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EP-4735592-A2 - THERAPEUTIC METHOD FOR MODULATING COMPARTMENTED CYCLIC ADENOSINE MONOPHOSPHATE IN REACTIVE ASTROCYTES

EP4735592A2EP 4735592 A2EP4735592 A2EP 4735592A2EP-4735592-A2

Abstract

Compartmentalization of cAMP signaling in reactive astrocytes is shown to promote neuroprotective astrocyte proliferation and neuronal survival for therapeutic applications.

Inventors

  • GOLDBERG, JEFFREY L.
  • CAMERON, Evan G.
  • TOTH, Anna Bettina

Assignees

  • The Board of Trustees of the Leland Stanford Junior University

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1 . A method of increasing neuroprotective activity of astrocytes in a subject, the method comprising: contacting the astrocytes with an effective dose of an agent that (i) increases nuclear cyclic adenosine monophosphate (cAMP) and/or (ii) decreases cytoplasmic cAMP.
  2. 2. The method of claim 1 , wherein the agent comprises a nuclear localized adenylyl cyclase (AC) or a fragment thereof, which increases nuclear cAMP levels.
  3. 3. The method of claim 2, wherein the AC is operably linked to a nuclear localization signal.
  4. 4. The method of claim 2 or claim 3, wherein the AC is soluble AC (sAC) or yeast CYR.
  5. 5. The method of claim 4, wherein the sAC is truncated sAC.
  6. 6. The method of any of claims 1 -5, wherein the AC is constitutively active, or activated by an exogenous signal.
  7. 7. The method of claim 1 , wherein the agent comprises a cytoplasmic localized cAMP sponge, which decreases cytoplasmic cAMP levels.
  8. 8. The method of claim 7, wherein the cAMP sponge comprises a high-affinity cAMP- binding portions of the regulatory subunits of protein kinase A (PKA), or unmodified, constitutively or partially active phosphodiesterases (PDEs).
  9. 9. The method of claim 8, wherein the high-affinity cAMP-binding portions of the regulatory subunits of protein kinase A (PKA) is the C-terminus of PRKAR1 B.
  10. 10. The method of any of claims 6-9, wherein the cAMP sponge is operably linked to a nuclear export signal.
  11. 1 1 . The method of any of claims 1 -10, wherein the subject has or is predicted to have an optic neuropathy.
  12. 12. The method of claim 1 1 , wherein the optic neuropathy is pre-glaucoma, glaucoma, ischemic optic neuropathy, diabetic retinopathy, dominant optic atrophy, traumatic optic neuropathy, optic neuritis, nutritional optic neuropathy, or toxic optic neuropathy.
  13. 13. The method of any of claims 1 -12, wherein the astrocytes are optic nerve head (ONH) astrocytes.
  14. 14. The method of any of claims 1-13, wherein the agent comprises a polynucleotide encoding a protein that (i) increases nuclear cyclic adenosine monophosphate (cAMP) and/or (ii) decreases cytoplasmic cAMP, operably linked to a promoter active in ONH astrocytes.
  15. 15. The method of claim 14, wherein the polynucleotide further comprises a first viral inverted terminal repeat sequence, and a second viral inverted terminal repeat sequence; wherein the polynucleotide sequence is encapsulated by a viral capsid to form a viral particle.
  16. 16. The method of claim 15, wherein the viral inverted terminal repeat sequences are AAV2 inverted terminal repeats.
  17. 17. The method of any of claims 14-16, wherein the promoter is gfaABCi D or a variant thereof.
  18. 18. The method of any of claims 15-17, wherein the viral capsid is AAV5.
  19. 19. The method of any of claims 15-18, wherein the effective dose comprises at least 1 x10 8 viral particles per ml.
  20. 20. The method of any of claims 1 -19, wherein the agent is administered via intravitreal injection.

