EP4735592A2 - Therapeutic method for modulating compartmented cyclic adenosine monophosphate in reactive astrocytes - Google Patents

Abstract

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

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 disclosure comprises a polynucleotide transgene encoding a polypeptide that increases nuclear cAMP and/or depletes cytoplasmic cAMP in astrocytes. The sequence is operably linked to a promoter active in ONH and retinal nerve fiber layer (NFL) astrocytes. In some embodiments, the transgene is expressed specifically in retinal and ONH astrocytes and not in microglia, ganglion cells (RGCs) or photoreceptors (PRCs). In some embodiments, expression of the transgene sequence is controlled by a modified glial fibrillary acidic protein (GFAP) promoter, gfaABCi D or a variant thereof. In other embodiments a different astrocyte specific promoter is used, e.g. a human gaf2 promoter, a human ALDH1 L1 promoter, a human EAATi promoter, etc. In other embodiments, a promoter activated by an exogenous signal is used.

[0011] The transgene sequence may comprise a first viral inverted terminal repeat sequence, a promoter, a transgene, a posttranslational regulatory element, a polyadenylation sequence and a second viral inverted terminal repeat sequence. In some embodiments the nucleic acid sequence is encapsulated by a viral capsid. The therapeutic nucleic acid may be provided in a vector, e.g. an AAV packaging plasmid. In some embodiments an effective unit dose of the composition is provided for therapeutic purposes.

[0012] In some embodiments, the AAV vector comprises AAV2 inverted terminal repeats (ITRs). In some embodiments, other AAV ITRs may be used which include but are not limited to AAV1 , AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, etc. In an embodiment, the nucleic acid composition is packaged in a viral particle. In some embodiments, the viral capsid (or particle) is an AAV5 viral capsid. In some embodiments, other AAV viral capsids may be used which include but are not limited to AAV1 , AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, etc. In a preferred embodiment, the gene therapy is packaged in a single stranded, double stranded, or self-complementary AAV vector.

[0013] In some embodiments, an effective dose for efficient optic nerve head astrocyte genetic modification comprises at least > 10⁸ viral particles/mL. In some embodiments, an effective dose comprises 10⁸-10⁹ viral particles/mL, 1 O⁹-1 O¹⁰ viral particles/mL, 10^l0-10¹¹ viral particles/mL, 10¹¹-10¹² viral particles/mL, 10¹²-10¹³ viral particles/mL or > 10¹³ viral particles/mL.

[0014] In some embodiments the therapeutic composition is administered to an individual for the treatment of optic neuropathies. In some embodiments, the composition is delivered through intravitreal administration. In some embodiment, the composition is administered for the treatment or prophylaxis of conditions that lead to inner retina degeneration including, without limitation, glaucoma, ischemic optic neuropathy, diabetic retinopathy, traumatic injury, etc.

BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 : Soluble adenylyl cyclase is a critical regulator of astrocyte proliferation. A. Pharmacologically inhibiting sAC, and not transmembrane ACs, suppresses astrocyte proliferation. Representative images of proliferating (EdU+) human astrocytes treated with DMSO (vehicle), the pan-transmembrane inhibitor, dDADO (10 pM), or sAC-specific inhibitors, KH7 (10 |_iM) or 2HE (10 pM). Scale bar is 50 pm. B. KH7 and 2HE significantly inhibit astrocyte proliferation in a concentration-dependent manner. One-way ANOVA with multiple comparisons; n.s. non-significant, * p<0.05; “ p<0.01. C. KH7- and 2HE-mediated inhibition of astrocyte proliferation is not associated with cell death at concentrations (1 -10 pM) that inhibit astrocyte proliferation. One- way ANOVA with multiple comparisons; n.s. non-significant, * p<0.05; ** p<0.01 . D. Domain structure of sAC isoforms, full-length sAC (sAC^,u"; 180kDa) and truncated sAC (sAC‘; 48kDa), showing loxP excision sites and qPCR primers detecting all sAC isoforms (sAC^a") and full-length sAC (sAC^,u"). C1 : catalytic domain-1 ; C2: catalytic domain-2; P-loop: phosphate binding domain; ZFD: zinc finger domain; TPR; tetratricopeptide repeat protein-protein interaction domain. E. Quantification of sAC mRNA expression in mouse astrocytes after 15 days in vitro (15DIV) verus 30DIV and after cre-mediated sAC KO (KO) relative to controls (CTRL). One-way ANOVA with multiple comparisons; n.s. non-significant, * p<0.05, ** p<0.01 . F. Western blots showing sAC^fu" and sAC’ protein expression decreases after sAC KO and with time in culture. G. Cre-mediated deletion leads to a significant decrease in sAC protein relative to control determined by densitometry of the sAC* band normalized to Ponceau or GAPDH. Paired t-test; * p<0.05. H. Representative images of sAC^fl/fl mouse astrocyte proliferation 15DIV and 30DIV following AAV2-dCre-GFP (CTRL) and AAV2-Cre- GFP (KO) transduction. Scale bar is 50 pm. I. Quiescent and genetic loss of sAC inhibits astrocyte proliferation, in vitro. One-way ANOVA with multiple comparisons; n.s. nonsignificant, “ p<0.01 .

[0016] FIG. 2: Soluble adenylyl cyclase promotes neuroprotective astrocyte proliferation and retinal ganglion cell survival after optic nerve crush injury in mice. A. Experimental design used to investigate the effects of sAC KO on optic nerve astrocyte reactivity. B. Representative images of GFAP-cre/ZEG (CTRL) and GFAP- cre/ZEG/sAC^,wl (sAC KO) optic nerves 7 days’ post crush injury showing broad cre-dependent eGFP expression in reactive optic nerve astrocytes. Astrocyte proliferation quantified by EdU incorporation in recombinant eGFP and Sox9-positive astrocytes every 500 pm starting 200 pm from the lesion core (*) moving towards the optic chiasm. Scale bar is 500 pm. C. Magnified images of 500 pm regions showing reactive astrocyte proliferation in CTRL and sAC KO optic nerves. Scale bar is 20 pm. D. Loss of sAC inhibits recombinant (sAC KO, eGFP+) reactive astrocyte proliferation throughout the optic nerve relative to control (CTRL, eGFP+). Non-recombinant astrocyte proliferation (sAC KO, eGFP-) is unaffected by loss of sAC in recombinant astrocytes. Paired t-test; n.s. non-significant, * p<0.05. E. Loss of sAC in reactive optic nerve astrocytes exacerbates RGC death after optic nerve crush injury. Representative retinal flat mounts (top) and 20X magnification (bottom) from control and sAC KO mice stained with the pan retinal ganglion cell (RGC) marker, RBPMS. Scale bar is 500 pm and 50 pm, respectively. F. Quantification of sAC KO effects on RGC survival after optic nerve crush compared to controls. Unpaired t-test; ** p<0.01 . G. Reactive astrocyte proliferation in the optic nerve positively correlates with RGC survival in the retina. Pearson correlation shown. H. Representative images of Iba1 immunoreactivity and proliferation in control and sAC KO optic nerves 7 days after crush injury. Scale is 500 m. I-J. Quantification of Iba1 + immunoreactivity and proliferation within the lesion core (dashed boxes from H.). K-L. Quantification of Iba1 + cells and proliferation outside the lesion core. M. Iba1 + cell density in the optic nerve and RGC survival in the retina are inversely correlated. Pearson correlation shown. N-O. Loss of sAC in reactive optic nerve astrocytes induces complement component 3 (C3) expression. Representative images of C3 staining in CTRL and sAC KO optic nerve astrocytes 500 pms from the crush site. Scale bar is 10 pm. P. Quantification of C3 intensity demonstrating a significant increase in average C3 expression in GFP+ sAC KO astrocytes relative to controls. Unpaired t-test; ** p<0.01 . Q. sAC KO leads to a significant increase in neurotoxic C3-postive reactive astrocytes using the mean C3 intensity from controls in P as a threshold. Unpaired t- test; ** p<0.01 .

