WO2024044659A1 - Constitutively active g protein-coupled receptor compositions and methods of use thereof - Google Patents

Constitutively active g protein-coupled receptor compositions and methods of use thereof Download PDF

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Publication number
WO2024044659A1
WO2024044659A1 PCT/US2023/072779 US2023072779W WO2024044659A1 WO 2024044659 A1 WO2024044659 A1 WO 2024044659A1 US 2023072779 W US2023072779 W US 2023072779W WO 2024044659 A1 WO2024044659 A1 WO 2024044659A1
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WIPO (PCT)
Prior art keywords
amino acid
cagpcr
certain embodiments
acid sequence
seq
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PCT/US2023/072779
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French (fr)
Inventor
John Diener
Andrew KRUSE
Lindsay Clark
Ximena BARROS-ALVAREZ
Seymour DE PICCIOTTO
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Tectonic Therapeutic, Inc.
Modernatx, Inc.
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Publication of WO2024044659A1 publication Critical patent/WO2024044659A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered

Definitions

  • the present disclosure relates to methods of inhibiting immune cell activation or inflammatory cytokine production by an immune cell in a subject.
  • the methods involve introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified G s alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell.
  • CAGPCR constitutively active modified G protein-coupled receptor
  • CAGaS constitutively active modified G s alpha subunit
  • Inflammation is a highly complex protective response of the mammalian immune system that is initiated at sites of infection or injury.
  • the main goal of this process is to remove the danger stimulus and damaged cells, resolve inflammation, and support a repair of the damaged tissue.
  • innate immune activation can be prolonged and become misdirected at healthy cells leading to chronic inflammation and disease.
  • Central to the inflammatory response and its regulation is the cellular innate immune response that in its early stages encompasses an initial neutrophil influx into the tissue followed by an immigration of monocytes and cells of the adaptive immune system. Inflammatory monocytes differentiate and form a major cellular component, the macrophages.
  • Macrophages respond strongly and early to antigenic challenges in the tissue and the local cytokine environment. Their central role in the regulation of an effective immune response is being increasingly appreciated and their dysregulation in this early phase can lead to various human diseases that are associated with chronic inflammation. Thus, controlling macrophage-driven immune responses is an important preventative and therapeutic goal in many chronic inflammatory pathologies.
  • the present disclosure provides methods of inhibiting immune cell activation or inflammatory cytokine production by an immune cell in a subject.
  • the methods generally comprise introducing into an immune cell of a subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified G s alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell.
  • CAGPCR constitutively active modified G protein-coupled receptor
  • CAGaS constitutively active modified G s alpha subunit
  • the present disclosure provides a method of inhibiting immune cell activation in a subject, comprising introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protem-coupled receptor (CAGPCR) and/or a constitutively active modified G s alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting immune cell activation in the subject.
  • CAGPCR constitutively active modified G protem-coupled receptor
  • CAGaS constitutively active modified G s alpha subunit
  • the present disclosure provides a method of inhibiting inflammatory cytokine production by an immune cell in a subject, introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified G s alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting inflammatory cytokine production by the immune cell in the subject.
  • CAGPCR constitutively active modified G protein-coupled receptor
  • CAGaS constitutively active modified G s alpha subunit
  • the immune cell is a myeloid cell, optionally wherein the immune cell endogenously expresses a Gi-coupled GPCR.
  • the subject is a human. In certain embodiments, the subject has an inflammatory disease.
  • the CAGPCR is a constitutively active G s -coupled GPCR.
  • the CAGPCR is derived from a GPCR selected from the group consisting of an autoinhibitory GPCR, a self-activated GPCR, an adhesion GPCR, and GPR65.
  • the CAGPCR further comprises a heterologous signal sequence.
  • the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-6.
  • the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1.
  • the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2.
  • the heterologous signal sequence comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3.
  • the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4.
  • the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5.
  • the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6.
  • the CAGPCR is derived from an autoinhibitory GPCR. In certain embodiments, the CAGPCR is derived from a mutated autoinhibitory GPCR. In certain embodiments, the mutated autoinhibitory GPCR comprises one or more amino acid substitutions.
  • the CAGPCR lacks one or more leucine-rich repeats (LRRs) as compared to the naturally-occurring GPCR. In certain embodiments, the CAGPCR does not comprise a LRR. In certain embodiments, the CAGPCR comprises a deletion in the ectodomain as compared to the naturally -occurring GPCR. In certain embodiments, the CAGPCR lacks an ectodomain. In certain embodiments, the CAGPCR is derived from RXFP1.
  • LRRs leucine-rich repeats
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 10-27.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 20.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23.
  • the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 26.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27.
  • the CAGPCR is derived from a self-activated GPCR. In certain embodiments, the CAGPCR is derived from GPR52. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 37.
  • the CAGPCR is derived from an adhesion GPCR.
  • the adhesion GPCR is selected from the group consisting of LEC1, LEC2, LEC3, ELTD1, EMR1, EMR2, EMR3, mEMR4, CD97, GPR124, GPR125, CelsRl, CelsR2, CelsR3, GPR133, GPR144, GPR110, GPR111, GPR113, GPR115, GPR116, BAI1, BAI2, BA13.
  • the CAGPCR is derived from ADGRG2.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 29-31.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 29.
  • the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 30.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 31.
  • the CAGPCR is derived from ADGRG4.
  • the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 33-35.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 33.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 34.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 35.
  • the CAGPCR is derived from GPR65.
  • the CAGPCR is a mutated GPR65 comprising one or more amino acid substitutions selected from the group consisting of D60N, SI 01 A, and D268A.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38, wherein the amino acid at amino acid position 60 of SEQ ID NO: 38 is N, the amino acid at amino acid position 101 of SEQ ID NO: 38 is A, and/or the amino acid at amino acid position 286 of SEQ ID NO: 38 is A.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 39-53.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 39.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 40.
  • the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 41.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 42.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 43.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 44.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 45.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 46.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequence set forth in SEQ ID NO: 47.
  • the CAGPCR compnses an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 48.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 49.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 50.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 51.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 52.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 53
  • the CAGaS is a mutated G s alpha subunit comprising one or more amino acid substitutions selected from the group consisting of R201H, R201C, Q227R, and Q227H.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 54, wherein the amino acid at amino acid position 201 of SEQ ID NO: 54 is H or C, and/or the amino acid at amino acid position 227 of SEQ ID NO: 54 is R or H.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 55-58.
  • the CAGaS comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 55.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 56.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 57.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 58
  • the polynucleotide is comprised within a viral vector.
  • the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picomavirus, lentivirus, herpes simplex virus, vaccinia virus, pox virus, and alphavirus.
  • AAV adeno-associated virus
  • the viral vector is a single-stranded AAV.
  • the viral vector is a self- complementary AAV.
  • the polynucleotide is comprised within a non-viral vector.
  • the non-viral vector is a transposon-based vector.
  • the non-viral vector is a PiggyBac-based vector, or a Sleeping Beauty-based vector.
  • the polynucleotide is a messenger RNA (mRNA). In certain embodiments, the polynucleotide is a modified messenger RNA (mmR A). In certain embodiments, substantially all uridines in the mmRNA are modified uridines.
  • mRNA messenger RNA
  • mmR A modified messenger RNA
  • substantially all uridines in the mmRNA are modified uridines.
  • the modified uridines are selected from the group consisting of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio- 5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy uridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1- propynyl-pseudoundme, 5-taurinomethyluridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl- 2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 -methyl-pseudouridine, 4-thio-l- methyl-pseudouridine, 2-thio-l -methyl-pseudouridine,
  • the polynucleotide is in a lipid nanoparticle formulation.
  • the lipid nanoparticle formulation comprises a cationic lipid, a sterol, and/or a PEG-lipid.
  • the lipid nanoparticle formulation comprises a cationic lipid, a sterol, and a PEG-lipid.
  • FIGs. 1A-1C are graphs showing the level of TNFa production in THP-1 cells (FIG. 1A), human monocyte-derived macrophages (HMDM; FIG. IB), and mouse bone marrow-derived macrophages (BMDM; FIG. 1C), in response to liposaccharides (LPS) at the indicated pH.
  • FIG. 1 A shows the level of TNFa production in THP-1 cells as a percentage of response to LPS, normalized to the pH 7.4 condition.
  • FIGs. IB and 1C shows the level of TNFa production in THP-1 cells in pg/mL.
  • 2A-2B are graphs showing the level of cyclic AMP (cAMP) production (as a percentage of the level of cAMP production induced by wild type GPR65) in cells expressing the various GPR65 and mutants as indicated, in response to varying pH.
  • cAMP cyclic AMP
  • the present disclosure provides constitutively active modified G protein-coupled receptors (CAGPCRs) and methods of using the same.
  • CAGaS constitutively active modified G s alpha subunit
  • expression of a CAGPCR and/or CAGaS in target immune cells modulates second messenger signaling (e.g., cyclic adenosine monophosphate [cAMP] signaling) in the immune cells, resulting in the inhibition of immune cell activation and/or inflammatory cytokine production by the immune cells.
  • second messenger signaling e.g., cyclic adenosine monophosphate [cAMP] signaling
  • expression of a CAGPCR and/or CAGaS in an immune cell may lead to increased cAMP production and associated downstream signaling.
  • intracellular cAMP levels reduce the production of pro-inflammatory mediators and increase the production of anti-inflammatory factors in numerous immune cells.
  • expression of a CAGPCR and/or CAGaS in target immune cells may find use in the treatment of various inflammatory diseases.
  • G protein-coupled receptor or "GPCR” are known in the art and refer to polypeptides comprising seven, transmembrane-spanning alpha helices of between 22 to 24 amino acids, which mediate heterotrimeric G protein signaling. Each transmembrane helix is identified by number, i.e., transmembrane- 1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are joined by regions of amino acids between TM2 and TM3, TM4 and TM5, and TM6 and TM7, on the exterior, or extracellular side, of the cell membrane, referred to as extracellular loops 1, 2 and 3 (ECL1, ECL2 and ECL3), respectively.
  • TM1 transmembrane- 1
  • TM2 transmembrane-2
  • ECL1 extracellular loops 1, 2 and 3
  • the transmembrane helices are also joined by regions of amino acids between TM1 and TM2, TM3 and TM4, and TM5 and TM6 on the interior, or intracellular side, of the cell membrane, referred to as intracellular loops 1, 2 and 3 (ICL1, ICL2 and ICL3), respectively.
  • the seven TM helices may be referred to collectively as a 7TM region of the GPCR.
  • the "carboxy" (“C”) terminus of the receptor lies in the intracellular space within the cell, and the "amino" (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
  • GPCR structure and classification is generally well known in the art, and further discussion of GPCRs may be found in Probst, DNA Cell Biol.
  • adhesion GPCR refers to a certain class of GPCR characterized by seven transmembrane domains and an extracellular region comprising a GPCR autoproteolysis-inducing (GAIN) domain. It does not necessarily need to but can additionally contain one or more adhesion domains within its extracellular region.
  • GAIN GPCR autoproteolysis-inducing
  • the term "naturally-occurring" in reference to a GPCR means a GPCR that is naturally produced (e.g., by a wild type mammal such as a human). Such GPCRs are found in nature.
  • a “modified” GPCR e.g., a constitutively active modified GPCR refers to a naturally-occurring GPCR that has been modified such that it is non-naturally occurring (i.e., is no longer found in nature).
  • the term “polynucleotide,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides linked via a phosphodi ester bond.
  • the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein.
  • the methods of “treatment” employ administration of a polynucleotide to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.
  • the term “subject” includes any human or non-human animal. In certain embodiments, the subject is a human or non-human mammal. In certain embodiments, the subject is a human.
  • the method disclosed herein employ a constitutively active modified G protein-coupled receptor (CAGPCRs).
  • CAGPCRs constitutively active modified G protein-coupled receptor
  • GPCRs can be modified to become constitutively active through mutation, or through the generation of GPCR variants, e.g., by deleting one or more regions in the N-terminal extracellular region (or ectodomain) of the GPCR.
  • a CAGPCR of the present disclosure comprises a deletion in the ectodomain as compared to the naturally-occurring GPCR from which it is modified from.
  • the CAGPCR lacks an ectodomain.
  • the CAGPCR is a mutated GPCR comprising one or more amino acid substitutions that result in increased signaling.
  • the CAGPCR further comprises a heterologous signal sequence.
  • the CAGPCR further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (P2AR).
  • the CAGPCR further comprises an influenza hemagglutinin signal sequence (HASigSeq).
  • HSigSeq influenza hemagglutinin signal sequence
  • Such heterologous sequences may also be referred to as leader sequences, and their presence in the CAGPCR, in certain embodiments, aids in the expression of the CAGPCR. Exemplary signal/leader sequences are described in Table 1.
  • the CAGPCR may further comprise a detectable label, e.g., a fluorescent label, an enzyme, a radiolabel, or other detectable group.
  • a detectable label e.g., a fluorescent label, an enzyme, a radiolabel, or other detectable group.
  • the detectably labeled CAGPCR can be detected using standard techniques based on a characteristic of the detectable label, such as its enzymatic activity, radioactivity, or fluorescence.
  • Exemplary detectable labels include, without limitation, the FLAG octapeptide DYKDDDK (SEQ ID NO: 7) and FLAG-K8A peptide DYKDDDA (SEQ ID NO: 8).
  • CAGPCRs useful in the present disclosure constitutively activate cAMP signaling.
  • a CAGPCR may be derived from any GPCR that functions to directly or indirectly stimulate cAMP signaling.
  • the CAGPCR is derived from a G s a-coupled GPCR.
  • a CAGPCR of the present disclosure is derived from a G s alpha subunit (G s a)-coupled GPCR.
  • G s a-coupled GPCRs are known in the art, and include, without limitation, 5-HT receptors; ACTH receptor (also known as MC2R); adenosine receptor types A2 a and A2t; arginine vasopressin receptor 2; P-adrenergic receptor ty pes Pi, P2, and P3; calcitonin receptor; calcitonin gene-related peptide receptor; cannabinoid receptor 2; corticotropin-releasing hormone receptor; dopamine receptors Dl-like family; FSH-receptor; gastric inhibitory polypeptide receptor; glucagon receptor; growth hormone releasing hormone receptor; histamine H2 receptor; luteinizing hormone / choriogonadotropin receptor; melanocortin receptors (e g., MC1R,
  • the CAGPCRs of the present disclosure can be derived from any known GPCR, including, without limitation, human GPCRs and non-human orthologs thereof (e.g. from mammalians such as mouse, rat, Macaca mulatta or Macaca fascicularis, other vertebrates such as Xenopus tropicalis and Danio rerio, and invertebrates such as Drosophila melanogaster or Caenorhabditis elegans).
  • the CAGPCR of the present disclosure is derived from a human GPCR.
  • a CAGPCR of the present disclosure is derived from an auto- inhibitory GPCR.
  • An auto-inhibitory GPCR suitable for modification into a CAGPCR of the present disclosure generally does not have significant basal activity, but signals in response to the binding of a ligand to its ectodomain. Such auto-inhibitory GPCRs likely use separate mechanisms to prevent continuous self-activation of the seven transmembrane domain.
  • Auto-inhibitory GPCRs are known in the art and include, without limitation, RXFP1, adhesion GPCRs (aGPCRs), and protease activated receptors (PARs).
