kirita Y, et al. Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2. Nat Commun. 2021; 12(l):3802], SARS-CoV-2 evolution has occurred in a way that retains tight binding to its primary cell entry receptor, angiotensinconverting enzyme 2 [Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol.
  • CDY14HL-Fc4 (hAce2-Variant2-IgG) contains six (6) amino acid substitutions that improve neutralization of CoV2 variants by 300-fold vs un-engineered ACE2 as well as ablate its endogenous angiotensin-cleaving activity (as described in Examples 1-4, and 7 above). Furthermore, though it was engineered to improve activity against SARS-CoV-2, CDY14HL- Fc4 also maintains tight binding or neutralizing activity for the distantly -related sarbecoviruses WIVl-CoV and SARS-CoVl. This suggests that this decoy may be a useful tool to combat future pandemics from as-yet pre-emergent ACE2-dependent coronaviruses.
  • CDY14HL-Fc4 Maintains Tight Binding to Diverse SARS-CoV-2 Variants
  • Decoy or un-engineered ACE2 are incubated with the yeast, stained with antibodies, and the relative level of decoy binding is assessed by flow cytometry.
  • CDY14HL-Fc bound the ancestral (Wuhan-Hui) RBD with an apparent affinity of 0. 14nM (FIG. 37B).
  • FIG. 37B shows representative decoy binding data for the RBD from the ancestral (Wuhan-Hu 1) SARS-CoV-2 strain.
  • FIG 36 shows Engineered ACE2 decoys bind diverse ACE2-dependent CoVs, plotted as decoy fluorescence for each specified CoV strain. This is in good agreement with the picomolar binding affinity we previously measured for the engineered decoy:RBD interaction using surface plasmon resonance [Examples 1-4, and 7], Since we first described CDY14HL-Fc [Examples 1-4, and 7], several SARS-CoV-2 Variants of Concern (VoC) have emerged with far greater transmissibility and clinical sequalae than the original Wuhan strain with most of this evolution occurring at the RBD:ACE2 interface (image not shown) [Campbell F, Archer B, Laurenson-Schafer H, Jinnai Y, Konings F, Batra N, et al.
  • VoC SARS-CoV-2 Variants of Concern
  • the SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines.
  • bioRxiv. 2021:2021.08.22.457114 Remarkably, CDY14HL-Fc4 maintains sub- nanomolar binding affinity for all VoC RBDs, including Iota, Delta +, Lambda and Mu, and the “Delta 4+” RBD (table immediately below).
  • CDY14HL Maintains Potent Neutralization for Diverse SARS-CoV-2 Variants
  • FIG 33 shows viral neutralization assay using lentiviruses pseudotyped with the ancestral (Wuhan Hui) or Omicron variant spike protein.
  • CDY14HL-Fc4 neutralizes omicron more potently than the ancestral strain (18 ng/ml vs 35 ng/ml, summarized in table immediately below). We extended this approach to include all VoCs not previously evaluated.
  • CDY14HL-Fc4 neutralizes all SARS-CoV-2 strain pseudotypes tested, including lambda, kappa, delta, delta +, mu and zeta, with IC50 values near or below the potency of the ancestral strain, Wuhan, against which it was engineered (summarized in table immediately below). Table of CDY14HL neutralization IC50 values collected for SARS-CoV-2 variant pseudotypes along with the RBD mutations of each variant.
  • FIG. 38B shows a schematic representation measuring the competition between decoy and endogenous ACE2 receptor using yeast-displayed RBDs.
  • FIG 38C shows relative levels of decoy binding to diverse RBDs under several conditions as assessed by the yeast-display system. Similar to SARS-CoV2, all sarbecovirus RBDs retain >30% binding in the competition assay. Taken together with sub- nanomolar binding affinity, these data predict broad and potent neutralization of the entire class of ACE2-dependent beta-CoVs.
  • the lone alpha-CoV in our study, NL63, retains a lower fraction of decoy binding in the competition assay (23%, FIG 38C). Further study is performed to determine if this indicates a lower neutralizing potency of the decoy for the genetically distinct ACE2-dependent alpha-CoVs.
  • the affinity -matured, soluble ACE2 decoy termed CDY14HL-Fc4 binds and neutralizes SARS-CoV2 strains from the early pandemic as well as the related pandemic sarbecovirus, SARS-CoVl [Examples 1-4, and 7],
  • SARS-CoVl pandemic sarbecovirus
  • the original strategy for deploying the decoy was in the context of prevention of SARS-CoV2 infection. This was accomplished through the creation of an AAV vector expressing the decoy that is administered via a nasal administration to engineer proximal airway cells to express neutralizing levels of the decoy at the airway surface where the virus enters. This approach is expected to be particularly useful in immune compromised patients who do not generate protective immunity following active vaccination.