[0017] FIG. 3: Distinct subcellular pools of cAMP differentially regulate astrocyte proliferation, in vitro. A. Genetically encoded cAMP “sponges” derived from the PKAR1 p regulatory subunit fused to mCherry alone or mCherry and a nuclear exclusion (NES), nuclear localization (NLS) or plasma membrane (PM) signal sequences. B. Illustration of predicted cAMP sponge localization. C. Representative images of proliferating (EdU) mouse astrocytes expressing mCherry (control), and non-targeted (Ubiq), cytosolic (NES), nuclear (NLS) and plasma membrane (PM) targeted sponges. Scale bar is 50pm. D. Quantification of compartment specific cAMP depletion on mouse astrocyte proliferation (colored bars/+) compared to mCherry (dotted black) and inactive mutant controls (grey bars/-). Buffering cAMP exclusively in the nucleus (NLS+) and cytoplasm (NES+) significantly inhibits and promotes astrocyte proliferation, respectively. Buffering cAMP across all compartments with a sponge lacking a targeting sequence (Ubiq+) also significantly inhibits proliferation (but to a lesser degree than the NLS-sponge) and a plasma membrane (PM+) targeted sponge has no effect on proliferation. Paired t-tests; n.s. non-significant, * p<0.05 ; ** p<0.01. E. Buffering cAMP exclusively in the nucleus induces G2/M cell cycle arrest. Percent of mouse astrocytes in G2/M expressing mCherry alone (control), nuclear (NLS+), cytosolic (NES+) and non-targeted (Ubiq+) sponges determined by propidium iodide staining. One-way ANOVA with multiple comparisons; n.s. non-significant, * p<0.05, ** p<0.01. F. Buffering cAMP in the nucleus of proliferating mouse astrocytes induces nuclear p21^Cip expression. Representative images of p2i^c'P immunofluorescence in proliferating mouse astrocytes expressing mCherry alone (control), nuclear (NLS+), cytosolic (NES+) and non- targeted (Ubiq+) sponges. Scale bar is 50pm. G. Quantification of cAMP sponge effects on p21 ^Cip expression in proliferating astrocytes. Buffering nuclear cAMP (NLS+) significantly promotes p21 ^Cip expression, whereas buffering cAMP the cytoplasm (NES+) or throughout all compartments (Ubiq+) inhibits and promotes nuclear p21 ^Cip expression, respectively. One-way ANOVA with multiple comparisons; n.s. non-significant, * p<0.05. [0018] FIG. 4: Intravitreal injection of AAV5.gfaABCi D specifically transduces optic nerve head (ONH) astrocytes in mice. A. Schematic of retinal features and AAV5.gfaABC(1 )DtdTomato intravitreal injection. B-C. Retinal cross section and flat-mount showing AAV5.gfaABC(1 )D.tdT preferentially transduces cells in the optic nerve head (ONH) region. Scale in B and C is 500 pm. D-E. Magnified dashed region from B showing intravitreal injection of AAV5-gfaABC(1 )D preferentially transduces ONH astrocytes. ONH defined by the area between the glial lamina and optic disc rim -200 pm from the center of the retina. Scale in D and E is 50 pm. F. Magnified ONH region from retinal flat mount (dashed from C). Scale is 20pm. G. Magnified retinal region (dotted from B) showing a small subset of nerve fiber layer (NFL; solid arrow) astrocytes and Muller glial (MG; open arrow) are transduced by AAV5- gfaABC(1 )D.tdT. Scale is 50 pm. H. NFL astrocytes defined by GFAP and Sox9 within the NFL layer. Muller glia defined by Sox9 and DAPI in the inner nuclear layer (INL). Photoreceptors defined by DAPI in the outer nuclear later (ONL) were not transduced. Scale is 50 pm. I. Magnified retinal region from flat mount (dotted in C) showing AAV5.gfaABC(1 )D.tdT transduces a small subset of NFL astrocytes identified by GFAP staining, but not retinal ganglion cells (RGCs) stained with RNA-binding protein with multiple splice (RBPMS). Scale is 20 pm. J. ONH stained for Iba1 showing microglia are not transduced following intravitreal injection of AAV5.gfaABCiD. Scale is 100 pm. K. Individual channels (from J) showing lba+ immunoreactivity and tdTomato do not colocalize. Scale is 100 pm. L. Quantification of AAV5. gfaABCi D.tdTomato expression in the major retinal cell-types. One-way ANOVA with multiple comparisons; “ p<0.01 .

[0019] FIG. 5: Compartmented cAMP in optic nerve head astrocytes differentially regulates microglial activation and RGC survival after optic nerve crush (ONC) injury, in vivo. A. Nuclear (NLS-sp) and cytoplasmic (NES-sp) cAMP sponges and a nuclear sACt (NLS-sAC) construct packaged in AAV5.gfaABC(1 )D. B. Experimental design used to investigate the effects of compartment-specific cAMP signaling in reactive optic nerve head (ONH) astrocytes after ONC injury. C. Representative retinal flat mount images of AAV5.gfaABC(1 )D. tdTomato, NLS- sp, NLS-sAC, NES-sp injected retinas stained for Iba1 . Scale is 500 pm. D-E. Magnified ONH regions showing localization and expression of each construct and effects on local Iba1 + cell infiltration. F. Representative images of RBPMS staining in the proximal region following AAV5-mediated cAMP manipulation in ONH astrocytes. G. Buffering cytoplasmic and elevating nuclear cAMP in ONH astrocytes inhibits retinal immune cell infiltration after ONC injury. Quantification of total retinal Iba1 + immunoreactivity following compartment-specific cAMP modulation in ONH astrocytes. Scale bar is 50 pm. H. Effects of compartment- specific cAMP modulation in reactive astrocytes on immune cell infiltration are highest proximal to the ONH region. Quantification of Iba1 + cell density in the ONH region following compartmented cAMP elevation and depletion in ONH astrocytes. One-way ANOVA with multiple comparisons; n.s. non-significant, * p<0.05, ** p<0.01. Scale bar is 50 pm. I. Quantification of RGC survival following NLS-sp, NLS-AC, and NES-sp expression relative to tdTomato control. One-way ANOVA with multiple comparisons; n.s. non-significant, * p<0.05, ** p<0.01. Scale bar is 50 pm. J. ONH astrocyte specific cAMP-mediated RGC survival and local ONH immune cell infiltration are inversely related. Pearson correlation shown.