  • the CAGPCR is an auto-inhibitory GPCR that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is an auto-inhibitory GPCR that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is an auto- inhibitory GPCR that lacks an ectodomain. In certain embodiments, the CAGPCR comprises only the 7TM region of an auto-inhibitory GPCR , or a functional fragment thereof. In certain embodiments, the CAGPCR is an auto-inhibitory GPCR that lacks an ectodomain and comprises a sequence derived from a heterologous GPCR.
  • the CAGPCR comprises an auto-inhibitory GPCR that lacks an ectodomain fused to an N-terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR comprises only the 7TM region of an auto-inhibitory GPCR fused to an N-terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR is a mutated auto-inhibitory GPCR comprising one or more amino acid substitutions that results in increased activity' (e.g., downstream signaling).
  • the CAGPCR is derived from RXFP 1.
  • RXFP l is a member of the leucine-rich repeat-containing GPCRs (LGRs), where the leucine-rich repeats (LRRs) function as the extracellular ligand-binding domain for certain protein agonists (e.g., glycoprotein hormones, R-spondins, and relaxins).
  • LGRs leucine-rich repeat-containing GPCRs
  • LRRs leucine-rich repeats
  • RXFP1 is a unique member of the LGR family as it contains an additional LDLa module in the ectodomain.
  • RXFP1 is a G s -coupled GPCR that when activated, leads to increased cAMP signaling.
  • An exemplary wild type RXFP1 amino acid sequence is set forth in SEQ ID NO: 9.
  • RXFP1 wild type amino acid sequence and RXFPl-denved CAGPCR ammo acid sequences are set forth in Table 2.
  • the CAGPCR is a mutated RXFP1 comprising one or more amino acid substitutions that results in increased cAMP signaling.
  • the CAGPCR is a mutated RXFP1 comprising an I396A and/or S397A substitution.
  • the CAGPCR is a mutated RXFP1 comprising an 1396 A substitution.
  • the CAGPCR is a mutated RXFP1 comprising an S397A substitution.
  • the CAGPCR is a mutated RXFP1 comprising an I396A and S397A substitution.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9, wherein the amino acid at amino acid position 396 of SEQ ID NO: 9 is A, and/or the amino acid at amino acid position 397 of SEQ ID NO: 9 is A.
  • the CAGPCR is RXFP1 that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is RXFP1 that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is RXFP1 that lacks one or more LRRs. In certain embodiments, the CAGPCR is RXFP1 that does not comprise a LRR. In certain embodiments, the CAGPCR is RXFP1 that lacks an ectodomain. In certain embodiments, the CAGPCR comprises only the 7TM region of RXFP1, or a functional fragment thereof. Exemplary RXFP 1 -derived CAGPCR amino acid sequences are set forth in Table 2.
  • the CAGPCR further comprises a heterologous signal/leader sequence.
  • the CAGPCR further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (
  • the CAGPCR further comprises an influenza hemagglutinin signal sequence (HASigSeq).
  • HSigSeq influenza hemagglutinin signal sequence
  • Exemplary signal/leader sequences are set forth in Table 1. Table 2: Exemplary RXFP1 Sequences
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 10-27.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequence set forth in SEQ ID NO: 11.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 20.
  • the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 26.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27.
  • Adhesion GPCRs are amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27.
  • a CAGPCR of the present disclosure is derived from an adhesion GPCR (aGPCR).
  • aGPCRs are a class of GPCRs characterized by seven transmembrane domains and an extracellular region (or ectodomain) comprising a GAIN domain.
  • aGPCRs further comprise one or more adhesion domains within its extracellular region.
  • the GPCR autoproteolysis-inducing domain refers to a region within the extracellular region of an aGPCR which is composed of an N-terminal subdomain A and a C-terminal subdomain B.
  • Subdomain A of the GAIN domain is comprised of 3 to 6 alpha-helices.
  • Subdomain B of the GAIN domain is comprised of 13 p-strands and can additionally contain several small alpha-helices.
  • the GAIN domain of an aGPCR is auto-inhibitory, and cleavage thereof results in activation of the aGPCR.
  • GAIN domains include domains which do not exert autoproteolytic activity, but are classified as GAIN domains on the basis of alignments and/or sequence similarity to other proteins known to have a GAIN domain.
  • Some aGPCRs contain autoproteolytic domains in addition to the GAIN domain, and some aGPCRs are processed by other endogenous proteases.
  • aGPCRs possessing all three modes of cleavage may be used to generate the CAGPCRs of the present disclosure.
  • aGPCRs are known in the art, and include, without limitation, Group I aGPCRs LPHN1, LPHN2, LPHN3, and ETL; Group II aGPCRs CD97, EMR1, EMR2, EMR3, and EMR4; Group III aGPCRs GPR123, GPR124, and GPR125; Group IV aGPCRs CELSR1, CELSR2, and CELSR3; Group V aGPCRs GPR133 and GPR144; Group VI aGPCRs GPR110, GPR111, GPR113, GPR115, and GPR116; Group VII aGPCRs B All, BAE, and BAB; Group VIII aGPCRs GPR56, GPR97, GPR112, GPR114, GPR126, and GPR64; and ungrouped aGPCRs VLGR1 and GPR128.
  • a CAGPCR of the present disclosure is derived from an aGPCR selected from the group consisting of LEC1, LEC2, LEC3, ELTD1, EMR1, EMR2, EMR3, mEMR4, CD97, GPR124, GPR125, CelsRl, CelsR2, CelsR3, GPR133, GPR144, GPR110, GPR111, GPR113, GPR115, GPR116, BAI1, BAE, BAI3, GPR64, GPR97, GPR112, GPR114, GPR126, GPR128, VLGR1.
  • the CAGPCR is an aGPCR that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is an aGPCR that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is an aGPCR that lacks an ectodomain. In certain embodiments, the CAGPCR comprises the 7TM region and tethered agonist sequence (i.e., tethered ligand) of an aGPCR, or a functional fragment thereof. In certain embodiments, the CAGPCR is a mutated aGPCR comprising one or more amino acid substitutions that results in increased activity (e.g., downstream signaling).
  • the CAGPCR is derived from an aGPCR and further comprises a heterologous signal/leader sequence.
  • the CAGPCR is derived from an aGPCR and further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (P2AR).
  • the CAGPCR is derived from an aGPCR and further comprises an influenza hemagglutinin signal sequence (HASigSeq).
  • HSigSeq influenza hemagglutinin signal sequence
  • Exemplary signal/leader sequences are set forth in Table 1.
  • the CAGPCR is derived from ADGRG2.
  • An exemplary wild type ADGRG2 amino acid sequence is set forth in SEQ ID NO: 28.
  • ADGRG2 wild type amino acid sequence and ADGRG2-derived CAGPCR amino acid sequences are set forth in Table 3.
  • the CAGPCR is derived from ADGRG4.
  • An exemplary wild type ADGRG4 amino acid sequence is set forth in SEQ ID NO: 32.
  • ADGRG4 wild type amino acid sequence and ADGRG4-derived CAGPCR amino acid sequences are set forth in Table 4.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 29-31, and 33- 35.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequence set forth in SEQ ID NO: 29.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 30.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 31.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 32.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 33.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 34.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 35.
  • a CAGPCR of the present disclosure is derived from a selfactivated GPCR.
  • Self-activated GPCRs have very high basal activity signaling at close to its maximum response (Emax value) without any agonist bound.
  • the CAGPCR is a self-activated GPCR that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is a self-activated GPCR that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is a self-activated GPCR that lacks an ectodomain. In certain embodiments, the CAGPCR comprises only the 7TM region of a self-activated GPCR, or a functional fragment thereof. In certain embodiments, the CAGPCR is a self-activated GPCR that lacks an ectodomain and comprises a sequence derived from a heterologous GPCR.
  • the CAGPCR comprises a self-activated GPCR that lacks an ectodomain fused to an N-terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR comprises only the 7TM region of a self-activated GPCR fused to an N- terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR is a mutated self-activated GPCR comprising one or more amino acid substitutions that results in increased activity (e.g., downstream signaling).
  • the CAGPCR is derived from a self-activated GPCR and further comprises a heterologous signal/leader sequence.
  • the CAGPCR is derived from a self-activated GPCR and further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor ( 2AR).
  • the CAGPCR is derived from a selfactivated GPCR and further comprises an influenza hemagglutinin signal sequence (HASigSeq). Exemplary signal/leader sequences are set forth in Table 1.
  • the CAGPCR is derived from GPR52 (also known as the psychosine receptor).
  • GPR52 is a self-activating orphan GPCR in which its intrinsic activity is governed by its own ECL2.
  • GPR52 comprises an unstructured N-terminus.
  • An exemplary wild type GPR52 amino acid sequence is set forth in SEQ ID NO: 36.
  • GPR52 wild type amino acid sequence and GPR52-derived CAGPCR amino acid sequences are set forth in Table 5.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
  • a CAGPCR of the present disclosure is derived from a GPCR in which one or more amino acid substitutions results in increased activity (e.g., increased downstream signaling; increased cAMP signaling).
  • GPR65 (also known as TDAG8) is a GPCR that senses extracellular pH. Levels of cAMP were found to be elevated in neutral to acidic extracellular pH in cells expressing GPR65. GPR65 senses pH by protonation of histidine residues on its extracellular domain.
  • a CAGPCR of the present disclosure is derived from GPR65.
  • the CAGPCR is a mutated GPR65 comprising one or more amino acid substitutions that result in increased cAMP signaling.
  • the CAGPCR is a mutated GPR65 comprising an D60N, S101A, and/or a D286A substitution.
  • the CAGPCR is a mutated GPR65 comprising a D60N substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a S101A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a D286A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a D60N and S101A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a D60N and D286A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a SI 01 A and D286A substitution. Tn certain embodiments, the CAGPCR is a mutated GPR65 comprising a D60N, S101 A, and a D286A substitution.
  • the CAGPCR is derived from GPR65 and further comprises a heterologous signal/leader sequence.
  • the CAGPCR is derived from GPR65 and further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (P2AR).
  • the CAGPCR is derived from GPR65 and further comprises an influenza hemagglutinin signal sequence (HASigSeq). Exemplary signal/leader sequences are set forth in Table 1.
  • GPR65 wild type amino acid sequence is set forth in SEQ ID NO: 38.
  • GPR65 wild type amino acid sequence and GPR65-derived CAGPCR amino acid sequences are set forth in Table 6.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38, wherein the amino acid at amino acid position 60 of SEQ ID NO: 38 is N, the amino acid at amino acid position 101 of SEQ ID NO: 38 is A, and/or the amino acid at amino acid position 286 of SEQ ID NO: 38 is A.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 39.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 40.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 41.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 42.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 43.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 44.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 45.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 46.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 47.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 48.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 49.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 50.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 51.
  • the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 52.
  • the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 53.
  • CAGuS Constitutively Active G s Alpha Subunit
  • the method disclosed herein employ a constitutively active modified G s Alpha Subunit (CAGaS) protein.
  • CAGaS constitutively active modified G s Alpha Subunit
  • the G a s protein can be modified to become constitutively active through mutation.
  • a CAGaS is a mutated G a s protein comprising one or more amino acid substitutions that result in increased signaling.
  • the CAGaS is a mutated G a s protein comprising a R201H or R201C substitution, and/or a Q227R or Q227H substitution.
  • the CAGaS is a mutated G a s protein comprising a R201H substitution.
  • the CAGaS is a mutated G 0 .s protein comprising a R201C substitution.
  • the CAGaS is a mutated G a s protein comprising a Q227R substitution.
  • the CAGaS is a mutated G a s protein comprising a Q227H substitution.
  • the CAGaS is a mutated G 0 .s protein comprising a R201H and Q227R substitution. In certain embodiments, the CAGaS is a mutated G a s protein comprising a R201H and Q227H substitution. In certain embodiments, the CAGaS is a mutated G a s protein comprising a R201C and Q227R substitution. In certain embodiments, the CAGaS is a mutated G 0 .s protein comprising a R201C and Q227H substitution.
  • G a s protein amino acid sequence is set forth in SEQ ID NO: 54.
  • G a s protein wild type amino acid sequence and CAGaS amino acid sequences are set forth in Table 7.
  • the CAGaS comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 54, wherein the amino acid at amino acid position 201 of SEQ ID NO: 54 is H or C, and/or the amino acid at amino acid position 227 of SEQ ID NO: 54 is R or H.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 55-58.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 55.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 56.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 57.
  • the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 58.
  • the methods disclosed herein employ a polynucleotide that encodes any of constitutively active modified G protein-coupled receptors (CAGPCRs) or constitutively active modified G s Alpha Subunit (CAGaS) proteins described herein.
  • the polynucleotide is a DNA molecule.
  • the polynucleotide is an RNA molecule.
  • the polynucleotide described herein can be transcribed from an expression vector (e.g., a recombinant expression vector).
  • the polynucleotide is comprised within a vector.
  • the vector is a non-viral vector.
  • Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministnngs, and oligonucleotides (e.g., mRNA, naked DNA).
  • the non-viral vector is a DNA plasmid vector.
  • the non-viral vector is a transposon-based vector.
  • the non-viral vector is a PiggyBac-based vector, or a Sleeping Beauty-based vector.
  • the vector is a viral vector.
  • Viral vectors can be replication competent or replication incompetent. Viral vectors can be integrating or non-integrating. A number of viral based systems have been developed for gene transfer into mammalian cells, and a suitable viral vector can be selected by a person of ordinary skill in the art.
  • Exemplary viral vectors include, but are not limited to, adenovirus vectors (e.g, adenovirus 5), adeno-associated virus (AAV) vectors (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9), retrovirus vectors (e.g, MMSV, MSCV), lentivirus vectors (e.g., HIV-1, HIV-2), gammaretrovirus vectors, herpes virus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g.
  • adenovirus vectors e.g, adenovirus 5
  • AAV adeno-associated virus
  • retrovirus vectors e.g., MMSV, MSCV
  • lentivirus vectors e.g., HIV-1, HIV-2
  • gammaretrovirus vectors e.g., herpes virus vectors (e.g., HSV1, HSV2),
  • the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picomavirus, lentivirus, herpes simplex virus, vaccinia virus, pox virus, and alphavirus.
  • AAV adeno-associated virus
  • the viral vector is a single-stranded AAV Tn certain embodiments, the viral vector is a self- complementary AAV.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see. e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthal
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus; and the like.
  • the polynucleotide is integrated into the genome of the target cell (e.g., immune cell). In certain embodiments, the polynucleotide is integrated via random integration, a site-specific integration, or a biased integration. In certain embodiments, the sitespecific integration can be non-assisted or assisted. In certain embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In some embodiments, the site-directed nuclease comprises the polynucleotide with 5' and 3' nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration.
  • the polynucleotide with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology -mediated end joining, ornonhomologous end-joining.
  • the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism.
  • Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.
  • the site-specific integration occurs at a site that disrupts expression of a target gene.
  • disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.
  • the site-specific integration occurs at a site that results in enhanced expression of a target gene.
  • enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.
  • enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the polynucleotide.
  • enzymes create single-strand breaks.
  • enzymes create double-strand breaks.
  • examples of break-inducing enzymes include but are not limited to: transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVERTM, and CPF1.
  • break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, as a nucleoprotein complex with a guide RNA (gRNA).
  • gRNA guide RNA
  • the polynucleotide is RNA, e.g., in vitro synthesized RNA.
  • Methods for in vitro synthesis of RNA are known to those of skill in the art. Any known method can be used to synthesize RNA comprising a nucleic acid sequence encoding a CAGPCR of the present disclosure.