  • the decoy is also useful as a therapeutic protein for treatment and prevention in high-risk groups, following parenteral administration.
  • COVID-19 has illustrated how powerful the drive for viral fitness can be in circumventing immunity generated from previous infection, vaccines or antibody therapeutics. This rapid evolution is substantially amplified in the setting of a world-wide pandemic caused by a highly transmissible virus.
  • the use of a decoy protein that is based on a soluble version of a viral receptor significantly restricts virus escape since any mutation that diminished decoy binding will diminish viral fitness.
  • RVP-701L Wuhan (lot CL-114B), RVP-763L Delta (lot CL-267A), RVP-736L Zeta (lot CL- 255A), RVP-730L Kappa (lot CL-247A), RVP-768L Omicron (lot CL-297A), RVP-767L Mu (lot CL-274A), RVP-766L Lambda (lot CL-259A), and RVP-765L Delta + (lot CL-258A).
  • RVP-701L Wuhan (lot CL-114B), RVP-763L Delta (lot CL-267A), RVP-736L Zeta (lot CL- 255A), RVP-730L Kappa (lot CL-247A), RVP-768L Omicron (lot CL-297A), RVP-767L Mu (lot CL-274A), RVP-766L Lambda (lot CL-259A), and RVP-765L Delta + (lot CL-258A).
  • ACE2 Integral Molecular
  • the RBD sequences of the CoVs were taken from spike protein coding sequences downloaded from the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • MEGA X Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018;35(6): 1547-9
  • the nucleic acid sequences of the CoV RBDs were taken from NCBI: CoV-2 (NC_045512.2), NL63 (AY567487), LYRal l (KF569996), Rs4048 (KY417144), Rs4231 (KY417146), Rs7327 (KY417151), RsSHC014 (KC881005), WIV16 (KT444582.1), BANAL-236 (MZ937003), BANAL-103 (MZ937001), Rs4874 (KY417150), and RaTG13 (MN996532.2).
  • Plasmids were transformed into EBY100 using the Frozen-EZ Yeast Transformation II Kit (Zymo).
  • CDY14HL-Fcl For the titration of CDY14HL-Fcl, we incubated the yeast with 1: 10 dilution series of CDY14HL-Fcl at 25°C for 6 hr. The yeast were analyzed on an ACEA NovoCyte flow cytometer. We determined the level of CDY14HL-Fcl binding by taking the mean FITC signal for 500 RBD+ yeast cells collected for each condition. We fitted the decoy concentration versus the decoy binding signal in GraphPadPrism using a three-parameter fit to the binding isotherm.
  • the mutant hAce2 soluble decoy protein therapeutic constructs are: GTP14HL-Fcl (CDY14HL-hIgGl-Fc; hAce2-GTP14-HL-IgGl; original engineered Ace2 with 7 mutations), MR27HL-Fcl (MR27HL-hIgGl-Fc; 5 mutations (same as GTP14HL decoy plus reversion at aa 49 and 79)), GTP14HL-Fcl* (CDY14HL-hIgGl-Fc; hAce2-GTP14-HL-IgGl), and MR27HL-Fcl* (MR27HL-hIgGl-Fc) (*shortened hinge/linker between decoy and Fc region).
  • the injectable hAce2 decoy protein drugs were generated in CHO Cell line (CHO stable cell lines were created for stable production of hAce2 decoy proteins).
  • leader peptides native vs. heterologous
  • promoters CMV, EFl, EEF2
  • selection marker stringency high and low
  • addition of co-transfection of additional glycosylation enzymes to promote sialyation
  • CHO pools were created in which 500 ml fed-batch cultures were generated, tittered, purified and assessed for activity across a variety of metrics. Briefly, for the GMP-suitable manufacturing process a stable cell line created. Horizon Discovery Limited HD-BIOP3 GS KO CHO-K1 Cells were used for stable expression of constructs which were co-transfected with Leap-In Transposase mRNA into CHO cells. Chemically defined, animal component free cell culture media components were used. Some low pH instability were identified but process controls are implemented to manage for this. Fusion protein has been undergone tangential flow filtration concentration and buffer exchange to maintain monomer form (i.e., aggregation is a commonly reported problem with fusion proteins). Additional downstream operations steps are added for orthogonal adventitious viral inactivation and/or removal. The manufacturing process development allows for a high stability formulation for ease of standard temperature storage.