[0020] FIG. 6. Increased nuclear cAMP promotes astrocyte proliferation. A. Nuclear constitutively active AC (NLS-AC^oa) construct design. B. Proliferation quantified by EdU incorporation in cultured astrocytes transfected with control and NLS-AC^ca plasmids. C. NLS- AC^ca significantly increases proliferation in cultured astrocytes relative to control. Unpaired t- test, ****p<0.0001 .

[0021] FIG. 7. Increased nuclear cAMP promotes RGC survival after ONC. A. Experimental design used to investigate the effect of nuclear-localized constitutively active AC in ONH astrocytes after optic nerve injury. NLS-AC^ca construct was packaged in AAV5.gfaABC(1 )D and injected intravitreally, followed by ONC injury at 7 days post-injection, and collection of the retina at 14 days post-injury. B. Representative retinal flat mount images showing that AAV5.gfaABC(1 )D.tdTomato control and NLS-AC^ca viruses are targeted to the ONH. C. Representative images of Iba1 intensity at the ONH. NLS-AC^ca decreased microglial activation at the ONH relative to control. Unpaired t-test, “ p<0.01. D. Representative images of RBPMS+ cell density 500pm proximal to the ONH. NLS-AC^ca increased RGC survival after injury relative to control. Unpaired t-test, *p<0.05.

DETAILED DESCRIPTION

DEFINITIONS

[0022] Before embodiments of the present disclosure are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.

[0024] It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound but also a combination of two or more compounds, reference to "a substituent" includes a single substituent as well as two or more substituents, and the like.

[0025] In describing and claiming the present invention, certain terminology will be used in accordance with the definitions set out below. It will be appreciated that the definitions provided herein are not intended to be mutually exclusive. Accordingly, some chemical moieties may fall within the definition of more than one term.

[0026] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0027] As used herein, the phrases “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. These examples are provided only as an aid for understanding the disclosure, and are not meant to be limiting in any fashion.

[0028] Adenylyl cyclase. cAMP is generated from ATP by adenylyl cyclases (ACs). In humans, this family contains nine transmembrane ACs transcribed from 9 different genes, and one gene encoding a soluble AC (sAC). By alternative splicing, several sAC isoforms are generated. Full-length sAC includes an N-terminus with the two catalytic domains (-1 ,100 amino acids spanning 33 exons). Exclusion of exon 12 generates a truncated isoform, sACt (amino acids 1-490), which contains just the two sAC catalytic domains.

[0029] sAC is encoded in a single Homo sapiens gene identified as ADCY10 or Adenylate cyclase 10 (soluble). The functional mammalian sAC consists of two heterologous catalytic domains (C1 and C2), forming the 50 kDa amino terminus of the protein. The additional -140 kDa C terminus of the enzyme includes an autoinhibitory region, canonical P-loop, potential heme-binding domain, and leucine zipper-like sequence, which are a form of putative regulatory domains. A truncated form of the enzyme only includes the C1 and C2 domains and it is referred to herein as the minimal functional sAC variant. This sAC-truncated form has cAMP-forming activity much higher than its full-length type. These sAC variants are stimulated by HCO3- and respond to all known selective sAC inhibitors. Sequences of human adenylate cyclase 10 are known in the art, e.g., mRNA Genbank References: NM 001297772.2, NM 001 167749.3, and NM 018417.6; and protein Genbank References: NP 060887.2, NP_001284701 .1 , and NP_001161221.1. An exemplary amino acid sequence of human soluble adenylate cyclase 10 is: MNTPKEEFQDWPIVRIAAHLPDLIVYGHFSPERPFMDYFDGVLMFVDISGFTAMTEKFSSAM YMDRGAEQLVEILNYHISAIVEKVLIFGGDILKFAGDALLALWRVERKQLKNIITVVIKCSLEIH GLFETQEWEEGLDIRVKIGLAAGHISMLVFGDETHSHFLVIGQAVDDVRLAQNMAQMNDVIL SPNCWQLCDRSMIEIESVPDQRAVKVNFLKPPPNFNFDEFFTKCTTFMHYYPSGEHKNLLR LACTLKPDPELEMSLQKYVMESILKQIDNKQLQGYLSELRPVTIVFVNLMFEDQDKAEEIGPA IQDAYMHITSVLKIFQGQINKVFMFDKGCSFLCVFGFPGEKVPDELTHALECAMDIFDFCSQ VHKIQTVSIGVASGIVFCGIVGHTVRHEYTVIGQKVNLAARMMMYYPGIVTCDSVTYNGSNL PAYFFKELPKKVMKGVADSGPLYQYWGRTEKVMFGMACLICNRKEDYPLLGRNKEINYFM YTMKKFLISNSSQVLMYEGLPGYGKSQILMKIEYLAQGKNHRIIAISLNKISFHQTFYTIQMFM ANVLGLDTCKHYKERQTNLRNKVMTLLDEKFYCLLNDIFHVQFPISREISRMSTLKKQKQLEI LFMKILKLIVKEERIIFIIDEAQFVDSTSWRFMEKLIRTLPIFIIMSLCPFVNIPCAAARAVIKNRN TTYIVIGAVQPNDISNKICLDLNVSCISKELDSYLGEGSCGIPFYCEELLKNLEHHEVLVFQQT ESEEKTNRTWNNLFKYSIKLTEKLNMVTLHSDKESEEVCHLTSGVRLKNLSPPTSLKEISLIQ LDSMRLSHQMLVRCAAIIGLTFTTELLFEILPCWNMKMMIKTLATLVESNIFYCFRNGKELQK ALKQNDPSFEVHYRSLSLKPSEGMDHGEEEQLRELENEVIECHRIRFCNPMMQKTAYELW LKDQRKAMHLKCARFLEEDAHRCDHCRGRDFIPYHHFTVNIRLNALDMDAIKKMAMSHGFK TEEKLILSNSEIPETSAFFPENRSPEEIREKILNFFDHVLTKMKTSDEDIIPLESCQCEEILEIVIL PLAHHFLALGENDKALYYFLEIASAYLIFCDNYMAYMYLNEGQKLLKTLKKDKSWSQTFESA TFYSLKGEVCFNMGQIVLAKKMLRKALKLLNRIFPYNLISLFLHIHVEKNRHFHYVNRQAQES PPPGKKRLAQLYRQTVCLSLLWRIYSYSYLFHCKYYAHLAVMMQMNTALETQNCFQIIKAYL DYSLYHHLAGYKGVWFKYEVMAMEHIFNLPLKGEGIEIVAYVAETLVFNKLIMGHLDLAIELG SRALQMWALLQNPNRHYQSLCRLSRCLLLNSRYPQLIQVLGRLWELSVTQEHIFSKAFFYF VCLDILLYSGFVYRTFEECLEFIHQYENNRILKFHSGLLLGLYSSVAIWYARLQEWDNFYKFS NRAKNLLPRRTMTLTYYDGISRYMEGQVLHLQKQIKEQSENAQASGEELLKNLENLVAQNT TGPVFCPRLYHLMAYVCILMGDGQKCGLFLNTALRLSETQGNILEKCWLNMNKESWYSTS ELKEDQWLQTILSLPSWEKIVAGRVNIQDLQKNKFLMRANTVDNHF (SEQ ID NO: 1 ). An exemplary nucleic acid sequence of human soluble adenylate cyclase 10 is SEQ ID NO: 2.