  • the polynucleotide is a modified RNA that contains one or more modified nucleosides
  • modified RNAs include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. They may also include RNAi- inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
  • the modified RNA is one or more modified messenger RNAs (mmRNAs).
  • mmRNAs modified messenger RNAs
  • the mmRNAs do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
  • the mmRNAs further express a protein-binding partner or a receptor on the surface of the cell, which functions to target the cell to a specific tissue space or to interact with a specific moiety, either in vivo or in vitro.
  • Suitable protein-binding partners include antibodies and functional fragments thereof, scaffold proteins, or peptides.
  • the mmRNAs may further encode a cell-penetrating polypeptide.
  • “cell -penetrating polypeptide” refers to a polypeptide which may facilitate the cellular uptake of molecules.
  • the cell-penetrating polypeptide may contain one or more detectable labels.
  • the polypeptides may be partially labeled or completely labeled throughout.
  • the mmRNA may encode the detectable label completely, partially or not at all.
  • the cell-penetrating peptide may also include a signal sequence.
  • a “signal sequence” refers to a sequence of amino acid residues bound at the amino terminus of a nascent protein during protein translation. The signal sequence may be used to signal the secretion of the cell-penetrating polypeptide.
  • Polynucleotides may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription, enzymatic or chemical cleavage of a longer precursor, etc.
  • Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D C : IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).
  • modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared from readily available starting materials using the following general methods and procedures. It is understood that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • the manufacturing process can be monitored according to any suitable method know n in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., iH or BC) infrared spectroscopy, spectrophotometry (e.g., UV -visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., iH or BC) infrared spectroscopy, spectrophotometry (e.g., UV -visible), or mass spectrometry
  • chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
  • mmRNAs generally comprise a translatable region and one, two, or more than two different modifications.
  • the chemical modifications can be located on the nucleobase of the nucleotide.
  • the chemical modifications can be located on the sugar moiety of the nucleotide.
  • the chemical modifications can be located on the phosphate backbone of the nucleotide.
  • RNAs Preparation of modified nucleosides and nucleotides used in the manufacture or synthesis of modified RNAs can involve the protection and deprotection of various chemical groups.
  • the need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art.
  • the chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
  • Modified nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al. Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al. Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al. Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9): 1729-1740, 1992, each of which are incorporated by reference in their entirety.
  • Modified mRNAs need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the polynucleotide sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a mRNA sequence such that the function of the modified mRNA is not substantially decreased. A modification may also be a 5' or 3' terminal modification. Modified mRNAs may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
  • the mmRNAs may contain a modified pyrimidine such as uracil or cytosine.
  • a modified pyrimidine such as uracil or cytosine.
  • at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide may be replaced with a modified uracil.
  • the modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the mRNA may be replaced with a modified cytosine.
  • the modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy uridine, 3-melhyluridine, 5-carboxymethyl-uridine, 1 -carboxy methyl-pseudouri dine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1 -taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l -taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 - methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl
  • modified nucleosides include 5 -aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5 -hydroxy methylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5 -methylcytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-
  • modified nucleosides include 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio- N6-(cis-hydroxyisopentenyl) adenosine, N6-glycmylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- di
  • modified nucleosides include inosine, 1 -methyl-inosine, wyosine, wybutosme, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
  • a modified nucleoside is 5'-0-(l-Thiophosphate)-Adenosine, 5'-0-(l-Thiophosphate)-Cytidine, 5'-0-(l-Thiophosphate)-Guanosine, 5'-0-(l-Thiophosphate)- Uridine or 5'-0-(l-Thiophosphate)-Pseudouridine.
  • the modified mRNA comprises one or more modified uridines. In certain embodiments, the modified mRNA comprises one or more 1-methyl- pseudouri dines.
  • RNA molecules are optional, and may be beneficial in some embodiments.
  • a 5' untranslated region (UTR) and/or a 3'UTR may be included, wherein either or both may independently contain one or more different nucleoside modifications.
  • nucleoside modifications may also be present in the translatable region.
  • polynucleotides comprise a Kozak sequence.
  • the polynucleotide comprises one or more additional elements. Additional elements include, but are not limited to, promoters, enhancers, polyadenylation (poly A) sequences, and selection genes.
  • Suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated as part of the present disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the methods disclosed herein employ pharmaceutical compositions comprising a polynucleotide disclosed herein.
  • the pharmaceutical compositions described herein are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington ’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as L1POFECTINTM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • vesicles such as L1POFECTINTM, Life Technologies, Carlsbad, CA
  • the dose of the polynucleotide administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering the polynucleotide that encode them may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).
  • Various delivery systems are known and can be used to administer the pharmaceutical composition disclosed herein, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • any pharmaceutical composition described herein can be delivered subcutaneously or intravenously with a standard needle and synnge.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition disclosed herein.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see, Langer, supra,' Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g, by dissolving, suspending, or emulsifying any of the polynucleotides described herein in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g, ethanol), a polyalcohol (e.g, propylene glycol, polyethylene glycol), a nonionic surfactant [e g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g, ethanol
  • a polyalcohol e.g, propylene glycol, polyethylene glycol
  • a nonionic surfactant e g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the formulations include one or more cell penetration agents, e.g., transfection agents.
  • an mmRNA is mixed or admixed with a transfection agent (or mixture thereof) and the resulting mixture is employed to transfect cells.
  • Preferred transfection agents are cationic lipid compositions, particularly monovalent and polyvalent cationic lipid compositions, more particularly LIPOFECTIN®, LIPOFECTACE®, LIPOFECTAMINETM, CELLFECTIN®, DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER, and dendrimer compositions, particularly G5-G10 dendrimers, including dense star dendrimers, PAMAM dendrimers, grafted dendrimers, and dendrimers known as dendrigrafts and SUPERFECT®.
  • a ribonucleic acid is conjugated to a nucleic acid-binding group, for example a polyamine and more particularly a spermine, which is then introduced into the cell or admixed with a transfection agent (or mixture thereof) and the resulting mixture is employed to transfect cells.
  • a nucleic acid-binding group for example a polyamine and more particularly a spermine
  • a mixture of one or more transfection-enhancing peptides, proteins, or protein fragments including fusagenic peptides or proteins, transport or trafficking peptides or proteins, receptor-ligand peptides or proteins, or nuclear localization peptides or proteins and/or their modified analogs (e.g., spermine modified peptides or proteins) or combinations thereof are mixed with and complexed with a ribonucleic acid to be introduced into a cell, optionally being admixed with transfection agent and the resulting mixture is employed to transfect cells.
  • transfection-enhancing peptides, proteins, or protein fragments including fusagenic peptides or proteins, transport or trafficking peptides or proteins, receptor-ligand peptides or proteins, or nuclear localization peptides or proteins and/or their modified analogs (e.g., spermine modified peptides or proteins) or combinations thereof are mixed with and complexed with a ribonucleic acid to be introduced into a cell
  • a component of a transfection agent e.g., lipids, cationic lipids or dendrimers
  • a component of a transfection agent is covalently conjugated to selected peptides, proteins, or protein fragments directly or via a linking or spacer group.
  • peptides or proteins that are fusagenic, membrane-permeabilizing, transport or trafficking, or which function for celltargeting.
  • the peptide- or protein-transfection agent complex is combined with a ribonucleic acid and employed for transfection.
  • the formulations include a pharmaceutically acceptable earner that causes the effective amount of mmRNA to be substantially retained in a target tissue containing the cell.
  • the formulation may include at least an mmRNA and a deliver ⁇ ' agent.
  • the delivery agent may comprise lipidoid-based formulations allowed for localized and systemic delivery of mmRNA.
  • compositions for generation of an in vivo depot containing an engineered ribonucleotide contains a bioerodible, biocompatible polymer, a solvent present in an amount effective to plasticize the polymer and form a gel therewith, and an engineered ribonucleic acid.
  • the composition also includes a cell penetration agent as described herein.
  • the composition also contains a thixotropic amount of a thixotropic agent mixable with the polymer so as to be effective to form a thixotropic composition.
  • Further compositions include a stabilizing agent, a bulking agent, a chelating agent, or a buffering agent.
  • sustamed-release delivery depots such as for administration of a mmRNA to an environment (meaning an organ or tissue site) in a patient.
  • Such depots generally contain a mmRNA and a flexible chain polymer where both the mmRNA and the flexible chain polymer are entrapped within a porous matrix of a crosslinked matrix protein.
  • the pore size is less than 1 mm, such as 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, or less than 100 nm.
  • the flexible chain polymer is hydrophilic.
  • the flexible chain polymer has a molecular weight of at least 50 kDa, such as 75 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, or greater than 500 kDa.
  • the flexible chain polymer has a persistence length of less than 10%, such as 9, 8, 7, 6, 5, 4, 3, 2, 1 or less than 1% of the persistence length of the matrix protein.
  • the flexible chain polymer has a charge similar to that of the matrix protein.
  • the flexible chain polymer alters the effective pore size of a matrix of crosslinked matrix protein to a size capable of sustaining the diffusion of the mmRNA from the matrix into a surrounding tissue comprising a cell into which the mmRNA is capable of entering.
  • Lipidoid-based formulations are also provided herein, allowing for localized and systemic delivery of mmRNA.
  • the synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21 ; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864- 1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which are incorporated herein by reference in their entireties).
  • complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, result in an effective delivery of mmRNA, as judged by the production of an encoded protein (e.g., CAGPCR), following the injection of an mmRNA-formulated lipidoids via localized and systemic routes of administration.
  • Modified mRNA-lipidoid complexes can be administered by vanous means disclosed herein.
  • lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 herein incorporated by reference), use of lipidoid oligonucleotides to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.
  • lipidoid formulations may have a similar component molar ratio.
  • Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of the mmRNA molecule for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc.
  • the component molar ratio may include, but is not limited to, 50% lipid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG.
  • the lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 (including variants and derivatives), DLin-MC3-DMA and analogs thereof.
  • lipidoid formulations for the localized deliver ⁇ ' of nucleic acids to cells may also not require all of the formulation components which may be required for systemic delivery, and as such may comprise the lipidoid and the mmRNA.
  • combinations of different lipidoids may be used to improve the efficacy of mmRNA-directed protein.
  • a modified rnRNA may be formulated by mixing the mmRNA with the lipidoid at a set ratio prior to addition to cells.
  • In vivo formulations may require the addition of extra ingredients to facilitate circulation throughout the body.
  • a standard formulation process used for siRNA-lipidoid formulations may be used as a starting point
  • Initial mmRNA-lipidoid formulations consist of particles composed of 42% lipidoid, 48% cholesterol and 10% PEG, with further optimization of ratios possible. After formation of the particle, mmRNA is added and allowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays.
  • nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety).
  • particle size Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety.
  • PEG polyethylene glycol
  • Formulations with the different lipidoids including, but not limited to penta[3-(l- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-SLAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401 :61 (2010)), C12-200 (including derivatives and variants), MD1, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA and DLin-MC3-DMA, can be tested for in vivo activity.
  • TETA-SLAP penta[3-(l- laurylaminopropionyl)]-triethylenetetramine hydrochloride
  • C12-200 including derivatives and variants
  • MD1, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA and DLin-MC3-DMA can be tested for in vivo activity.
  • the lipidoid referred to herein as “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670; both of which are herein incorporated by reference in their entirety.
  • the lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotide, primary construct, or mmRNA.
  • formulations with certain lipidoids include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (Cl 4 alkyl chain length).
  • formulations with certain lipidoids include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG- DMG.
  • mmRNA The ratio of mmRNA to lipidoid used to test for in vitro transfection is tested empirically at different lipidoid: mmRNA ratios. Previous work using siRNA and lipidoids have utilized 2.5: 1, 5: 1, 10: 1, and 15: 1 lipidoid: siRNA wt:wt ratios. Given the longer length of mmRNA relative to siRNA, a lower wt:wt ratio of lipidoid to mmRNA is likely to be effective. In addition, for comparison mmRNA are also formulated using RNAiMax (Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison Wis.) cationic lipid delivery vehicles.
  • RNAiMax Invitrogen, Carlsbad, Calif.
  • TRANSIT-mRNA Microl Bio, Madison Wis.
  • the pharmaceutical composition comprises amodifiedmRNA formulated in a lipid nanoparticle formulation comprising a lipid selected from the group consisting of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200; a cholesterol; and a PEG-lipid.
  • the modified mRNA and lipid nanoparticle are formulated at a total lipid to mRNA weight ratio of 10: 1, 15: 1, 20: 1, or 30: 1.
  • the lipid is DLin-KC2-DMA or 98N12-5.
  • the lipid nanoparticle formulation comprises about 42% lipid, about 48% cholesterol, and about 10% PEG- lipid.
  • the lipid nanoparticle formulation comprises about 50% lipid, about 38.5% cholesterol, and about 1.5% PEG-lipid.
  • the lipid nanoparticle has a mean particle size between 86 nm and 155 nm.
  • the lipid nanoparticle has a polydisperity index between 0.02 and 0.17.
  • the lipid nanoparticle formulation further comprises a formulation buffer for in vivo delivery , wherein the formulation buffer has a pH of 6.5 and comprises sodium chloride, calcium chloride, and Na + -phosphate.
  • the formulation buffer comprises 150 mM sodium chloride, 2 rnM calcium chloride, and 2 mM Na+-phosphate.
  • the present disclosure provides a method of inhibiting immune cell activation in a subject.
  • the present disclosure provides a method of inhibiting inflammatory cytokine production by an immune cell in a subject.
  • the methods of the present disclosure comprise introducing into an immune cell of the subj ect a polynucleotide encoding a constitutively active modified G-coupled G protein-coupled GPCR (CAGPCR) and/or a constitutively active modified G s Alpha Subunit (CAGaS) protein.
  • CAGPCR constitutively active modified G-coupled G protein-coupled GPCR
  • CAGaS constitutively active modified G s Alpha Subunit
  • the CAGPCR and/or CAGaS Upon introduction of the polynucleotide encoding a CAGPCR and/or CAGaS, the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting immune cell activation or inflammatory cytokine production by the immune cell in the subject.
  • GPCRs play important roles in inflammation, and inflammatory cells such as leukocytes, macrophages, and monocytes, express a large number of endogenous GPCRs that recognize classic chemoattractants and chemokines. Such endogenous GPCRs are critical to the migration of phagocytes and their accumulation at sites of inflammation, where the cells can exacerbate inflammation. Many endogenous GPCRs present in inflammatory cells also mediate transcription factor activation, resulting in the synthesis and secretion of pro-inflammatory factors. In certain embodiments, activation of endogenous GPCRs expressed by inflammatory cells (e g., pro- inflammatory immune cells) leads to a reduction in intracellular levels of cAMP.
  • inflammatory cells e g., pro- inflammatory immune cells
  • expression of a CAGPCR of the present disclosure in immune cells may shift the balance of signaling within the immune cell towards signaling that results in anti-inflammatory functions, for example, leads to the increase in intracellular levels of cAMP.
  • the immune cell endogenously expresses a Gi-coupled GPCR.
  • methods of the present disclosure are for inhibiting pro- inflammatory cytokine production by an immune cell of the subject.
  • Pro-inflammatory cytokines are secreted from Thl cells, CD4 + cells, macrophages, and dendritic cells.
  • Pro-inflammatory cytokines are known in the art, and include, without limitation, IL-1 (e.g., IL-1 P), IL-2, IL-6, IL-8, IL-12, IL- 17, IL-18, TNF-a, IFN-y, and GM-CSF.