Description

THERAPEUTIC METHOD FOR MODULATING COMPARTMENTED CYCLIC ADENOSINE MONOPHOSPHATE IN REACTIVE ASTROCYTES GOVERNMENT SUPPORT RESEARCH [0001] This invention was made with Government support under contracts EY025915 (FELLOWSHIP), EY026877, and EY029903 awarded by the National Institutes of Health. The Government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS [0002] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63/524,446 filed June 30, 2023, the disclosure of which application is herein incorporated by reference. SEQUENCE LISTING [0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN- 21 18WO_SEQ_LIST” created June 28, 2024 and having a size of 69,534 Bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. BACKGROUND [0004] Understanding the molecular and cellular pathways that regulate protective versus harmful astrocyte reactivity remains a major goal in basic and translational neuroscience. In the central nervous system (CNS), reactive astrocytes’ interactions with microglia, neurons, and peripheral cells strongly influence the balance of neurodegeneration and repair. The nature of the underlying insult, e.g. neuroinflammatory versus ischemic/traumatic, regulates reactive astrocytosis responsible for neuronal recovery versus loss of function, previously simplified to neurotoxic (so-called “A1 ”) and neuroprotective (so-called “A2”) reactive astrocytes. Neurotoxic astrocytes defined by expression of complement factor C3 promote lipid-mediated neuronal death induced by activated microglia. In contrast, neuroprotective 03- negative astrocytes are thought to promote neuronal survival and regeneration through upregulation and release of neurotrophic factors and other signaling molecules poorly defined. [0005] Some distinctions between neuroprotective and neurotoxic astrocyte phenotypes have been identified, including the importance of neuroprotective astrocytes’ proliferation and generation of a glial scar that sequesters inflammatory cells and inhibits neurodegenerative processes in the CNS. Little data link molecular pathways in astrocyte subtypes to these cellular phenotypes, although STAT3 signaling in vivo and cyclic adenosine monophosphate (cAMP) signaling in vitro and in vivo have been implicated in some glial-specific functions such as proliferation, differentiation, astrocyte-neuron coupling, and vulnerability to oxidative stress. [0006] The visual system serves as a useful model to investigate the mechanisms that regulate astrocyte reactivity due to its well-characterized structure, including a pure white matter tract formed by the centrally projecting axons of retinal ganglion cell (RGC) neurons. This model has also proven effective to study traumatic and glaucomatous optic neuropathies whose pathophysiology and progression have been associated with, and in some cases, driven by neurotoxic astrocyte reactivity. SUMMARY [0007] Optic nerve head astrocytes have been implicated in the pathogenesis of optic neuropathies from glaucoma to trauma and others. Cyclic-AMP generated by soluble adenylyl cyclase (sAC) is critical for neuroprotective astrocyte proliferation and retinal ganglion cell (RGC) survival after optic nerve injury. Modulating cAMP in specific reactive astrocyte populations provides therapeutic benefits in different disease contexts including, without limitation, in glaucoma where optic nerve head astrocytes are specifically implicated. [0008] Specifically, it is shown herein that these effects can be mediated through compartment-specific cAMP. Nuclear and cytoplasmic pools of cAMP oppose one another in the regulation of astrocyte proliferation, whereby nuclear cAMP promotes and cytoplasmic cAMP inhibits proliferation. Increased cAMP in the nucleus can increase neuroprotective astrocyte proliferation, decrease microglial activity, and promote RGC survival, e.g. after injury, in glaucoma, etc. The present disclosure demonstrates manipulation of compartmented pools of cAMP in reactive astrocytes for therapeutic applications. [0009] Therapeutic compositions and methods are provided for increasing neuroprotective activity of reactive astrocytes by modulating compartmented cAMP. Increasing nuclear, or depleting cytosolic, cAMP can increase reactive astrocyte proliferation and neuroprotective activity and is useful in therapy. In some embodiments the astrocytes are optic nerve head (ONH) astrocytes. In some embodiments the methods are used in the treatment of traumatic or glaucomatous optic neuropathies. In some embodiments a viral vector and promoter combination is used to target astrocytes, e.g. ONH astrocytes, for delivery of a therapeutic coding sequence, e.g. a nuclear targeted adenylyl cyclase; a cytosolic targeted cAMP “sponge”; etc. [0010] In some embodiments, an astrocyte-targeted agent of the