[0030] An exemplary mus musculus amino acid sequence of soluble adenylate cyclase 10 is: MSARRQELQDRAIVKIAAHLPDLIVYGDFSPERPSVKCFDGVLMFVDISGFTAMTEKFSTAM YMDRGAEQLVEILNYYISAIVEKVLIFGGDILKFAGDALLALWKVERKQLKNIITVVIKCSLEIH GLFEAKEAEEGLDIRVKIGLAAGHITMLVFGDETRNYFLVIGQAVDDVRLAQNMAQMNDVIL SPNCWQLCDRSMIEIERIPDQRAVKVSFLKPPPTFNFDEFFTKCMGFMDYYPSGDHKNFLR LACMLESDPELELSLQKYVMEIILKQIDDKQLRGYLSELRPVTIVFVNLMFKEQDKVEVIGSAI QAACVHITSVLKVFRGQINKVFMFDKGCSFLCVFGFPGEKAPDEITHALESAVDIFDFCSQV HKIRTVSIGVASGIVFCGIVGHTVRHEYTVIGQKVNIAARMMMYYPGIVSCDSVTYDGSNLPA YFFKELPKKVMKGVADPGPVYQCLGLNEKVMFGMAYLICNRYEGYPLLGRVREIDYFMST MKDFLMTNCSRVLMYEGLPGYGKSQVLMEIEYLASQHENHRAVAIALTKISFHQNFYTIQIL MANVLGLDTCKHYKERQTNLQNRVKTLLDEKFHCLLNDIFHVQFPVSREMSRMSKIRKQKQ LEALFMKILAQTVREERIIFIIDEAQFVDGTSWAFIEKLIRSMPIFIVMSLAPFSEVPCAAANAIM KNRNTTYITLGTMQPQEIRDKVCVDLSVSSIPRELDSYLVEGSCGIPYYCEELLKNLDHHRVL LFQQAETEQKTNVTWNNMFKHSVRPTDDMQLFTSISEGQKEVCYLVSGVRLNNLSPPASL KEISLVQLDSMSLSHQMLVRCAAIIGLTFTTELLFEILPCWNMKMMIKALATLVESNVFNCFR SSKDLQLALKQNVPTFEVHYRSLALKLKEGLTYGEEEELREMEGEVVECRILRFCRPIMQKT AYELWLKDQKKVLHLKCARFLEESAHRCNHCRNVDFIPYHHFIVDIRLNTLDMDTVKRMVTS QGFKIDEEEAIFSKSELPRKYKFPENLSITEIREKILHFFDNVILKMKSSPNDIIPLESCQCKELL QIVILPLAQHFVALEENNKALYYFLELASAYLILGDNYNAYMYLGEGERLLKSLTNEDSWSQT FEYATFYSLKAEVCFNMGQMVLAKKMLRKALKLLNRMFPCNLLTLTFQMHVEKNRLSHFM NQHTQEGSVPGKKLAQLYLQASCFSLLWRIYSLNFFFHYKYYGHLAAMMEMNTSLETQND FQIIKAYLDFSLYHHLAGYQGVWFKYEILVMEQLLNLPLKGEAIEIMAYTADTLGHIKFLMGHL DLAIELGSRAHRMWSLLRNPNKYQMVLCRLSKPLFLKSRYKHLVQVLGWLWDLSVTEEDIF SKAFFYFVCLDIMLYSGFIYRTFEECLEFIHHNEDNRILKFQSGLLLGLYSCIAVWYARLQEW DNFNKFSDRAKHLVTRRTPTVLYYEGISRYMEGQVLHLQKQIEEQAENAQDSGVEILKALET LVAQNTTGPVFYPRLYHLMAYVCILMGDGHSCDFFLNTALELSETHGNLLEKCWLSMSKE WWYSASELTGDQWLQTVLSLPSWDKIVSGKGGQRKRSWSWFCPPNFSMVSWSQPQCA (SEQ ID NO: 3). An exemplary mus musculiis nucleic acid sequence of soluble adenylate cyclase 10 is SEQ ID NO: 4.