  • the immune cell is a myeloid cell.
  • the immune cell is an immune cell involved in pro-inflammatory responses.
  • immune cells include, without limitation, lymphocytes (e.g., CD4 + T cells), leukocytes, macrophages, and monocytes.
  • inflammatory disease or disorder refers to a disease, disorder or pathological condition where the pathology results, in whole or in part, from, e.g., a change in number, change in rate of migration, or change in activation, of cells of the immune system.
  • Cells of the immune system include, e.g., T cells, B cells, monocytes or macrophages, innate lymphoid cells, antigen presenting cells (APCs), dendritic cells, microglia, NK cells, neutrophils, eosinophils, mast cells, or any other cell specifically associated with the immunology, for example, cytokine-producing endothelial or epithelial cells.
  • T cells e.g., T cells, B cells, monocytes or macrophages, innate lymphoid cells, antigen presenting cells (APCs), dendritic cells, microglia, NK cells, neutrophils, eosinophils, mast cells, or any other cell specifically associated with the immunology, for example, cytokine-producing endothelial or epithelial cells.
  • APCs antigen presenting cells
  • dendritic cells e.g., microglia, NK cells, neutrophils, eosinophils
  • the “inflammatory disease or disorder” is an immune disorder or condition selected from the group consisting of asthma, (including steroid resistant asthma, steroid sensitive asthma, eosinophilic asthma or non-eosinophilic asthma), allergy, anaphylaxis, multiple sclerosis, inflammatory bowel disorder (e.g.
  • COPD chronic obstructive pulmonary disease
  • ACOS COPD overlap syndrome
  • eosinophilic esophagitis chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, lupus, atopic dermatitis, psoriasis, scleroderma and other fibrotic diseases, sjogren's syndrome, vasculitis (behcef s disease, Giant cell arteritis, Henoch-Schonlein purpura and Churg Strauss syndrome), inflammatory pain and arthritis.
  • the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, and psoriatic arthritis.
  • multiple doses of the polynucleotide may be administered to a subject over a defined time course.
  • the methods according to this aspect of the disclosure comprise sequentially administering to a subject multiple doses of the polynucleotide.
  • sequentially administering means that each dose of the polynucleotide is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months).
  • the present disclosure provides methods which comprise sequentially administering to the patient a single initial dose of the polynucleotide, followed by one or more secondary doses of the polynucleotide, and optionally followed by one or more tertiary doses of the polynucleotide.
  • the terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the polynucleotide.
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose;
  • the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of the polynucleotide, but generally may differ from one another in terms of frequency of administration.
  • the amounts of the polynucleotide contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • two or more (e.g, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
  • each secondary and/or tertiary dose is administered 1 to 26 (e.g, 1, 1/2, 2, 2 2, 3, 3/ 2 , 4, 4/2, 5, 5/ 2 , 6, 6/2, 7, 7/ 2 , 8, 8/2, 9, 9/ 2 , 10, IO/2, 11, H/2, 12, 12/ 2 , 13, 13/2, 14, 14/2, 15, 15/2, 16, I6/2, 17, 17/2, 18, I8/2, 19, 19/ 2 , 20, 20/ 2 , 21, 21 / 2 , 22, 22/ 2 , 23, 23/, 24, 24/, 25, 25/, 26, 26/, or more) weeks after the immediately preceding dose.
  • the phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of the polynucleotide, which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of the polynucleotide.
  • a single secondary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient.
  • only a single tertiary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
  • each secondary dose may be administered at the same frequency as the other secondary doses.
  • each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose.
  • each tertiary dose may be administered at the same frequency as the other tertiary doses.
  • each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose.
  • the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • one or more of the polynucleotides are administered to a subject as a weight-based dose.
  • a “weight-based dose” e.g., a dose in mg/kg is a dose of the protein or peptides that will change depending on the subject’s weight.
  • one or more of the polynucleotides is administered to a subject as a fixed dose.
  • a “fixed dose” e.g., a dose in mg
  • one dose of the polynucleotide is used for all subjects regardless of any specific subject-related factors, such as weight.
  • a fixed dose of the polynucleotide is based on a predetermined weight or age.
  • kits comprises one or more of the polynucleotides.
  • the kit may further include reagents or instructions for using the polynucleotides in a subject. It may also include one or more buffers.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. However, various combinations of components may be comprised in a vial.
  • the kits of the present disclosure also typically include a means for containing the polynucleotides, and any other reagent containers in close confinement for commercial sale.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • LPS is the major component of Gram-negative bactena cell walls and can cause an acute inflammatory response by triggering the release of a vast number of inflammatory cytokines in various cell types.
  • THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport.
  • THP-1 cells are known to express wild-type (naturally-occurring) RXFP1 and are know n to be sensitive to liposaccharide (LPS). It has been shown that stimulation of THP-1 cells by LPS induces the production of pro-inflammatory cytokines such as interleukin-1 (3 ( I L- 1 (3), tumour necrosis factor (TNF-a) and interleukin-6 (IL-6). Further, THP-1 cells are know n to express wild-type GPR65 (also known as TDAG8 and the psychosine receptor), a pH sensing GPCR that stimulates cAMP signaling in response to acidic pH.
  • GPR65 also known as TDAG8 and the psychosine receptor
  • THP-1 cells as well as primary human monocyte-derived macrophages (HMDM), and mouse bone marrow-derived macrophages (BMDM), were tested for sensitivity to LPS.
  • Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and contamination with Mycoplasma was periodically excluded.
  • FCS fetal calf serum
  • 1x10 6 cells were incubated for 24 hours in growth medium containing 100 ng/ml LPS from Escherichia coli O26/B6. Cells were then pelleted, washed with PBS and re-suspended in fresh medium before plating into wells of 24-well plates that have been pre-coated with an anti -TNFa antibody. Expression of TNFa was measured by standard ELISA.
  • FIGs. 1A-1C incubation of THP-1 cells (FIG. 1A), HMDM (FIG. IB), and BMDM (FIG. 1C) with LPS resulted in increased production of the pro-inflammatory cytokine TNFa.
  • TNFa pro-inflammatory cytokine TNFa
  • HMDM HMDM
  • BMDM BMDM
  • relaxin a ligand for RXFP1
  • a constitutively active modified GPCR will be generated from RXFP1.
  • Exemplary amino acid sequences of RXFPl-CAGPCRs that will be tested are set forth in Table 2.
  • the RXFP1-CAGPCR will be expressed in THP-1 cells to confirm that RXFP1-CAGPCR expression can decrease the LPS- induced immune cytokine expression and/or secretion by THP-1 cells, in the absence or presence of relaxin.
  • Example 1 LPS induced production of TNFa in THP-1 cells. Decreasing the pH of medium to acidic pH significantly decreased the LPS-induced production of TNFa, indicative of activated GPR65-induced cAMP signaling.
  • GPR65-derived constitutively active modified GPCRs will reproduce the effect of wild type (WT) GPR65.
  • WT wild type
  • GPR65-CAGPCRs Exemplary amino acid sequences of GPR65-CAGPCRs are set forth in Table 8.
  • GPR65 (WT), GPR65 (D60N), GPR65 (D286A), GPR65 (D286N), and GPR65 (S101A) were tested as follows, and the cAMP response to varying pH was measured (FIGs. 2A and 2B). Briefly, HEK293 cells were seeded into a 96-well tissue culture plate followed by transient co- transfection with GPR65 (WT) or GPR65-CAGPCR constructs, and a pGloSensor-22F plasmid. Transfected cells were stimulated by maintaining cells in buffer at a defined pH, inducing G s - mediated cAMP signaling.
  • cAMP was assayed using the activity of the GloSensor biosensor, a mutant luciferase fused to a cAMP binding domain that leads to production of light in the presence of its substrate luciferin.
  • the readout of relative luminescent units (RLU) was used as a proxy for cAMP response.
  • GPR65-CAGPCR will be expressed in THP-1 cells to confirm that GPR65- CAGPCR expression can decrease the LPS-induced immune cytokine expression and/or secretion by THP-1 cells, at different pH.

Abstract

Provided herein are methods of inhibiting immune cell activation or inflammatory cytokine production by an immune cell in a subject. The methods involve introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified Gs alpha subunit (CAGαS), such that the CAGPCR and/or CAGαS is expressed in the immune cell.

Description

CONSTITUTIVELY ACTIVE G PROTEIN-COUPLED RECEPTOR COMPOSITIONS AND METHODS OF USE THEREOF
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63/373,377, filed August 24, 2022, the entire disclosure of which is hereby incorporated herein by reference.
REFERENCE TO SEQUENCE LISTING
[0002] This application contains a sequence listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety (said ST.26 copy, created on August 22, 2023, is named “201730_seqlist.xml” and is 75,010 bytes in size)
FIELD
[0001] The present disclosure relates to methods of inhibiting immune cell activation or inflammatory cytokine production by an immune cell in a subject. The methods involve introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified Gs alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell.
BACKGROUND
[0002] Inflammation is a highly complex protective response of the mammalian immune system that is initiated at sites of infection or injury. The main goal of this process is to remove the danger stimulus and damaged cells, resolve inflammation, and support a repair of the damaged tissue. However, if this process that is normally targeted at an infectious stimuli or injured tissue remains unresolved, innate immune activation can be prolonged and become misdirected at healthy cells leading to chronic inflammation and disease. Central to the inflammatory response and its regulation is the cellular innate immune response that in its early stages encompasses an initial neutrophil influx into the tissue followed by an immigration of monocytes and cells of the adaptive immune system. Inflammatory monocytes differentiate and form a major cellular component, the macrophages. Macrophages respond strongly and early to antigenic challenges in the tissue and the local cytokine environment. Their central role in the regulation of an effective immune response is being increasingly appreciated and their dysregulation in this early phase can lead to various human diseases that are associated with chronic inflammation. Thus, controlling macrophage-driven immune responses is an important preventative and therapeutic goal in many chronic inflammatory pathologies.
[0003] Accordingly, there is a need in the art for improved methods of modulating these immune responses.
SUMMARY
[0004] The present disclosure provides methods of inhibiting immune cell activation or inflammatory cytokine production by an immune cell in a subject. The methods generally comprise introducing into an immune cell of a subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified Gs alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell.
[0005] In one aspect, the present disclosure provides a method of inhibiting immune cell activation in a subject, comprising introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protem-coupled receptor (CAGPCR) and/or a constitutively active modified Gs alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting immune cell activation in the subject.
[0006] In another aspect, the present disclosure provides a method of inhibiting inflammatory cytokine production by an immune cell in a subject, introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified Gs alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting inflammatory cytokine production by the immune cell in the subject.
[0007] In certain embodiments, the immune cell is a myeloid cell, optionally wherein the immune cell endogenously expresses a Gi-coupled GPCR.
[0008] In certain embodiments, the subject is a human. In certain embodiments, the subject has an inflammatory disease.
[0009] In certain embodiments, the CAGPCR is a constitutively active Gs-coupled GPCR. In certain embodiments, the CAGPCR is derived from a GPCR selected from the group consisting of an autoinhibitory GPCR, a self-activated GPCR, an adhesion GPCR, and GPR65.
[0010] In certain embodiments, the CAGPCR further comprises a heterologous signal sequence. In certain embodiments, the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-6. Tn certain embodiments, the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the heterologous signal sequence comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6.
[0011] In certain embodiments, the CAGPCR is derived from an autoinhibitory GPCR. In certain embodiments, the CAGPCR is derived from a mutated autoinhibitory GPCR. In certain embodiments, the mutated autoinhibitory GPCR comprises one or more amino acid substitutions.
[0012] In certain embodiments, the CAGPCR lacks one or more leucine-rich repeats (LRRs) as compared to the naturally-occurring GPCR. In certain embodiments, the CAGPCR does not comprise a LRR. In certain embodiments, the CAGPCR comprises a deletion in the ectodomain as compared to the naturally -occurring GPCR. In certain embodiments, the CAGPCR lacks an ectodomain. In certain embodiments, the CAGPCR is derived from RXFP1. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 10-27. Tn certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27.
[0013] In certain embodiments, the CAGPCR is derived from a self-activated GPCR. In certain embodiments, the CAGPCR is derived from GPR52. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 37.
[0014] In certain embodiments, the CAGPCR is derived from an adhesion GPCR. In certain embodiments, the adhesion GPCR is selected from the group consisting of LEC1, LEC2, LEC3, ELTD1, EMR1, EMR2, EMR3, mEMR4, CD97, GPR124, GPR125, CelsRl, CelsR2, CelsR3, GPR133, GPR144, GPR110, GPR111, GPR113, GPR115, GPR116, BAI1, BAI2, BA13. GPR64, GPR97, GPR112, GPR114, GPR126, GPR128, VLGR1.
[0015] In certain embodiments, the CAGPCR is derived from ADGRG2. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 29-31. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 31.
[0016] In certain embodiments, the CAGPCR is derived from ADGRG4. In certain embodiments, the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 33-35. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 35.
[0017] In certain embodiments, the CAGPCR is derived from GPR65. In certain embodiments, the CAGPCR is a mutated GPR65 comprising one or more amino acid substitutions selected from the group consisting of D60N, SI 01 A, and D268A. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38, wherein the amino acid at amino acid position 60 of SEQ ID NO: 38 is N, the amino acid at amino acid position 101 of SEQ ID NO: 38 is A, and/or the amino acid at amino acid position 286 of SEQ ID NO: 38 is A. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 39-53. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequence set forth in SEQ ID NO: 47. In certain embodiments, the CAGPCR compnses an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 51. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 52. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 53
[0018] In certain embodiments, the CAGaS is a mutated Gs alpha subunit comprising one or more amino acid substitutions selected from the group consisting of R201H, R201C, Q227R, and Q227H. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 54, wherein the amino acid at amino acid position 201 of SEQ ID NO: 54 is H or C, and/or the amino acid at amino acid position 227 of SEQ ID NO: 54 is R or H. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 55-58. In certain embodiments, the CAGaS comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 55. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 58
[0019] In certain embodiments, the polynucleotide is comprised within a viral vector. In certain embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picomavirus, lentivirus, herpes simplex virus, vaccinia virus, pox virus, and alphavirus. In certain embodiments, the viral vector is a single-stranded AAV. In certain embodiments, the viral vector is a self- complementary AAV.
[0020] In certain embodiments, the polynucleotide is comprised within a non-viral vector. In certain embodiments, the non-viral vector is a transposon-based vector. In certain embodiments, the non-viral vector is a PiggyBac-based vector, or a Sleeping Beauty-based vector.
[0021] In certain embodiments, the polynucleotide is a messenger RNA (mRNA). In certain embodiments, the polynucleotide is a modified messenger RNA (mmR A). In certain embodiments, substantially all uridines in the mmRNA are modified uridines. In certain embodiments, the modified uridines are selected from the group consisting of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio- 5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy uridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1- propynyl-pseudoundme, 5-taurinomethyluridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl- 2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 -methyl-pseudouridine, 4-thio-l- methyl-pseudouridine, 2-thio-l -methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihy drops eudouridine, 2-methoxyundine, 2-methoxy-4-thio-undine, 4-methoxy-pseudoundine, 4- methoxy-2-thio-pseudouridine, and pseudouridine. In certain embodiments, the modified uridines are 1-methyl-pseudouri dines.