  • FIG. 35 shows exemplary pool hAce2 decoy protein expression levels (mg/L) in a 10-day process in a 10L Stirred Tank Controlled Bioreactor. These results show about 1.2 g/L of protein purified using a one or two step process with higher percent monomer purity and good recovery (-90%).
  • FIGs. 29A to 29D shows results of the pharmacokinetic examination of the engineered hAce2 decoy IgGl Fc fusion protein in vivo in hamsters.
  • FIG. 29A shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected serum samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc decoy fusion at doses of 3 mg/kg, 10 mg/kg, and 30 mg/kg.
  • FIG. 29A shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected serum samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc decoy fusion at doses of 3 mg/kg, 10 mg/kg, and 30 mg/kg.
  • FIG. 29B shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected NJF samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc decoy fusion at doses of 3 mg/kg, 10 mg/kg, and 30 mg/kg.
  • FIG. 29C shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected serum samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc or GTP14HL-Fcl decoy fusion from various pools of protein samples, when administered at a dose of 3 mg/kg.
  • FIG. 29C shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected serum samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc or G
  • 29D shows concentration of the engineered hAce2 decoy Fcl fusion protein, as measured with mass spectrometry in collected NLF samples, post intraperitoneal administration of hAceMR27HL-Variant-IgGl Fc or GTP14HL-Fcl decoy fusion from various pools of protein samples, when administered at a dose of 3 mg/kg.
  • Pharmacokinetic endpoints are serum decoy protein levels (peak and halflife), decoy point levels in BALF /NLF as measured by mass spectrometry. Additional endpoints are neutralization assay on BALF / NLF and serum, spike binding assay on BALF / NLF and serum.
  • Levels of genomic viral RNA, subgenomic viral RNA, and TCID50 are determined on the following samples: lung, nasal turbinates, and trachea. Additionally, oral swabs are performed, from samples of which both viral load and TCID50 assay are completed (i.e., on the same swab). To minimize the unwanted side effects in the lung pathology read out, BALF samples are not collected. Serum is collected on day 2 post dosing and at termination of the study to confirm levels of the engineered decoy protein. Weights are measured to give insight into the effectiveness of the decoy protein.
  • a DSI transmitter is surgically implanted to allow for remote collection of data (telemetry; blood pressure, heart rate, temperature).
  • Safety endpoints are clinical signs, body weight, clinical pathology (hematology, coagulation, and clinical chemistry), urinalysis, cytokines, blood pressure, heart rate, ECG, angiotensin system (Ang II, Ang 1-7, Ang 1-9), anti-decoy antibodies, ELISPOTs, full histopathology.
  • FIG. 32A shows serum decoy levels in NHP following administration by IV infusion of hAce2 decoy protein (MR27HL-Fcl) at a dose of 30 mg/kg (broken x axis for showing early timepoints (Hours)).
  • FIG. 32B shows potency of decoy in serum samples of NHP following administration by IV infusion of hAce2 decoy protein (MR27HL-Fcl) at a dose of 30 mg/kg, plotted as a reporter virus activity over decoy MS concentration (ng/ml) at 1 hour, 7 hours, and 24 hours post administration. Potency of decoy was measured by CoV2 pseudotype neutralization assay (described herein).
  • Weinreich DM Sivapalasingam S, Norton T, Ah S, Gao H, Bhore R, et al. REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid- 19. N Engl J Med. 2021;384(3):238-51. 53. Weinreich DM, Sivapalasingam S, Norton T, Ali S, Gao H, Bhore R, et al. REGEN-COV Antibody Combination and Outcomes in Outpatients with Covid- 19. N Engl J Med. 2021;385(23):e81.

Abstract

L'invention concerne une protéine Ace2 humaine soluble mutante (hAce2) utile dans la prévention d'une infection par des bêtacoronavirus, y compris le SARS-CoV2, ainsi que des compositions utiles dans le traitement d'une maladie associée au bêtacoronavirus, comprenant, par exemple, la COVID -19. L'invention concerne également des compositions les contenant formulées pour une administration intranasale et/ou intrapulmonaire, des méthodes de production de celles-ci et des dosages. L'invention concerne en outre l'utilisation des compositions de rAAV pour prévenir les symptômes d'une infection au COVID-19 chez l'homme.
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