[0031] An exemplary saccharomyces cerevisiae amino acid sequence of soluble adenylate cyclase, also referred to as CYR, is: MSSKPDTGSEISGPQRQEEQEQQIEQSSPTEANDRSIHDEVPKVKKRHEQNSGHKSRRNS AYSYYSPRSLSMTKSRESITPNGMDDVSISNVEHPRPTEPKIKRGPYLLKKTLSSLSMTSAN STHDDNKDHGYALNSSKTHNYTSTHNHHDGHHDHHHVQFFPNRKPSLAETLFKRFSGSNS HDGNKSGKESKVANLSLSTVNPAPANRKPSKDSTLSNHLADNVPSTLRRKVSSLVRGSSV HDINNGIADKQIRPKVVAQSENTLHSSDVPNSKRSHRKSFLLGSTSSSSTRRGSNVSSMTN SDSASMATSGSHVLQHNVSNVSPTTKSKDSVNSESADHTNNKSEKVTPEYNENIPENSNS DNKREATTPTIETPISCKPSLFRLDTNLEDVTDITKTVPPTAVNSTLNSTHGTETASPKTVIMP EGPRKSVSMADLSVAAAAPNGEFTSTSNDRSQWVAPQSWDVETKRKKTKPKGRSKSRRS SIDADELDPMSPGPPSKKDSRHRKNRHSRHHHDRKDNESMVTAGDSNSSFVDICKENVPN DSKTALDTKSVNRLKSNLAMSPPSIRYAPSNLDGDYDTSSTSSSLPSSSISSEDTSSCSNSS SYTNAYMEANREQDNKTPILNKTKSYTKKFTSSSVNMNSPDGAQSSGLVLQDEKDDEVEC QLEHYYKDFSDLDPKRHYAIRIFNTDDTFTTLSCTPATTVEEIIPALKRKFNITAQGNFQISLKV GKLSKILRPTSKPILIERKLLLLNGYRKSDPLHIMGIEDLSFVFKFLFHPVTPSHLTPEQEQRIM RSEFVHVDLRNMDLTTPPIIFYQHTSEIESLDVSNNANIFLPLEFIESSIKLLSLRMVNIRASKF PSNITEAYKLVSLELQRNFIRKVPNSIMKLSNLTILNLQCNELESLPAGFVELKNLQLLDLSLN KFMHYPEVINYCTNLLQIDLSYNKIQSLPQSTKYLVKLAKMNLSHNKLNFIGDLSEMTNLRTL NLRYNRISSIKTNASNLQNLFLTDNRISNFEDTLPKLRALEIQENPITSISFKDFYPKNMTSLTL NKAQLSSIPRELLTKLSFLEKLELNQNNLTRLPQEISKLTKLVFLSVARNKLEYIPPELSQLKSL RTLDLHSNNIRDFVDGMENLELTSLNISSNAFGNSSLENSFYHNMSYGSKLSKSLMFFIAAD NQFDDAMWPLFNCFVNLKVLNLSYNNFSDVSHMKLESITELYLSGNKLTTLSGDTVLKWSS LKTLMLNSNQMLSLPAELSNLSQLSVFDVGANQLKYNISNYHYDWNWRNNKELRYLNFSG NRRFEIKSFISHDIDADLSDLTVLPQLKVLGLMDVTLNTTKVPDENVNFRLRTTASIINGMRY GVADTLGQRDYVSSRDVTFEKFRGNDDECLLCLHDSKNQNADYGHNISRIVRDIYDKILIRQ LERYGDDTDDNIKTALRFSFLQLNKEINGMLNSVDNGADVANLSYADLLSGACSTVIYIRGK KLFAANLGDCMAILSKNNGDYQTLTKQHLPTKREEYERIRISGGYVNNGKLDGVVDVSRAV GFFDLLPHIHASPDISVVTLTKADEMLIVATHKLWEYMDVDTVCDIARENSTDPLRAAAELKD HAMAYGCTENITILCLALYENIQQQNRFTLNKNSLMTRRSTFEDTTLRRLRPEISPPTGNLAM VFTDIKNSTFLWELFPNAMRTAIKTHNDIMRRQLRIYGGYEVKTEGDAFMVAFPTPTSGLTW CLSVQLKLLDAQWPEEITSVQDGCQVTDRNGNIIYQGLSVRMGIHWGCPVPELDLVTQRM DYLGPMVNKAARVQGVADGGQIAMSSDFYSEFNKIMKYHERVVKGKESLKEVYGEEIIGEV LEREIAMLESIGWAFFDFGEHKLKGLETKELVTIAYPKILASRHEFASEDEQSKLINETMLFRL RVISNRLESIMSALSGGFIKLDSRTEGSYIKFNPKVENGIMQSISEKDALLFFDHVITRIESSVA LLHLRQQRCSGLEICRNDKTSARSNIFNVVDELLQMVKNAKDLST (SEQ ID NO: 33). An exemplary saccharomyces cerevisiae nucleic acid sequence of soluble adenylate cyclase, also referred to as CYR, is SEQ ID NO: 34. In some embodiments, human soluble adenylate cyclase 10 is a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the human soluble adenylate cyclase (sAC) is a variant of human sAC. In some embodiments, the variant of human sAC is a truncated human sAC. In some embodiments, the variant human sAC only comprises the catalytic domain of human sAC. In some embodiments, the variant of human sAC only comprises amino acid residues 1 -469 of SEQ ID NO: 1. In some embodiments, the mus musculus soluble adenylate cyclase (sAC) is a variant of mus musculus sAC. In some embodiments, the variant of mus musculus sAC is a truncated human sAC. In some embodiments, the variant mus musculus sAC only comprises the catalytic domain of human sAC. In some embodiments, the variant of mus musculus sAC only comprises amino acid residues 1 -469 of SEQ ID NO: 3.

[0032] cAMP is a second messenger that can signal at different locations in a single cell. For this purpose, a tight spatial and temporal control of the cAMP concentration is critical. Whereas tmACs are restricted to membranes, sAC can be localized throughout the cell and is found in mitochondria, nuclei, centrioles, the mitotic spindle as well as cilia. At these locations, ACs are anchored together with PKA by scaffold proteins, allowing local utilization. Further, the diffusion of cAMP is confined by the degradation of cAMP by PDEs and the apical actin web. These mechanisms together with the local production and utilization of cAMP create “microdomains” of cAMP signaling. As used here, compartmented cAMP may refer to a locally higher concentration in the nucleus, or the cytoplasm.

[0033] sAC is directly activated by HCO3 , leading to increased substrate turnover and reduced substrate inhibition and by Ca²⁺, enhancing substrate binding. In this way, small changes in the intracellular concentration of bicarbonate and calcium ions may significantly affect local cAMP levels.