[0022] In certain embodiments, the polynucleotide is in a lipid nanoparticle formulation. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a sterol, and/or a PEG-lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a sterol, and a PEG-lipid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGs. 1A-1C are graphs showing the level of TNFa production in THP-1 cells (FIG. 1A), human monocyte-derived macrophages (HMDM; FIG. IB), and mouse bone marrow-derived macrophages (BMDM; FIG. 1C), in response to liposaccharides (LPS) at the indicated pH. FIG. 1 A shows the level of TNFa production in THP-1 cells as a percentage of response to LPS, normalized to the pH 7.4 condition. FIGs. IB and 1C shows the level of TNFa production in THP-1 cells in pg/mL. [0024] FIGs. 2A-2B are graphs showing the level of cyclic AMP (cAMP) production (as a percentage of the level of cAMP production induced by wild type GPR65) in cells expressing the various GPR65 and mutants as indicated, in response to varying pH.
DETAILED DESCRIPTION
[0025] The present disclosure provides constitutively active modified G protein-coupled receptors (CAGPCRs) and methods of using the same. The present disclosure also provides constitutively active modified Gs alpha subunit (CAGaS) proteins and methods of using the same. In certain embodiments, expression of a CAGPCR and/or CAGaS in target immune cells modulates second messenger signaling (e.g., cyclic adenosine monophosphate [cAMP] signaling) in the immune cells, resulting in the inhibition of immune cell activation and/or inflammatory cytokine production by the immune cells. For example, expression of a CAGPCR and/or CAGaS in an immune cell may lead to increased cAMP production and associated downstream signaling. As known in the art, intracellular cAMP levels reduce the production of pro-inflammatory mediators and increase the production of anti-inflammatory factors in numerous immune cells. As such, expression of a CAGPCR and/or CAGaS in target immune cells may find use in the treatment of various inflammatory diseases.
Definitions
[0026] The terms "G protein-coupled receptor" or "GPCR" are known in the art and refer to polypeptides comprising seven, transmembrane-spanning alpha helices of between 22 to 24 amino acids, which mediate heterotrimeric G protein signaling. Each transmembrane helix is identified by number, i.e., transmembrane- 1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are joined by regions of amino acids between TM2 and TM3, TM4 and TM5, and TM6 and TM7, on the exterior, or extracellular side, of the cell membrane, referred to as extracellular loops 1, 2 and 3 (ECL1, ECL2 and ECL3), respectively. The transmembrane helices are also joined by regions of amino acids between TM1 and TM2, TM3 and TM4, and TM5 and TM6 on the interior, or intracellular side, of the cell membrane, referred to as intracellular loops 1, 2 and 3 (ICL1, ICL2 and ICL3), respectively. The seven TM helices may be referred to collectively as a 7TM region of the GPCR. The "carboxy" ("C") terminus of the receptor lies in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor lies in the extracellular space outside of the cell. GPCR structure and classification is generally well known in the art, and further discussion of GPCRs may be found in Probst, DNA Cell Biol. 1992 11 : 1-20; Marchese et al Genomics 23: 609-618, 1994; and the following books: Jurgen Wess (Ed) Structure-Function Analysis of G Protein-Coupled Receptors published by Wiley-Liss (1 st edition; Oct. 15, 1999); Kevin R. Lynch (Ed) Identification and Expression of G Protein-Coupled Receptors published by John Wiley & Sons (March 1998) and Tatsuya Haga (Ed), G Protein-Coupled Receptors, published by CRC Press (Sep. 24, 1999); and Steve Watson (Ed) G-Protein Linked Receptor Factsbook, published by Academic Press (1st edition; 1994). [0027] As used herein, the term “adhesion GPCR” or “aGPCR” refers to a certain class of GPCR characterized by seven transmembrane domains and an extracellular region comprising a GPCR autoproteolysis-inducing (GAIN) domain. It does not necessarily need to but can additionally contain one or more adhesion domains within its extracellular region.
[0028] As used herein, the term "naturally-occurring" in reference to a GPCR means a GPCR that is naturally produced (e.g., by a wild type mammal such as a human). Such GPCRs are found in nature. As used herein, a “modified” GPCR (e.g., a constitutively active modified GPCR) refers to a naturally-occurring GPCR that has been modified such that it is non-naturally occurring (i.e., is no longer found in nature).
[0029] As used herein, the term “polynucleotide,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides linked via a phosphodi ester bond. [0030] As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration of a polynucleotide to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
[0031] As used herein, the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.
[0032] As used herein, the term “subject” includes any human or non-human animal. In certain embodiments, the subject is a human or non-human mammal. In certain embodiments, the subject is a human.
Constitutively Active G Protein-Coupled Receptors
[0033] In certain embodiments, the method disclosed herein employ a constitutively active modified G protein-coupled receptor (CAGPCRs).
[0034] GPCRs can be modified to become constitutively active through mutation, or through the generation of GPCR variants, e.g., by deleting one or more regions in the N-terminal extracellular region (or ectodomain) of the GPCR. In certain embodiments, a CAGPCR of the present disclosure comprises a deletion in the ectodomain as compared to the naturally-occurring GPCR from which it is modified from. In certain embodiments, the CAGPCR lacks an ectodomain. In certain embodiments, the CAGPCR is a mutated GPCR comprising one or more amino acid substitutions that result in increased signaling.
[0035] In certain embodiments, the CAGPCR further comprises a heterologous signal sequence. In certain embodiments, the CAGPCR further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (P2AR). In certain embodiments, the CAGPCR further comprises an influenza hemagglutinin signal sequence (HASigSeq). Such heterologous sequences may also be referred to as leader sequences, and their presence in the CAGPCR, in certain embodiments, aids in the expression of the CAGPCR. Exemplary signal/leader sequences are described in Table 1.
Table 1: Exemplary Leader Sequences
Figure imgf000014_0001
[0036] In certain embodiments, the CAGPCR may further comprise a detectable label, e.g., a fluorescent label, an enzyme, a radiolabel, or other detectable group. The detectably labeled CAGPCR can be detected using standard techniques based on a characteristic of the detectable label, such as its enzymatic activity, radioactivity, or fluorescence. Exemplary detectable labels include, without limitation, the FLAG octapeptide DYKDDDK (SEQ ID NO: 7) and FLAG-K8A peptide DYKDDDA (SEQ ID NO: 8).
[0037] CAGPCRs useful in the present disclosure constitutively activate cAMP signaling. As such, a CAGPCR may be derived from any GPCR that functions to directly or indirectly stimulate cAMP signaling. In certain embodiments, the CAGPCR is derived from a Gsa-coupled GPCR.
[0038] In certain embodiments, a CAGPCR of the present disclosure is derived from a Gs alpha subunit (Gsa)-coupled GPCR. Gsa-coupled GPCRs are known in the art, and include, without limitation, 5-HT receptors; ACTH receptor (also known as MC2R); adenosine receptor types A2a and A2t; arginine vasopressin receptor 2; P-adrenergic receptor ty pes Pi, P2, and P3; calcitonin receptor; calcitonin gene-related peptide receptor; cannabinoid receptor 2; corticotropin-releasing hormone receptor; dopamine receptors Dl-like family; FSH-receptor; gastric inhibitory polypeptide receptor; glucagon receptor; growth hormone releasing hormone receptor; histamine H2 receptor; luteinizing hormone / choriogonadotropin receptor; melanocortin receptors (e g., MC1R, MC2R, MC3R, MC4R, MC5R); olfactory receptors; parathyroid hormone receptor 1; prostaglandin receptor types D2 and 12; secretin receptor; thyrotropin receptor; and trace amine-associated receptor 1.
[0039] The CAGPCRs of the present disclosure can be derived from any known GPCR, including, without limitation, human GPCRs and non-human orthologs thereof (e.g. from mammalians such as mouse, rat, Macaca mulatta or Macaca fascicularis, other vertebrates such as Xenopus tropicalis and Danio rerio, and invertebrates such as Drosophila melanogaster or Caenorhabditis elegans). In certain embodiments, the CAGPCR of the present disclosure is derived from a human GPCR.
Auto-inhibitory GPCRs
[0040] In certain embodiments, a CAGPCR of the present disclosure is derived from an auto- inhibitory GPCR. An auto-inhibitory GPCR suitable for modification into a CAGPCR of the present disclosure generally does not have significant basal activity, but signals in response to the binding of a ligand to its ectodomain. Such auto-inhibitory GPCRs likely use separate mechanisms to prevent continuous self-activation of the seven transmembrane domain. Auto-inhibitory GPCRs are known in the art and include, without limitation, RXFP1, adhesion GPCRs (aGPCRs), and protease activated receptors (PARs).
[0041] In certain embodiments, the CAGPCR is an auto-inhibitory GPCR that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is an auto-inhibitory GPCR that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is an auto- inhibitory GPCR that lacks an ectodomain. In certain embodiments, the CAGPCR comprises only the 7TM region of an auto-inhibitory GPCR , or a functional fragment thereof. In certain embodiments, the CAGPCR is an auto-inhibitory GPCR that lacks an ectodomain and comprises a sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR comprises an auto-inhibitory GPCR that lacks an ectodomain fused to an N-terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR comprises only the 7TM region of an auto-inhibitory GPCR fused to an N-terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR is a mutated auto-inhibitory GPCR comprising one or more amino acid substitutions that results in increased activity' (e.g., downstream signaling).
[0042] In certain embodiments, the CAGPCR is derived from RXFP 1. RXFP l is a member of the leucine-rich repeat-containing GPCRs (LGRs), where the leucine-rich repeats (LRRs) function as the extracellular ligand-binding domain for certain protein agonists (e.g., glycoprotein hormones, R-spondins, and relaxins). RXFP1 is a unique member of the LGR family as it contains an additional LDLa module in the ectodomain. RXFP1 is a Gs-coupled GPCR that when activated, leads to increased cAMP signaling. An exemplary wild type RXFP1 amino acid sequence is set forth in SEQ ID NO: 9. RXFP1 wild type amino acid sequence and RXFPl-denved CAGPCR ammo acid sequences are set forth in Table 2.
[0043] In certain embodiments, the CAGPCR is a mutated RXFP1 comprising one or more amino acid substitutions that results in increased cAMP signaling. In certain embodiments, the CAGPCR is a mutated RXFP1 comprising an I396A and/or S397A substitution. In certain embodiments, the CAGPCR is a mutated RXFP1 comprising an 1396 A substitution. In certain embodiments, the CAGPCR is a mutated RXFP1 comprising an S397A substitution. In certain embodiments, the CAGPCR is a mutated RXFP1 comprising an I396A and S397A substitution.
[0044] In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9, wherein the amino acid at amino acid position 396 of SEQ ID NO: 9 is A, and/or the amino acid at amino acid position 397 of SEQ ID NO: 9 is A.
[0045] In certain embodiments, the CAGPCR is RXFP1 that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is RXFP1 that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is RXFP1 that lacks one or more LRRs. In certain embodiments, the CAGPCR is RXFP1 that does not comprise a LRR. In certain embodiments, the CAGPCR is RXFP1 that lacks an ectodomain. In certain embodiments, the CAGPCR comprises only the 7TM region of RXFP1, or a functional fragment thereof. Exemplary RXFP 1 -derived CAGPCR amino acid sequences are set forth in Table 2.
[0046] In certain embodiments, the CAGPCR further comprises a heterologous signal/leader sequence. In certain embodiments, the CAGPCR further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (|32AR). In certain embodiments, the CAGPCR further comprises an influenza hemagglutinin signal sequence (HASigSeq). Exemplary signal/leader sequences are set forth in Table 1. Table 2: Exemplary RXFP1 Sequences
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
[0047] In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 10-27. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18. Tn certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27. Adhesion GPCRs
[0048] In certain embodiments, a CAGPCR of the present disclosure is derived from an adhesion GPCR (aGPCR). aGPCRs are a class of GPCRs characterized by seven transmembrane domains and an extracellular region (or ectodomain) comprising a GAIN domain. In certain embodiments, aGPCRs further comprise one or more adhesion domains within its extracellular region.
[0049] The GPCR autoproteolysis-inducing domain, or GAIN domain, refers to a region within the extracellular region of an aGPCR which is composed of an N-terminal subdomain A and a C-terminal subdomain B. Subdomain A of the GAIN domain is comprised of 3 to 6 alpha-helices. Subdomain B of the GAIN domain is comprised of 13 p-strands and can additionally contain several small alpha-helices. The GAIN domain of an aGPCR is auto-inhibitory, and cleavage thereof results in activation of the aGPCR.
[0050] GAIN domains include domains which do not exert autoproteolytic activity, but are classified as GAIN domains on the basis of alignments and/or sequence similarity to other proteins known to have a GAIN domain. Some aGPCRs contain autoproteolytic domains in addition to the GAIN domain, and some aGPCRs are processed by other endogenous proteases. In certain embodiments, aGPCRs possessing all three modes of cleavage (through the GAIN domain, through non-GAIN domains of the receptors, and/or through endogenous proteases) may be used to generate the CAGPCRs of the present disclosure.
[0051] aGPCRs are known in the art, and include, without limitation, Group I aGPCRs LPHN1, LPHN2, LPHN3, and ETL; Group II aGPCRs CD97, EMR1, EMR2, EMR3, and EMR4; Group III aGPCRs GPR123, GPR124, and GPR125; Group IV aGPCRs CELSR1, CELSR2, and CELSR3; Group V aGPCRs GPR133 and GPR144; Group VI aGPCRs GPR110, GPR111, GPR113, GPR115, and GPR116; Group VII aGPCRs B All, BAE, and BAB; Group VIII aGPCRs GPR56, GPR97, GPR112, GPR114, GPR126, and GPR64; and ungrouped aGPCRs VLGR1 and GPR128.
[0052] In certain embodiments, a CAGPCR of the present disclosure is derived from an aGPCR selected from the group consisting of LEC1, LEC2, LEC3, ELTD1, EMR1, EMR2, EMR3, mEMR4, CD97, GPR124, GPR125, CelsRl, CelsR2, CelsR3, GPR133, GPR144, GPR110, GPR111, GPR113, GPR115, GPR116, BAI1, BAE, BAI3, GPR64, GPR97, GPR112, GPR114, GPR126, GPR128, VLGR1.
[0053] In certain embodiments, the CAGPCR is an aGPCR that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is an aGPCR that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is an aGPCR that lacks an ectodomain. In certain embodiments, the CAGPCR comprises the 7TM region and tethered agonist sequence (i.e., tethered ligand) of an aGPCR, or a functional fragment thereof. In certain embodiments, the CAGPCR is a mutated aGPCR comprising one or more amino acid substitutions that results in increased activity (e.g., downstream signaling). [0054] In certain embodiments, the CAGPCR is derived from an aGPCR and further comprises a heterologous signal/leader sequence. In certain embodiments, the CAGPCR is derived from an aGPCR and further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (P2AR). In certain embodiments, the CAGPCR is derived from an aGPCR and further comprises an influenza hemagglutinin signal sequence (HASigSeq). Exemplary signal/leader sequences are set forth in Table 1.
[0055] In certain embodiments, the CAGPCR is derived from ADGRG2. An exemplary wild type ADGRG2 amino acid sequence is set forth in SEQ ID NO: 28. ADGRG2 wild type amino acid sequence and ADGRG2-derived CAGPCR amino acid sequences are set forth in Table 3. Table 3: Exemplary ADGRG2 Sequences
Figure imgf000025_0001
Figure imgf000026_0001
[0056] In certain embodiments, the CAGPCR is derived from ADGRG4. An exemplary wild type ADGRG4 amino acid sequence is set forth in SEQ ID NO: 32. ADGRG4 wild type amino acid sequence and ADGRG4-derived CAGPCR amino acid sequences are set forth in Table 4.