[0034] In some embodiments, a constitutively active AC is delivered to the cell of interest, see for example Geller et al. Proc Natl Acad Sci U S A 90, 7603-7607 (1993). An exemplary human constitutively active AC amino acid sequence is according to: MSPTSKPILIERKLLLLNGYRKSDPLHIMGIEDLSFVFKFLFHPVTPSHLTPEQEQRIMRSEFV HVDLRNMDLTTPPIIFYQHTSEIESLDVSNNANIFLPLEFIESSIKLLSLRMVNIRASKFPSNITE AYKLVSLELQRNFIRKVPNSIMKLSNLTILNLQCNELESLPAGFVELKNLQLLDLSLNKFMHY PEVINYCTNLLQIDLSYNKIQSLPQSTKYLVKLAKMNLSHNKLNFIGDLSEMTNLRTLNLRYN RISSIKTNASNLQNLFLTDNRISNFEDTLPKLRALEIQENPITSISFKDFYPKNMTSLTLNKAQL SSIPRELLTKLSFLEKLELNQNNLTRLPQEISKLTKLVFLSVARNKLEYIPPELSQLKSLRTLDL HSNNIRDFVDGMENLELTSLNISSNAFGNSSLENSFYHNMSYGSKLSKSLMFFIAADNQFDD AMWPLFNCFVNLKVLNLSYNNFSDVSHMKLESITELYLSGNKLTTLSGDTVLKWSSLKTLML NSNQMLSLPAELSNLSQLSVFDVGANQLKYNISNYHYDWNWRNNKELRYLNFSGNRRFEI KSFISHDIDADLSDLTVLPQLKVLGLMDVTLNTTKVPDENVNFRLRTTASIINGMRYGVADTL GQRDYVSSRDVTFEKFRGNDDECLLCLHDSKNQNADYGHNISRIVRDIYDKILIRQLERYGD DTDDNIKTALRFSFLQLNKEINGMLNSVDNGADVANLSYADLLSGACSTVIYIRGKKLFAANL GDCMAILSKNNGDYQTLTKQHLPTKREEYERIRISGGYVNNGKLDGVVDVSRAVGFFDLLP HIHASPDISVVTLTKADEMLIVATHKLWEYMDVDTVCDIARENSTDPLRAAAELKDHAMAYG CTENITILCLALYENIQQQNRFTLNKNSLMTRRSTFEDTTLRRLRPEISPPTGNLAMVFTDIKN STFLWELFPNAMRTAIKTHNDIMRRQLRIYGGYEVKTEGDAFMVAFPTPTSGLTWCLSVQL KLLDAQWPEEITSVQDGCQVTDRNGNIIYQGLSVRMGIHWGCPVPELDLVTQRMDYLGPM VNKAARVQGVADGGQIAMSSDFYSEFNKIMKYHERVVKGKESLKEVYGEEIIGEVLEREIAM LESIGWAFFDFGEHKLKGLETKELVTIAYPKILASRHEFASEDEQSKLINETMLFRLRVISNRL ESIMSALSGGFIKLDSRTEGSYIKFNPKVENGIMQSISEKDALLFFDHVITRIESSVALLHLRQ QRCSGLEICRNDKTSARSNIFNVVDELLQMVKNAKDLST (SEQ ID NO: 5). An exemplary human constitutively active AC nucleic acid sequence is according to SEQ ID NO: 6. An exemplary saccharomyces cerevisiae constitutively active AC amino acid sequence is according to: MSPTSKPILIERKLLLLNGYRKSDPLHIMGIEDLSFVFKFLFHPVTPSHLTPEQEQRIMRSEFV HVDLRNMDLTTPPIIFYQHTSEIESLDVSNNANIFLPLEFIESSIKLLSLRMVNIRASKFPSNITE AYKLVSLELQRNFIRKVPNSIMKLSNLTILNLQCNELESLPAGFVELKNLQLLDLSLNKFMHY PEVINYCTNLLQIDLSYNKIQSLPQSTKYLVKLAKMNLSHNKLNFIGDLSEMTNLRTLNLRYN RISSIKTNASNLQNLFLTDNRISNFEDTLPKLRALEIQENPITSISFKDFYPKNMTSLTLNKAQL SSIPRELLTKLSFLEKLELNQNNLTRLPQEISKLTKLVFLSVARNKLEYIPPELSQLKSLRTLDL HSNNIRDFVDGMENLELTSLNISSNAFGNSSLENSFYHNMSYGSKLSKSLMFFIAADNQFDD AMWPLFNCFVNLKVLNLSYNNFSDVSHMKLESITELYLSGNKLTTLSGDTVLKWSSLKTLML NSNQMLSLPAELSNLSQLSVFDVGANQLKYNISNYHYDWNWRNNKELRYLNFSGNRRFEI KSFISHDIDADLSDLTVLPQLKVLGLMDVTLNTTKVPDENVNFRLRTTASIINGMRYGVADTL GQRDYVSSRDVTFEKFRGNDDECLLCLHDSKNQNADYGHNISRIVRDIYDKILIRQLERYGD DTDDNIKTALRFSFLQLNKEINGMLNSVDNGADVANLSYADLLSGACSTVIYIRGKKLFAANL GDCMAILSKNNGDYQTLTKQHLPTKREEYERIRISGGYVNNGKLDGVVDVSRAVGFFDLLP HIHASPDISVVTLTKADEMLIVATHKLWEYMDVDTVCDIARENSTDPLRAAAELKDHAMAYG CTENITILCLALYENIQQQNRFTLNKNSLMTRRSTFEDTTLRRLRPEISPPTGNLAMVFTDIKN STFLWELFPNAMRTAIKTHNDIMRRQLRIYGGYEVKTEGDAFMVAFPTPTSGLTWCLSVQL KLLDAQWPEEITSVQDGCQVTDRNGNIIYQGLSVRMGIHWGCPVPELDLVTQRMDYLGPM VNKAARVQGVADGGQIAMSSDFYSEFNKIMKYHERVVKGKESLKEVYGEEIIGEVLEREIAM LESIGWAFFDFGEHKLKGLETKELVTIAYPKILASRHEFASEDEQSKLINETMLFRLRVISNRL ESIMSALSGGFIKLDSRTEGSYIKFNPKVENGIMQSISEKDALLFFDHVITRIESSVALLHLRQ QRCSGLEICRNDKTSARSNIFNVVDELLQMVKNAKDLST (SEQ ID NO: 7). An exemplary saccharomyces cerevisiae constitutively active AC amino acid sequence is according to SEQ ID NO: 8. In some embodiments a constitutively active AC is operably linked to a promoter active selectively in astrocytes. In other embodiments a constitutively active AC is operably linked to an inducible promoter activated by an exogenous signal, e.g. light, small molecules, etc. In some embodiments, the constitutively active AC is human constitutively active AC. In some embodiments, the constitutively active AC is saccharomyces cerevisiae constitutively active AC (CYR).

[0035] Alternatively, AC can be activated pharmacologically, e.g. using bicarbonate as a physiological activator. Elevating intracellular levels of bicarbonate through the addition of bicarbonate salts or using carbonic anhydrase inhibitors can stimulate sAC activity. Another method involves direct pharmacological activation using forskolin. Additionally, calcium ions (Ca²⁺) have been shown to activate sAC, with increased cytosolic calcium levels leading to sAC activation and subsequent cAMP production.

[0036] Nuclear Localization Signals. In some embodiments it is desirable to localize a protein, e.g. sAC, to the nucleus. Nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediate the transport of proteins from the cytoplasm into the nucleus. This NLS-dependent protein recognition, a process necessary for cargo proteins to pass the nuclear envelope through the nuclear pore complex, is facilitated by members of the importin superfamily.

[0037] Various NLS known on the art can be used for this purpose. For example, monopartite NLS are a single cluster composed of 4-8 basic amino acids, which generally contains 4 or more positively charged residues, that is, arginine (R) or lysine (K). The characteristic motif of MP NLS is usually defined as K (K/R) X (K/R), where X can be any residue. For example: PKKKRKV (SEQ ID NO: 9); PKLKRQ (SEQ ID NO: 10); RPRK (SEQ ID NO: 1 1 ); RRARRPRG (SEQ ID NO: 12); etc. Bipartite NLS are characterized by two clusters of 2-3 positively charged amino acids that are separated by a 9-12 amino-acid linker region, which contains several proline (P) residues. The consensus sequence can be expressed as R/K(X) -i2KRXK, e.g. KRPAATKKAGQAKKKK (SEQ ID NO: 13); GKRKLITSEEERSPAKRGRKS (SEQ ID NO: 14); KGKKGRTQKEKKAARARSKGKN (SEQ ID NO: 15);

RKRCAAGVGGGPAGCPAPGSTPLKKPRR (SEQ ID NO: 16);

RKPVTAQERQREREEKRRRRQERAKEREKRRQERER (SEQ ID NO: 17); etc. Additionally, many proteins have unusual NLS; for example FGNYNNQSSNFGPMKGGNFGGRSSGPY (SEQ ID NO: 18); TLLLRETMNNLGVSDHAVLSRKTPQPY (SEQ ID NO: 19); PGKMDKGEHRQERRDRPY (SEQ ID NO: 20); RKHKTNRKPR (SEQ ID NO: 21 ); NRRAKAKR (SEQ ID NO: 22); RNKKKK (SEQ ID NO: 23); RKVIK (SEQ ID NO: 24); etc.

[0038] Nuclear exclusion signal (or nuclear export signal, NES) In some embodiments it may be desirable to localize a protein outside of the nucleus, e.g. where a cAMP sponge is delivered to a cell. A NES is a short target peptide containing 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. It has the opposite effect of a nuclear localization signal, which targets a protein located in the cytoplasm for import to the nucleus. The NES is recognized and bound by exportins. Known NESs found the most common spacing of the hydrophobic residues to be LxxxLxxLxL, where "L" is a hydrophobic residue (often leucine) and "x" is any other amino acid. NESbase is a database of proteins with experimentally verified leucine-rich nuclear export signals (NES), see for example Xu et al. Mol Biol Cell. 2012 Sep;23(18):3677-93. doi: 10.1091/mbc.E12-01 -0046. Epub 2012 Jul 25. PMID: 22833565; PMCID: PMC3442415, herein specifically incorporated by reference. In some embodiments, the nuclear export signal comprises the amino acid sequence ALPPLERTLTL (SEQ ID NO: 25)

[0039] cAMP sponge. Cyclic AMP sponges are described, for example, by Lefkimmiatis, et al. PLoS One 4, 2-9 (2009) herein specifically incorporated by reference, and may comprise a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxyterminus of the regulatory subunit Rl(3 of protein kinase A (PKA or PRKAR1 B). Addition of targeting sequences permit localization of this fragment to a desired cellular compartment. Such a sponge exploits the high-affinity cAMP-binding portions of the regulatory subunits of protein kinase A (PKA) as a molecular approach for controlling intracellular elevations of cAMP. There are two classes of PKA regulatory subunits (Rl and RII) and each of these exist as two subtypes, a and p. The Rl subunits have the highest affinity for cAMP and consequently give rise to PKA holoenzymes with lower thresholds of activation as compared to the PKA-RII holoenzymes. The two cAMP binding domains are located in the carboxy terminus. A targeted high-affinity cAMP buffer is based on the carboxy-terminal cAMP-binding fragment of the regulatory subunit Rip. Over-expression of this “cAMP sponge” can buffer agonist-induced cAMP signals. The amino acid sequence of human cAMP-dependent protein kinase type I- beta regulatory subunit (PRKAR1 B) is: MASPPACPSEEDESLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILARQKSNSQSDSHDEEVSPTPPNPVVKARRRRGGVSAEVYTEEDAVSYVRKVIPKDYK TMTALAKAISKNVLFAHLDDNERSDIFDAMFPVTHIAGETVIQQGNEGDNFYVVDQGEVDVY VNGEWVTNISEGGSFGELALIYGTPRAATVKAKTDLKLWGIDRDSYRRILMGSTLRKRKMY EEFLSKVSILESLEKWERLTVADALEPVQFEDGEKIVVQGEPGDDFYIITEGTASVLQRRSPN EEYVEVGRLGPSDYFGEIALLLNRPRAATVVARGPLKCVKLDRPRFERVLGPCSEILKRNIQ RYNSFISLTV (SEQ ID NO: 26)

In some embodiments, the cAMP sponge comprises full length PRKAR1 B. In some embodiments, the cAMP sponge comprises only the C-terminus of PRKAR1 B. When the cAMP sponge only contains the C-terminus of PRKAR1 B, the cAMP sponge specifically omits the catalytic inhibitory domain of PRKAR1 B located in the N-terminus of PRKAR1 B. In some embodiments, the C-terminus is amino acid residues 133-380 of SEQ ID NO: 26. An exemplary amino acid sequence of the the C-terminus of PRKAR1 B is: KNVLFAHLDDNERSDIFDAMFPVTHIAGETVIQQGNEGDNFYVVDQGEVDVYVNGEWVTNI SEGGSFGELALIYGTPRAATVKAKTDLKLWGIDRDSYRRILMGSTLRKRKMYEEFLSKVSILE SLEKWERLTVADALEPVQFEDGEKIVVQGEPGDDFYIITEGTASVLQRRSPNEEYVEVGRL GPSDYFGEIALLLNRPRAATVVARGPLKCVKLDRPRFERVLGPCSEILKRNIQRYNSFISLT (SEQ ID NO: 27) In some embodiments, the cAMP sponge comprises a nuclear export signal.

[0040] The terms "active agent," “antagonist”, "inhibitor", "drug" and "pharmacologically active agent" are used interchangeably herein to refer to a chemical material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.

[0041] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect, such as reduction of viral titer. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e. , arresting its development; and (c) relieving the disease, i.e., causing regression of the disease (e.g., reduction in bacterial titers).

[0042] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; avians, and the like. "Mammal" means a member or members of any mammalian species, and includes, byway of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations.

[0043] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0044] The terms "polypeptide" and "protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and native leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, p-galactosidase, luciferase, etc.; and the like.

[0045] The terms "nucleic acid molecule" and “polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0046] A "therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to affect such treatment for the disease, condition, or disorder. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0047] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0048] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical formulation that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0049] As used herein, a "pharmaceutical formulation" is meant to encompass a formulation suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical formulation” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical formulation is pharmaceutical grade). Pharmaceutical formulations can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including for the purposes of the disclosed methods, intravitreal, topically applied to the eye, and the like.

Promoters and Vectors

[0050] Aspects of the present invention encompass expression cassettes and/or vectors comprising polynucleotide sequences of interest for expression in targeted cells. The polynucleotides can comprise promoters operably linked to the region of the polynucleotide that encodes e.g. a nuclear localized sAC, which may be constitutively active; a cytoplasmic localized cAMP sponge; etc. Targeted expression is accomplished using an astrocyte cell- selective or cell-specific promoter.