Table 4: Exemplary ADGRG4 Sequences
Figure imgf000026_0002
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
[0057] In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 29-31, and 33- 35. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 35.
Self-Activated GPCRs
[0058] In certain embodiments, a CAGPCR of the present disclosure is derived from a selfactivated GPCR. Self-activated GPCRs have very high basal activity signaling at close to its maximum response (Emax value) without any agonist bound.
[0059] In certain embodiments, the CAGPCR is a self-activated GPCR that lacks one or more regions in the ectodomain. In certain embodiments, the CAGPCR is a self-activated GPCR that comprises a deletion in the ectodomain. In certain embodiments, the CAGPCR is a self-activated GPCR that lacks an ectodomain. In certain embodiments, the CAGPCR comprises only the 7TM region of a self-activated GPCR, or a functional fragment thereof. In certain embodiments, the CAGPCR is a self-activated GPCR that lacks an ectodomain and comprises a sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR comprises a self-activated GPCR that lacks an ectodomain fused to an N-terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR comprises only the 7TM region of a self-activated GPCR fused to an N- terminal sequence derived from a heterologous GPCR. In certain embodiments, the CAGPCR is a mutated self-activated GPCR comprising one or more amino acid substitutions that results in increased activity (e.g., downstream signaling).
[0060] In certain embodiments, the CAGPCR is derived from a self-activated GPCR and further comprises a heterologous signal/leader sequence. In certain embodiments, the CAGPCR is derived from a self-activated GPCR and further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor ( 2AR). In certain embodiments, the CAGPCR is derived from a selfactivated GPCR and further comprises an influenza hemagglutinin signal sequence (HASigSeq). Exemplary signal/leader sequences are set forth in Table 1.
[0061] In certain embodiments, the CAGPCR is derived from GPR52 (also known as the psychosine receptor). GPR52 is a self-activating orphan GPCR in which its intrinsic activity is governed by its own ECL2. GPR52 comprises an unstructured N-terminus. An exemplary wild type GPR52 amino acid sequence is set forth in SEQ ID NO: 36. GPR52 wild type amino acid sequence and GPR52-derived CAGPCR amino acid sequences are set forth in Table 5.
Table 5: Exemplary GPR52 Sequences
Figure imgf000030_0001
Figure imgf000031_0001
[0062] In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, al least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 37.
Others
[0063] In certain embodiments, a CAGPCR of the present disclosure is derived from a GPCR in which one or more amino acid substitutions results in increased activity (e.g., increased downstream signaling; increased cAMP signaling).
[0064] For example, GPR65 (also known as TDAG8) is a GPCR that senses extracellular pH. Levels of cAMP were found to be elevated in neutral to acidic extracellular pH in cells expressing GPR65. GPR65 senses pH by protonation of histidine residues on its extracellular domain. In certain embodiments, a CAGPCR of the present disclosure is derived from GPR65. In certain embodiments, the CAGPCR is a mutated GPR65 comprising one or more amino acid substitutions that result in increased cAMP signaling. In certain embodiments, the CAGPCR is a mutated GPR65 comprising an D60N, S101A, and/or a D286A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a D60N substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a S101A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a D286A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a D60N and S101A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a D60N and D286A substitution. In certain embodiments, the CAGPCR is a mutated GPR65 comprising a SI 01 A and D286A substitution. Tn certain embodiments, the CAGPCR is a mutated GPR65 comprising a D60N, S101 A, and a D286A substitution.
[0065] In certain embodiments, the CAGPCR is derived from GPR65 and further comprises a heterologous signal/leader sequence. In certain embodiments, the CAGPCR is derived from GPR65 and further comprises an N-terminal sequence derived from the beta-2-adrenergic receptor (P2AR). In certain embodiments, the CAGPCR is derived from GPR65 and further comprises an influenza hemagglutinin signal sequence (HASigSeq). Exemplary signal/leader sequences are set forth in Table 1.
[0066] An exemplary wild type GPR65 amino acid sequence is set forth in SEQ ID NO: 38. GPR65 wild type amino acid sequence and GPR65-derived CAGPCR amino acid sequences are set forth in Table 6.
[0067] In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38, wherein the amino acid at amino acid position 60 of SEQ ID NO: 38 is N, the amino acid at amino acid position 101 of SEQ ID NO: 38 is A, and/or the amino acid at amino acid position 286 of SEQ ID NO: 38 is A.
Table 6: Exemplary GPR65 Sequences
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
[0068] In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 39-53. Tn certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 47. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 51. In certain embodiments, the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 52. In certain embodiments, the CAGPCR comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 53.
Constitutively Active Gs Alpha Subunit (CAGuS)
[0069] In certain embodiments, the method disclosed herein employ a constitutively active modified Gs Alpha Subunit (CAGaS) protein.
[0070] The Gas protein can be modified to become constitutively active through mutation. In certain embodiments, a CAGaS is a mutated Gas protein comprising one or more amino acid substitutions that result in increased signaling.
[0071] In certain embodiments, the CAGaS is a mutated Gas protein comprising a R201H or R201C substitution, and/or a Q227R or Q227H substitution. In certain embodiments, the CAGaS is a mutated Gas protein comprising a R201H substitution. In certain embodiments, the CAGaS is a mutated G0.s protein comprising a R201C substitution. In certain embodiments, the CAGaS is a mutated Gas protein comprising a Q227R substitution. In certain embodiments, the CAGaS is a mutated Gas protein comprising a Q227H substitution. In certain embodiments, the CAGaS is a mutated G0.s protein comprising a R201H and Q227R substitution. In certain embodiments, the CAGaS is a mutated Gas protein comprising a R201H and Q227H substitution. In certain embodiments, the CAGaS is a mutated Gas protein comprising a R201C and Q227R substitution. In certain embodiments, the CAGaS is a mutated G0.s protein comprising a R201C and Q227H substitution.
[0072] An exemplary wild type Gas protein amino acid sequence is set forth in SEQ ID NO: 54. Gas protein wild type amino acid sequence and CAGaS amino acid sequences are set forth in Table 7.
[0073] In certain embodiments, the CAGaS comprises an ammo acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 54, wherein the amino acid at amino acid position 201 of SEQ ID NO: 54 is H or C, and/or the amino acid at amino acid position 227 of SEQ ID NO: 54 is R or H.
Table 7: Exemplary CAGaS Sequences
Figure imgf000038_0001
Figure imgf000039_0001
[0074] In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 55-58. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 55. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 58.
Polynucleotides and Vectors
[0075] In certain embodiments, the methods disclosed herein employ a polynucleotide that encodes any of constitutively active modified G protein-coupled receptors (CAGPCRs) or constitutively active modified Gs Alpha Subunit (CAGaS) proteins described herein. In certain embodiments, the polynucleotide is a DNA molecule. In certain embodiments, the polynucleotide is an RNA molecule.
[0076] The polynucleotide described herein can be transcribed from an expression vector (e.g., a recombinant expression vector). In certain embodiments, the polynucleotide is comprised within a vector. In certain embodiments, the vector is a non-viral vector. Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministnngs, and oligonucleotides (e.g., mRNA, naked DNA). In certain embodiments, the non-viral vector is a DNA plasmid vector. In certain embodiments, the non-viral vector is a transposon-based vector. In certain embodiments, the non-viral vector is a PiggyBac-based vector, or a Sleeping Beauty-based vector.
[0077] In certain embodiments, the vector is a viral vector. Viral vectors can be replication competent or replication incompetent. Viral vectors can be integrating or non-integrating. A number of viral based systems have been developed for gene transfer into mammalian cells, and a suitable viral vector can be selected by a person of ordinary skill in the art. Exemplary viral vectors include, but are not limited to, adenovirus vectors (e.g, adenovirus 5), adeno-associated virus (AAV) vectors (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9), retrovirus vectors (e.g, MMSV, MSCV), lentivirus vectors (e.g., HIV-1, HIV-2), gammaretrovirus vectors, herpes virus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g. , SFV, SIN, VEE, Ml), flavivirus (e.g., Kunjin, West Nile, Dengue virus), rhabdo virus vectors (e.g., rabies virus, VSV), measles virus vector, Newcastle disease virus vectors, poxvirus vectors, and picomavirus vectors (e.g, Coxsackievirus). In certain embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picomavirus, lentivirus, herpes simplex virus, vaccinia virus, pox virus, and alphavirus. In certain embodiments, the viral vector is a single-stranded AAV Tn certain embodiments, the viral vector is a self- complementary AAV.
[0078] Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see. e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al.. Gene Ther 4:683 690, 1997. Rolling et al.. Hum Gene Ther 10:641648, 1999; Ali et al., Hum Mol Genet 5:591594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flott et al., PNAS (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see. e.g., Miyoshi et al., PNAS 94: 10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.
[0079] In certain embodiments, the polynucleotide is integrated into the genome of the target cell (e.g., immune cell). In certain embodiments, the polynucleotide is integrated via random integration, a site-specific integration, or a biased integration. In certain embodiments, the sitespecific integration can be non-assisted or assisted. In certain embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In some embodiments, the site-directed nuclease comprises the polynucleotide with 5' and 3' nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In certain embodiments, the polynucleotide with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology -mediated end joining, ornonhomologous end-joining. In certain embodiments the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus. [0080] Tn certain embodiments, the site-specific integration occurs at a site that disrupts expression of a target gene. In certain embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.
[0081] In certain embodiments, the site-specific integration occurs at a site that results in enhanced expression of a target gene. In certain embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.
[0082] In certain embodiments, enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the polynucleotide. In certain embodiments, enzymes create single-strand breaks. In certain embodiments, enzymes create double-strand breaks. In certain embodiments, examples of break-inducing enzymes include but are not limited to: transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVER™, and CPF1. In certain embodiments, break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, as a nucleoprotein complex with a guide RNA (gRNA).
[0083] In certain embodiments, the polynucleotide is RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art. Any known method can be used to synthesize RNA comprising a nucleic acid sequence encoding a CAGPCR of the present disclosure.
[0084] In certain embodiments, the polynucleotide is a modified RNA that contains one or more modified nucleosides Exemplary modified RNAs include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. They may also include RNAi- inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
[0085] In certain embodiments, the modified RNA is one or more modified messenger RNAs (mmRNAs). As described herein, in certain embodiments, the mmRNAs do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
[0086] In certain embodiments, the mmRNAs further express a protein-binding partner or a receptor on the surface of the cell, which functions to target the cell to a specific tissue space or to interact with a specific moiety, either in vivo or in vitro. Suitable protein-binding partners include antibodies and functional fragments thereof, scaffold proteins, or peptides.
[0087] In certain embodiments, the mmRNAs may further encode a cell-penetrating polypeptide. As used herein, “cell -penetrating polypeptide” refers to a polypeptide which may facilitate the cellular uptake of molecules. In certain embodiments, the cell-penetrating polypeptide may contain one or more detectable labels. The polypeptides may be partially labeled or completely labeled throughout. The mmRNA may encode the detectable label completely, partially or not at all. The cell-penetrating peptide may also include a signal sequence. As used herein, a “signal sequence” refers to a sequence of amino acid residues bound at the amino terminus of a nascent protein during protein translation. The signal sequence may be used to signal the secretion of the cell-penetrating polypeptide.
[0088] Polynucleotides may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription, enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D C : IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).
[0089] The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared from readily available starting materials using the following general methods and procedures. It is understood that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
[0090] The manufacturing process can be monitored according to any suitable method know n in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., iH or BC) infrared spectroscopy, spectrophotometry (e.g., UV -visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
[0091] mmRNAs generally comprise a translatable region and one, two, or more than two different modifications. In certain embodiments, the chemical modifications can be located on the nucleobase of the nucleotide. In certain embodiments, the chemical modifications can be located on the sugar moiety of the nucleotide. In certain embodiments, the chemical modifications can be located on the phosphate backbone of the nucleotide.
[0092] Preparation of modified nucleosides and nucleotides used in the manufacture or synthesis of modified RNAs can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
[0093] Modified nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al. Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al. Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al. Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9): 1729-1740, 1992, each of which are incorporated by reference in their entirety.
[0094] Modified mRNAs need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the polynucleotide sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a mRNA sequence such that the function of the modified mRNA is not substantially decreased. A modification may also be a 5' or 3' terminal modification. Modified mRNAs may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
[0095] For example, the mmRNAs may contain a modified pyrimidine such as uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide may be replaced with a modified uracil. The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the mRNA may be replaced with a modified cytosine. The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
[0096] In certain embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy uridine, 3-melhyluridine, 5-carboxymethyl-uridine, 1 -carboxy methyl-pseudouri dine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1 -taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l -taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 - methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l- deaza-pseudouridine, 2-thio-l -methyl-1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, modified nucleosides include 5 -aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5 -hydroxy methylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5 -methylcytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidme.
[0097] In certain embodiments, modified nucleosides include 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio- N6-(cis-hydroxyisopentenyl) adenosine, N6-glycmylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyl adenine, 2-methylthio-adenine, and 2-methoxy-adenine.
[0098] In certain embodiments, modified nucleosides include inosine, 1 -methyl-inosine, wyosine, wybutosme, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
[0099] In certain embodiments, the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
[00100] In certain embodiments, a modified nucleoside is 5'-0-(l-Thiophosphate)-Adenosine, 5'-0-(l-Thiophosphate)-Cytidine, 5'-0-(l-Thiophosphate)-Guanosine, 5'-0-(l-Thiophosphate)- Uridine or 5'-0-(l-Thiophosphate)-Pseudouridine.
[00101] In certain embodiments, the modified mRNA comprises one or more modified uridines. In certain embodiments, the modified mRNA comprises one or more 1-methyl- pseudouri dines.
[00102] Other components of an mRNA are optional, and may be beneficial in some embodiments. For example, a 5' untranslated region (UTR) and/or a 3'UTR may be included, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. In certain embodiments, polynucleotides comprise a Kozak sequence.
[00103] In some embodiments, the polynucleotide comprises one or more additional elements. Additional elements include, but are not limited to, promoters, enhancers, polyadenylation (poly A) sequences, and selection genes.
[00104] Examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
Pharmaceutical Compositions
[00105] In certain embodiments, the methods disclosed herein employ pharmaceutical compositions comprising a polynucleotide disclosed herein. The pharmaceutical compositions described herein are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington ’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as L1POFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also, Powell et al., “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.
[00106] The dose of the polynucleotide administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering the polynucleotide that encode them may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).
[00107] Various delivery systems are known and can be used to administer the pharmaceutical composition disclosed herein, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
[00108] Any pharmaceutical composition described herein can be delivered subcutaneously or intravenously with a standard needle and synnge. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition disclosed herein. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
[00109] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see, Langer, supra,' Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.
[00110] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g, by dissolving, suspending, or emulsifying any of the polynucleotides described herein in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g, ethanol), a polyalcohol (e.g, propylene glycol, polyethylene glycol), a nonionic surfactant [e g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
[00111] Regarding formulations of modified mRNAs, in certain embodiments, the formulations include one or more cell penetration agents, e.g., transfection agents. In one specific embodiment, an mmRNA is mixed or admixed with a transfection agent (or mixture thereof) and the resulting mixture is employed to transfect cells. Preferred transfection agents are cationic lipid compositions, particularly monovalent and polyvalent cationic lipid compositions, more particularly LIPOFECTIN®, LIPOFECTACE®, LIPOFECTAMINE™, CELLFECTIN®, DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER, and dendrimer compositions, particularly G5-G10 dendrimers, including dense star dendrimers, PAMAM dendrimers, grafted dendrimers, and dendrimers known as dendrigrafts and SUPERFECT®.
[00112] In a second specific transfection method, a ribonucleic acid is conjugated to a nucleic acid-binding group, for example a polyamine and more particularly a spermine, which is then introduced into the cell or admixed with a transfection agent (or mixture thereof) and the resulting mixture is employed to transfect cells. In a third specific embodiment, a mixture of one or more transfection-enhancing peptides, proteins, or protein fragments, including fusagenic peptides or proteins, transport or trafficking peptides or proteins, receptor-ligand peptides or proteins, or nuclear localization peptides or proteins and/or their modified analogs (e.g., spermine modified peptides or proteins) or combinations thereof are mixed with and complexed with a ribonucleic acid to be introduced into a cell, optionally being admixed with transfection agent and the resulting mixture is employed to transfect cells. Further, a component of a transfection agent (e.g., lipids, cationic lipids or dendrimers) is covalently conjugated to selected peptides, proteins, or protein fragments directly or via a linking or spacer group. Of particular interest in this embodiment are peptides or proteins that are fusagenic, membrane-permeabilizing, transport or trafficking, or which function for celltargeting. The peptide- or protein-transfection agent complex is combined with a ribonucleic acid and employed for transfection.
[00113] In certain embodiments, the formulations include a pharmaceutically acceptable earner that causes the effective amount of mmRNA to be substantially retained in a target tissue containing the cell.
[00114] In certain embodiments, the formulation may include at least an mmRNA and a deliver}' agent. In some embodiments, the delivery agent may comprise lipidoid-based formulations allowed for localized and systemic delivery of mmRNA.
[00115] Also provided are compositions for generation of an in vivo depot containing an engineered ribonucleotide. For example, the composition contains a bioerodible, biocompatible polymer, a solvent present in an amount effective to plasticize the polymer and form a gel therewith, and an engineered ribonucleic acid. In certain embodiments the composition also includes a cell penetration agent as described herein. In other embodiments, the composition also contains a thixotropic amount of a thixotropic agent mixable with the polymer so as to be effective to form a thixotropic composition. Further compositions include a stabilizing agent, a bulking agent, a chelating agent, or a buffering agent.
[00116] In other embodiments, provided are sustamed-release delivery depots, such as for administration of a mmRNA to an environment (meaning an organ or tissue site) in a patient. Such depots generally contain a mmRNA and a flexible chain polymer where both the mmRNA and the flexible chain polymer are entrapped within a porous matrix of a crosslinked matrix protein. Usually, the pore size is less than 1 mm, such as 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, or less than 100 nm. Usually the flexible chain polymer is hydrophilic. Usually the flexible chain polymer has a molecular weight of at least 50 kDa, such as 75 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, or greater than 500 kDa. Usually the flexible chain polymer has a persistence length of less than 10%, such as 9, 8, 7, 6, 5, 4, 3, 2, 1 or less than 1% of the persistence length of the matrix protein. Usually the flexible chain polymer has a charge similar to that of the matrix protein. In certain embodiments, the flexible chain polymer alters the effective pore size of a matrix of crosslinked matrix protein to a size capable of sustaining the diffusion of the mmRNA from the matrix into a surrounding tissue comprising a cell into which the mmRNA is capable of entering.
[00117] Lipidoid-based formulations are also provided herein, allowing for localized and systemic delivery of mmRNA. The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21 ; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864- 1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which are incorporated herein by reference in their entireties).
[00118] In certain embodiments, complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, result in an effective delivery of mmRNA, as judged by the production of an encoded protein (e.g., CAGPCR), following the injection of an mmRNA-formulated lipidoids via localized and systemic routes of administration. Modified mRNA-lipidoid complexes can be administered by vanous means disclosed herein.
[00119] The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 herein incorporated by reference), use of lipidoid oligonucleotides to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.
[00120] In one aspect, effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of the mmRNA molecule for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. For example, the component molar ratio may include, but is not limited to, 50% lipid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 (including variants and derivatives), DLin-MC3-DMA and analogs thereof. The use of lipidoid formulations for the localized deliver}' of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may also not require all of the formulation components which may be required for systemic delivery, and as such may comprise the lipidoid and the mmRNA.
[00121] In a further embodiment, combinations of different lipidoids may be used to improve the efficacy of mmRNA-directed protein.
[00122] In certain embodiments, a modified rnRNA may be formulated by mixing the mmRNA with the lipidoid at a set ratio prior to addition to cells. In vivo formulations may require the addition of extra ingredients to facilitate circulation throughout the body. To test the ability of these lipidoids to form particles suitable for in vivo work, a standard formulation process used for siRNA-lipidoid formulations may be used as a starting point Initial mmRNA-lipidoid formulations consist of particles composed of 42% lipidoid, 48% cholesterol and 10% PEG, with further optimization of ratios possible. After formation of the particle, mmRNA is added and allowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays.
[00123] In vivo delivery of nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety). As an example, small changes in the anchor chain length of polyethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy. Formulations with the different lipidoids, including, but not limited to penta[3-(l- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-SLAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401 :61 (2010)), C12-200 (including derivatives and variants), MD1, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA and DLin-MC3-DMA, can be tested for in vivo activity.
[00124] The lipidoid referred to herein as “98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879 and is incorporated by reference in its entirety.
[00125] The lipidoid referred to herein as “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670; both of which are herein incorporated by reference in their entirety. The lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotide, primary construct, or mmRNA. As an example, formulations with certain lipidoids, include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (Cl 4 alkyl chain length). As another example, formulations with certain lipidoids, include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG- DMG.
[00126] The ratio of mmRNA to lipidoid used to test for in vitro transfection is tested empirically at different lipidoid: mmRNA ratios. Previous work using siRNA and lipidoids have utilized 2.5: 1, 5: 1, 10: 1, and 15: 1 lipidoid: siRNA wt:wt ratios. Given the longer length of mmRNA relative to siRNA, a lower wt:wt ratio of lipidoid to mmRNA is likely to be effective. In addition, for comparison mmRNA are also formulated using RNAiMax (Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison Wis.) cationic lipid delivery vehicles.
[00127] The ability of lipidoid-formulated mmRNA to express the desired protein product can be confirmed by luminescence for luciferase expression, flow cytometry for expression, and by ELISA for secretion. [00128] Tn certain embodiments, the pharmaceutical composition comprises amodifiedmRNA formulated in a lipid nanoparticle formulation comprising a lipid selected from the group consisting of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200; a cholesterol; and a PEG-lipid. In certain embodiments, the modified mRNA and lipid nanoparticle are formulated at a total lipid to mRNA weight ratio of 10: 1, 15: 1, 20: 1, or 30: 1. In certain embodiments, the lipid is DLin-KC2-DMA or 98N12-5. In certain embodiments, the lipid nanoparticle formulation comprises about 42% lipid, about 48% cholesterol, and about 10% PEG- lipid. In certain embodiments, the lipid nanoparticle formulation comprises about 50% lipid, about 38.5% cholesterol, and about 1.5% PEG-lipid. In certain embodiments, the lipid nanoparticle has a mean particle size between 86 nm and 155 nm. In certain embodiments, the lipid nanoparticle has a polydisperity index between 0.02 and 0.17. In certain embodiments, the lipid nanoparticle formulation further comprises a formulation buffer for in vivo delivery , wherein the formulation buffer has a pH of 6.5 and comprises sodium chloride, calcium chloride, and Na+-phosphate. In certain embodiments, the formulation buffer comprises 150 mM sodium chloride, 2 rnM calcium chloride, and 2 mM Na+-phosphate.
Methods of Use
[00129] In one aspect, the present disclosure provides a method of inhibiting immune cell activation in a subject. In another aspect, the present disclosure provides a method of inhibiting inflammatory cytokine production by an immune cell in a subject. Generally, the methods of the present disclosure comprise introducing into an immune cell of the subj ect a polynucleotide encoding a constitutively active modified G-coupled G protein-coupled GPCR (CAGPCR) and/or a constitutively active modified Gs Alpha Subunit (CAGaS) protein. Upon introduction of the polynucleotide encoding a CAGPCR and/or CAGaS, the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting immune cell activation or inflammatory cytokine production by the immune cell in the subject.
[00130] GPCRs play important roles in inflammation, and inflammatory cells such as leukocytes, macrophages, and monocytes, express a large number of endogenous GPCRs that recognize classic chemoattractants and chemokines. Such endogenous GPCRs are critical to the migration of phagocytes and their accumulation at sites of inflammation, where the cells can exacerbate inflammation. Many endogenous GPCRs present in inflammatory cells also mediate transcription factor activation, resulting in the synthesis and secretion of pro-inflammatory factors. In certain embodiments, activation of endogenous GPCRs expressed by inflammatory cells (e g., pro- inflammatory immune cells) leads to a reduction in intracellular levels of cAMP. Without being bound by any theory, expression of a CAGPCR of the present disclosure in immune cells may shift the balance of signaling within the immune cell towards signaling that results in anti-inflammatory functions, for example, leads to the increase in intracellular levels of cAMP. In certain embodiments, the immune cell endogenously expresses a Gi-coupled GPCR.
[00131] In certain embodiments, methods of the present disclosure are for inhibiting pro- inflammatory cytokine production by an immune cell of the subject. Pro-inflammatory cytokines are secreted from Thl cells, CD4+ cells, macrophages, and dendritic cells. Pro-inflammatory cytokines are known in the art, and include, without limitation, IL-1 (e.g., IL-1 P), IL-2, IL-6, IL-8, IL-12, IL- 17, IL-18, TNF-a, IFN-y, and GM-CSF.
[00132] In certain embodiments, the immune cell is a myeloid cell. In certain embodiments, the immune cell is an immune cell involved in pro-inflammatory responses. Such immune cells are known in the art, and include, without limitation, lymphocytes (e.g., CD4+ T cells), leukocytes, macrophages, and monocytes.
[00133] Accordingly, methods of the present disclosure find use in treating a subject having an inflammatory disease or disorder. An “inflammatory disease or disorder”, as used herein, refers to a disease, disorder or pathological condition where the pathology results, in whole or in part, from, e.g., a change in number, change in rate of migration, or change in activation, of cells of the immune system. Cells of the immune system include, e.g., T cells, B cells, monocytes or macrophages, innate lymphoid cells, antigen presenting cells (APCs), dendritic cells, microglia, NK cells, neutrophils, eosinophils, mast cells, or any other cell specifically associated with the immunology, for example, cytokine-producing endothelial or epithelial cells. As used herein, in one embodiment, the “inflammatory disease or disorder” is an immune disorder or condition selected from the group consisting of asthma, (including steroid resistant asthma, steroid sensitive asthma, eosinophilic asthma or non-eosinophilic asthma), allergy, anaphylaxis, multiple sclerosis, inflammatory bowel disorder (e.g. Crohn's disease or ulcerative colitis), chronic obstructive pulmonary disease (COPD, which may or may not be related to, caused in part by, or resulting from, exposure to first or second hand cigarette smoke), asthma and COPD overlap syndrome (ACOS), eosinophilic esophagitis, chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, lupus, atopic dermatitis, psoriasis, scleroderma and other fibrotic diseases, sjogren's syndrome, vasculitis (behcef s disease, Giant cell arteritis, Henoch-Schonlein purpura and Churg Strauss syndrome), inflammatory pain and arthritis. In one embodiment, the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, and psoriatic arthritis.
[00134] In certain embodiments, multiple doses of the polynucleotide may be administered to a subject over a defined time course. The methods according to this aspect of the disclosure comprise sequentially administering to a subject multiple doses of the polynucleotide. As used herein, “sequentially administering” means that each dose of the polynucleotide is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure provides methods which comprise sequentially administering to the patient a single initial dose of the polynucleotide, followed by one or more secondary doses of the polynucleotide, and optionally followed by one or more tertiary doses of the polynucleotide.
[00135] The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the polynucleotide. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the polynucleotide, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amounts of the polynucleotide contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
[00136] In one exemplary embodiment, each secondary and/or tertiary dose is administered 1 to 26 (e.g, 1, 1/2, 2, 2 2, 3, 3/2, 4, 4/2, 5, 5/2, 6, 6/2, 7, 7/2, 8, 8/2, 9, 9/2, 10, IO/2, 11, H/2, 12, 12/2, 13, 13/2, 14, 14/2, 15, 15/2, 16, I6/2, 17, 17/2, 18, I8/2, 19, 19/2, 20, 20/2, 21, 21 /2, 22, 22/2, 23, 23/, 24, 24/, 25, 25/, 26, 26/, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of the polynucleotide, which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
[00137] The methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of the polynucleotide. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient. [00138] Tn embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
[00139] In one embodiment, one or more of the polynucleotides are administered to a subject as a weight-based dose. A “weight-based dose” (e.g., a dose in mg/kg) is a dose of the protein or peptides that will change depending on the subject’s weight.
[00140] In another embodiment, one or more of the polynucleotides, is administered to a subject as a fixed dose. A “fixed dose” (e.g., a dose in mg) means that one dose of the polynucleotide is used for all subjects regardless of any specific subject-related factors, such as weight. In one particular embodiment, a fixed dose of the polynucleotide is based on a predetermined weight or age.
Kits
[00141] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, the kit comprises one or more of the polynucleotides. The kit may further include reagents or instructions for using the polynucleotides in a subject. It may also include one or more buffers.
[00142] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. However, various combinations of components may be comprised in a vial. The kits of the present disclosure also typically include a means for containing the polynucleotides, and any other reagent containers in close confinement for commercial sale.
[00143] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
EXAMPLES
[00144] The following examples are offered by way of illustration, and not by way of limitation.
Example 1: Constitutively Active Modified GPCR Derived from RXFP1
[00145] LPS is the major component of Gram-negative bactena cell walls and can cause an acute inflammatory response by triggering the release of a vast number of inflammatory cytokines in various cell types.
[00146] THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. THP-1 cells are known to express wild-type (naturally-occurring) RXFP1 and are know n to be sensitive to liposaccharide (LPS). It has been shown that stimulation of THP-1 cells by LPS induces the production of pro-inflammatory cytokines such as interleukin-1 (3 ( I L- 1 (3), tumour necrosis factor (TNF-a) and interleukin-6 (IL-6). Further, THP-1 cells are know n to express wild-type GPR65 (also known as TDAG8 and the psychosine receptor), a pH sensing GPCR that stimulates cAMP signaling in response to acidic pH.
[00147] THP-1 cells, as well as primary human monocyte-derived macrophages (HMDM), and mouse bone marrow-derived macrophages (BMDM), were tested for sensitivity to LPS. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and contamination with Mycoplasma was periodically excluded. 1x106 cells were incubated for 24 hours in growth medium containing 100 ng/ml LPS from Escherichia coli O26/B6. Cells were then pelleted, washed with PBS and re-suspended in fresh medium before plating into wells of 24-well plates that have been pre-coated with an anti -TNFa antibody. Expression of TNFa was measured by standard ELISA.
[00148] As shown in FIGs. 1A-1C, incubation of THP-1 cells (FIG. 1A), HMDM (FIG. IB), and BMDM (FIG. 1C) with LPS resulted in increased production of the pro-inflammatory cytokine TNFa. For each type of cell, upon decreasing the pH of medium to acidic pH (pH 6.8 for THP-1 cells, and pH 6.3 for the primary cells), the induced production of TNFa was significantly decreased, indicative of activated GPR65-induced cAMP signaling. In FIG. 1A, the experiment was repeated three times, and **** refers to p < 0.0001 . In FIG. 1 B, the data is pooled from six donors, and **** refers to p < 0.0001. In FIG. 1C, **** refers to p < 0.0001.
[00149] Next, the ability of relaxin (a ligand for RXFP1) to decrease the LPS-induced immune cytokine expression and/or secretion by THP-1 cells, HMDM, and BMDM will be confirmed. Then, a constitutively active modified GPCR will be generated from RXFP1. Exemplary amino acid sequences of RXFPl-CAGPCRs that will be tested are set forth in Table 2. The RXFP1-CAGPCR will be expressed in THP-1 cells to confirm that RXFP1-CAGPCR expression can decrease the LPS- induced immune cytokine expression and/or secretion by THP-1 cells, in the absence or presence of relaxin.
Example 2: Constitutively Active Modified GPCR Derived from GPR65
[00150] As shown in Example 1, LPS induced production of TNFa in THP-1 cells. Decreasing the pH of medium to acidic pH significantly decreased the LPS-induced production of TNFa, indicative of activated GPR65-induced cAMP signaling.
[00151] To investigate whether GPR65-derived constitutively active modified GPCRs will reproduce the effect of wild type (WT) GPR65, GPR65-CAGPCRs will be generated. Exemplary amino acid sequences of GPR65-CAGPCRs are set forth in Table 8.
[00152] GPR65 (WT), GPR65 (D60N), GPR65 (D286A), GPR65 (D286N), and GPR65 (S101A) were tested as follows, and the cAMP response to varying pH was measured (FIGs. 2A and 2B). Briefly, HEK293 cells were seeded into a 96-well tissue culture plate followed by transient co- transfection with GPR65 (WT) or GPR65-CAGPCR constructs, and a pGloSensor-22F plasmid. Transfected cells were stimulated by maintaining cells in buffer at a defined pH, inducing Gs- mediated cAMP signaling. cAMP was assayed using the activity of the GloSensor biosensor, a mutant luciferase fused to a cAMP binding domain that leads to production of light in the presence of its substrate luciferin. The readout of relative luminescent units (RLU) was used as a proxy for cAMP response.
[00153] As shown in FIGs. 2A and 2B, and Table 8, expression of GPR65 (S101 A) resulted in the highest cAMP response, followed by GPR65 (D286N) and GPR65 (D60N).
Table 8: cAMP Signaling of Various GPR65-CAGPCRs
Figure imgf000057_0001
[00154] The GPR65-CAGPCR will be expressed in THP-1 cells to confirm that GPR65- CAGPCR expression can decrease the LPS-induced immune cytokine expression and/or secretion by THP-1 cells, at different pH.
* * *
[00155] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
[00156] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. [00157] Other embodiments are within the following claims.

Claims

1. A method of inhibiting immune cell activation in a subj ect, comprising introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protein- coupled receptor (CAGPCR) and/or a constitutively active modified Gs alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting immune cell activation in the subject.
2. A method of inhibiting inflammatory cytokine production by an immune cell in a subject, introducing into an immune cell of the subject a polynucleotide encoding a constitutively active modified G protein-coupled receptor (CAGPCR) and/or a constitutively active modified Gs alpha subunit (CAGaS), such that the CAGPCR and/or CAGaS is expressed in the immune cell, thereby inhibiting inflammatory cytokine production by the immune cell in the subject.
3. The method of claim 1 or 2, wherein the immune cell is a myeloid cell, optionally wherein the immune cell endogenously expresses a Gi-coupled GPCR.
4. The method of claim 3, wherein the subject is a human.
5. The method of claim 3 or 4, wherein the subject has an inflammatory disease.
6. The method of any one of claims 1-5, wherein the CAGPCR is a constitutively active Gs- coupled GPCR.
7. The method of any one of claims 1-6, wherein the CAGPCR is derived from a GPCR selected from the group consisting of an autoinhibitory GPCR, a self-activated GPCR, an adhesion GPCR, and GPR65.
8. The method of claim 6 or 7, wherein the CAGPCR further comprises a heterologous signal sequence.
9. The method of claim 8, wherein the heterologous signal sequence comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 1-6.
10. The method of any one of claims 1-9, wherein the CAGPCR is derived from an autoinhibitory GPCR.
11. The method of any one of claims 1-10, wherein the CAGPCR is derived from a mutated autoinhibitory GPCR.
12. The method of claim 11, wherein the mutated autoinhibitory GPCR comprises one or more amino acid substitutions.
13. The method of claim 10, wherein the CAGPCR lacks one or more leucine-rich repeats (LRRs) as compared to the naturally-occurring GPCR.
14. The method of claim 13, wherein the CAGPCR does not comprise a LRR.
15. The method of claim 13 or 14, wherein the CAGPCR comprises a deletion in the ectodomam as compared to the naturally-occurring GPCR.
16. The method of any one of claims 13-15, wherein the CAGPCR lacks an ectodomain.
17. The method of any one of claims 1-16, wherein the CAGPCR is derived from RXFP1.
18. The method of claim 17, wherein the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 10-27.
19. The method of any one of claims 1-9, wherein the CAGPCR is derived from a self-activated GPCR.
20. The method of claim 19, wherein the CAGPCR is derived from GPR52.
21 . The method of claim 19 or 20, wherein the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 37.
22. The method of any one of claims 1-9, wherein the CAGPCR is derived from an adhesion GPCR.
23. The method of claim 22, wherein the adhesion GPCR is selected from the group consisting of LEC1, LEC2, LEC3, ELTD1, EMR1, EMR2, EMR3, mEMR4, CD97, GPR124, GPR125, CelsRl, CelsR2, CelsR3, GPR133, GPR144, GPR110, GPR111, GPR113, GPR115, GPR116, BAI1, BAI2, BAI3, GPR64, GPR97, GPR112, GPR114, GPR126, GPR128, VLGR1.
24. The method of claim 22 or 23, wherein the CAGPCR is derived from ADGRG2.
25. The method of claim 24, wherein the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 29-31.
26. The method of claim 22 or 23, wherein the CAGPCR is derived from ADGRG4.
27. The method of claim 26, wherein the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 33-35.
28. The method of any one of claims 1-9, wherein the CAGPCR is derived from GPR65.
29. The method of claim 28, wherein the CAGPCR is a mutated GPR65 comprising one or more amino acid substitutions selected from the group consisting of D60N, SI 01 A, and D268A.
30. The method of claim 29, wherein the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38, wherein the amino acid at amino acid position 60 of SEQ ID NO: 38 is N, the amino acid at amino acid position 101 of SEQ ID NO: 38 is A, and/or the amino acid at amino acid position 286 of SEQ ID NO: 38 is A.
31. The method of claim 30, wherein the CAGPCR comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the ammo acid sequences set forth in SEQ ID NOs: 39-53.
32. The method of any one of claims 1-31, wherein the CAGaS is a mutated Gs alpha subunit comprising one or more amino acid substitutions selected from the group consisting of R201H, R201C, Q227R, and Q227H.
33. The method of claim 32, wherein the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the ammo acid sequence set forth in SEQ ID NO: 54, wherein the ammo acid at amino acid position 201 of SEQ ID NO: 54 is IT or C, and/or the amino acid at amino acid position 227 of SEQ ID NO: 54 is R or H.
34. The method of claim 33, wherein the CAGaS comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 55-58.
35. The method of any one of claims 1-34, wherein the polynucleotide is comprised within a viral vector.
36. The method of claim 35, wherein the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picomavirus, lentivirus, herpes simplex virus, vaccinia vims, pox vims, and alphavims
37. The method of claim 36, wherein the viral vector is a single-stranded AAV.
38. The method of claim 36, wherein the viral vector is a self-complementary AAV.
39. The method of any one of claims 1-34, wherein the polynucleotide is comprised within a non- viral vector.
40. The method of claim 39, wherein the non-viral vector is a transposon-based vector.
41. The method of claim 39 or 40, wherein the non-viral vector is a PiggyBac-based vector, or a Sleeping Beauty-based vector.
42. The method of any one of claims 1-34, wherein the polynucleotide is a messenger RNA (mRNA).
43. The method of claim 42, wherein the polynucleotide is a modified messenger RNA (mmRNA).
44. The method of claim 43, wherein substantially all uridines in the mmRNA are modified uridines.
45. The method of claim 44, wherein the modified uridines are selected from the group consisting of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5 -aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy uridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l -methyl- 1- deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihy drops eudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4- methoxy-2-thio-pseudouridine, and pseudouridine.
46. The method of claim 44 or 45, wherein the modified uridines are 1 -methyl-pseudouridines.
47. The method of any one of claims 42-46, wherein the polynucleotide is in a lipid nanoparticle formulation.
48. The method of claim 47, wherein the lipid nanoparticle formulation comprises a cationic lipid, a sterol, and/or a PEG-lipid.
49. The method of claim 47 or 48, wherein the lipid nanoparticle formulation comprises a cationic lipid, a sterol, and a PEG-lipid.
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003769A1 (en) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Adenovirus mediated transfer of genes to the gastrointestinal tract
WO1993009239A1 (en) 1991-11-08 1993-05-13 Research Corporation Technologies, Inc. Adeno-associated virus-2 basal vectors
WO1993019191A1 (en) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Defective recombinant adenoviruses expressing cytokines for use in antitumoral treatment
WO1994012649A2 (en) 1992-12-03 1994-06-09 Genzyme Corporation Gene therapy for cystic fibrosis
WO1994028938A1 (en) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy sponsorship
WO1995000655A1 (en) 1993-06-24 1995-01-05 Mc Master University Adenovirus vectors for gene therapy
WO1995011984A2 (en) 1993-10-25 1995-05-04 Canji, Inc. Recombinant adenoviral vector and methods of use
WO2005028667A1 (en) * 2003-09-19 2005-03-31 Riken Drug targeting g protein-coupled receptor expressed in human mast cells and method of screening the same
AU2004203102B2 (en) * 1998-10-12 2007-10-18 Arena Pharmaceuticals, Inc. Human G Protein-Coupled Receptors
WO2019036753A1 (en) * 2017-08-22 2019-02-28 Monash University Screening assays, modulators and modulation of activation of receptor for advanced glycation end-products (rage)
WO2020146795A1 (en) * 2019-01-11 2020-07-16 Omeros Corporation Methods and compositions for treating cancer

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003769A1 (en) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Adenovirus mediated transfer of genes to the gastrointestinal tract
WO1993009239A1 (en) 1991-11-08 1993-05-13 Research Corporation Technologies, Inc. Adeno-associated virus-2 basal vectors
WO1993019191A1 (en) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Defective recombinant adenoviruses expressing cytokines for use in antitumoral treatment
WO1994012649A2 (en) 1992-12-03 1994-06-09 Genzyme Corporation Gene therapy for cystic fibrosis
WO1994028938A1 (en) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy sponsorship
WO1995000655A1 (en) 1993-06-24 1995-01-05 Mc Master University Adenovirus vectors for gene therapy
WO1995011984A2 (en) 1993-10-25 1995-05-04 Canji, Inc. Recombinant adenoviral vector and methods of use
AU2004203102B2 (en) * 1998-10-12 2007-10-18 Arena Pharmaceuticals, Inc. Human G Protein-Coupled Receptors
WO2005028667A1 (en) * 2003-09-19 2005-03-31 Riken Drug targeting g protein-coupled receptor expressed in human mast cells and method of screening the same
WO2019036753A1 (en) * 2017-08-22 2019-02-28 Monash University Screening assays, modulators and modulation of activation of receptor for advanced glycation end-products (rage)
WO2020146795A1 (en) * 2019-01-11 2020-07-16 Omeros Corporation Methods and compositions for treating cancer

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"G Protein-Coupled Receptors", 24 September 1999, CRC PRESS
"Identification and Expression of G Protein-Coupled Receptors", March 1998, JOHN WILEY & SONS
"Methods in Molecular Biology", vol. 288, 2005, HUMANA PRESS, article "Oligonucleotide synthesis: methods and applications"
"Structure-Function Analysis of G Protein-Coupled Receptors", 15 October 1999, WILEY-LISS
AKINC ET AL., MOL THER, vol. 17, 2009, pages 872 - 879
AKINC ET AL., NAT BIOTECHNOL., vol. 26, 2008, pages 561 - 569
ALI ET AL., HUM GENE THER, vol. 9, 1998, pages 8186
ALI ET AL., HUM MOL GENET, vol. 5, 1996, pages 591594
BENNETT ET AL., INVEST OPTHALMOL VIS SCI, vol. 38, no. 2857, 1997, pages 2863
BORRAS ET AL., GENE THER, vol. 6, no. 515, 1999, pages 524
FLOTT ET AL., PNAS, vol. 90, 1993, pages 10613 - 10617
FUKUHARA ET AL., BIOCHEMISTRY, vol. 1, no. 4, 1962, pages 563 - 568
GOODSON, MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, 1984, pages 115 - 138
JOMARY ET AL., GENE THER, vol. 4, no. 683, 1997, pages 690
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
LI ET AL., INVEST OPTHALMOL VIS SCI, vol. 35, no. 2543, 1994, pages 2549
LIDAVIDSON, PNAS, vol. 92, no. 7700, 1995, pages 7704
LIUHUANG, MOLECULAR THERAPY, 2010, pages 669 - 670
LOVE ET AL., PROC NATL ACAD SCI USA., vol. 107, 2010, pages 1864 - 1869
MAHON ET AL., BIOCONJUG CHEM, vol. 21, 2010, pages 1448 - 1454
MARCHESE ET AL., GENOMICS, vol. 23, 1994, pages 609 - 618
MENDELSON ET AL., VIROL, vol. 166, 1988, pages 154 - 165
MIYOSHI ET AL., PNAS, vol. 94, no. 10319, 1997, pages 6921
MORDENTI ET AL., PHARMACEUT. RES, vol. 8, 1991, pages 1351
MURUGAIAH ET AL., ANALYTICAL BIOCHEMISTRY, vol. 401, 2010, pages 61
OGATA ET AL., JOURNAL OF ORGANIC CHEMISTRY, vol. 74, 2009, pages 2585 - 2588
POWELL ET AL.: "Compendium of excipients for parenteral formulations", J PHARM SCI TECHNOL, vol. 52, 1998, pages 238 - 311, XP009119027
PROBST, DNA CELL BIOL., vol. 11, 1992, pages 1 - 20
PURMAL ET AL., NUCLEIC ACIDS RESEARCH, vol. 22, no. 1, 1994, pages 72 - 78
ROLLING ET AL., HUM GENE THER, vol. 10, 1999, pages 641648
SAMULSKI ET AL., J. VIR, vol. 63, 1989, pages 3822 - 3828
SCHROEDER ET AL., J INTERN MED, vol. 267, 2010, pages 9 - 21
SIEGWART ET AL., PROC NATL ACAD SCI USA., vol. 108, 2011, pages 12996 - 3001
TAKAHASHI ET AL., J VIROL, vol. 73, no. 7812, 1999, pages 7816
WU ET AL., J. BIOL. CHEM., vol. 262, 1987, pages 4429 - 4432
XU ET AL., TETRAHEDRON, vol. 48, no. 9, 1992, pages 1729 - 1740

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