[0051] Promoters of interest for optic nerve head astrocyte specific expression include but are not limited to, full length human gaf2 promoter, full length gfa28 promoter, truncated human gaf2 promoter gfaABCiD, full length human ALDH1 L1 promoter, full length human EAAT1 promoter, any variants thereof, etc. In some embodiments the promoter is gfaABCiD. The gfaABCi D promoter may have a nucleic acid sequence having 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more or 100% nucleotide sequence identity to Aacatatcctggtgtggagtaggggacgctgctctgacagaggctcgggggcctgagctggctctgtgagctggggaggaggc agacagccaggccttgtctgcaagcagacctggcagcattgggctggccgccccccagggcctcctcttcatgcccagtgaatg actcaccttggcacagacacaatgttcggggtgggcacagtgcctgcttcccgccgcaccccagcccccctcaaatgccttccg agaagcccattgagcagggggcttgcattgcaccccagcctgacagcctggcatcttgggataaaagcagcacagcccccta ggggctgcccttgctgtgtggcgccaccggcggtggagaacaaggctctattcagcctgtgcccaggaaaggggatcagggg atgcccaggcatggacagtgggtggcagggggggagaggagggctgtctgcttcccagaagtccaaggacacaaatgggtg aggggagagctctccccatagctgggctgcggcccaaccccaccccctcaggctatgccagggggtgttgccaggggcaccc gggcatcgccagtctagcccactccttcataaagccctcgcatcccaggagcgagcagagccagagcaggttggagaggag acgcatcacctccgctgctcgc (SEQ ID NO: 28).

[0052] Variants of the above discussed promoters may also be used. In some instances, a suitable variant comprises a nucleotide sequence having 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more or 100% nucleotide sequence identity to their corresponding “reference” promoter, e.g. SEQ ID NO:28. A person of skill in the art will recognize that various promoters drive expression in various cell types, and will be able to decide on which promoter to use for their desired outcome.

[0053] Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein in astrocytes, one of the most abundant cell types in the vertebrate central nervous system (CNS) (Bignami et al., 1972; Eng et al., 1971 ). Its expression is used as a marker of astrocyte differentiation (Bonni et al., 1997), and its upregulation is the hallmark of gliosis in CNS injury (Eng and Ghirnikar, 1994; Ransom et al., 2003). Therefore, the regulatory elements of the GFAP gene have been studied to understand both developmental signaling for astrocyte maturation and the response of astrocytes to CNS injury (Bonni et al., 1997; Kahn et al., 1997; Martin et al., 2003). In addition, knowledge gained from the identification of GFAP regulatory elements has been used to develop promoters capable of targeting transgene expression to astrocytes (Brenner et al., 1994; de Leeuw et al., 2006). For example, the gfa2 promoter, which spans bp 22163 to 147 of the human GFAP gene, has been widely used for this purpose (reviewed in Su et al., 2004).

[0054] In a preferred embodiment, the truncated gfa2 promoter or a variant thereof is used. The GfaABCi D promoter is a compact GFAP promoter with the size of 681 bp. It was derived from the conventional 2.2 kb human GFAP promoter by deleting 5' nucleotides -2163 to -1757 and an internal segment from -1257 to -132. gfaABCi D previously displayed expression properties in transgenic mice indistinguishable from the 2.2 kb promoter. This promoter allows for greater flexibility in creating therapeutic AAV constructs with less transgene size restrictions, which can be a drawback of using AAV for gene delivery. In some embodiments a variant of the gfaABCiD may be used. A suitable gfaABCiD promoter variant comprises a nucleotide sequence having 30% or more, 40% or more, 50% or more,

[0055] 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,

95% or more, 99% or more or 100% nucleotide sequence identity to the gfaABCi D promoter. [0056] In other embodiments, an inducible promoter is used, many of which are known and used in the art, for example the tetracycline-inducible system, which consists of Tet-On and Tet-Off promoters that respond to the presence or absence of tetracycline or its analogs; the ecdysone-inducible system, which utilizes the ecdysone hormone or its derivatives to regulate gene expression; the Gal4/UAS system, based on the yeast Gal4 transcription factor and its binding site UAS; etc. Light-inducible promoters, also known as optogenetic promoters, are a class of inducible promoters that respond to specific wavelengths of light. These promoters are designed by fusing light-sensitive proteins, such as phytochromes, cryptochromes, or phototropins, to transcription factors or regulatory elements. Upon exposure to the appropriate light stimulus, these light-sensitive proteins undergo conformational changes, leading to the activation or repression of gene expression.

[0057] Utilizing a viral vehicle to deliver genetic material into cells allows direct targeting of pathogenic molecules and restoration of function. The retina is an advantageous target for gene therapy due to its easy access, confined non-systemic localization, partial immune privilege, and well-established definitive functional readouts. Because AAV is non-pathogenic and cannot reproduce itself without helper viruses, it has served as a primary vehicle for gene therapy. It is a single-stranded DNA virus that stably and efficiently infects a wide variety of cells in multiple tissues. AAV-mediated gene therapies specifically targeted to astrocytes within the ONH are disclosed herein.

[0058] In some embodiments, the vector is a recombinant adeno-associated virus (AAV) vector. AAV vectors are DNA viruses of relatively small size that can integrate, in a stable and site specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus. [0059] The application of AAV as a vector for gene therapy has been rapidly developed in recent years. Wild-type AAV could infect, with a comparatively high titer, dividing or nondividing cells, or tissues of mammal, including human, and also can integrate into in human cells at specific site (on the long arm of chromosome 19) (Kotin et al, Proc. Natl. Acad. Sci. U.S.A., 1990. 87: 221 1 -2215; Samulski et al, EMBO J., 1991. 10: 3941 -3950 the disclosures of which are hereby incorporated by reference herein in their entireties). AAV vector without the rep and cap genes loses specificity of site-specific integration, but may still mediate longterm stable expression of exogenous genes. AAV vector exists in cells in two forms, wherein one is episomic outside of the chromosome; another is integrated into the chromosome, with the former as the major form. Moreover, AAV has not been found to be associated with any human disease, nor any change of biological characteristics arising from the integration has been observed. There are sixteen serotypes of AAV reported in literature, respectively named AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAV14, AAV15, and AAV16, wherein AAV5 is originally isolated from humans (Bantel- Schaal, and H. zur Hausen. Virology, 1984. 134: 52-63), while AAV1 -4 and AAV6 are all found in the study of adenovirus (Ursula Bantel-Schaal, Hajo Delius and Harald zur Hausen. J. Viral., 1999. 73: 939-947).

[0060] AAV vectors may be prepared using any convenient methods. Adeno-associated viruses of any serotype are suitable (See, e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease" J. R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1 , 1974; P. Tattersall "The Evolution of Parvovirus Taxonomy" In Parvoviruses (J R Kerr, S F Cotmore. ME Bloom, RMLinden, C RParrish, Eds.) p 5-14, Rudder Arnold, London, UK (2006); and D E Bowles, J E Rabinowitz, R J Samulski "The Genus Dependovirus" (J R Kerr, SF Cotmore. ME Bloom, R M Linden, C R Parrish, Eds.) p 15-23, Rudder Arnold, London, UK (2006), the disclosures of which are hereby incorporated by reference herein in their entireties). Methods for purifying for vectors may be found in, for example, U.S. Pat. Nos. 6,566, 118, 6,989,264, and 6,995,006 and W0/1999/01 1764 titled "Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors", the disclosures of which are herein incorporated by reference in their entirety. Preparation of hybrid vectors is described in, for example, PCT Application No. PCTIUS2005/027091 , the disclosure of which is herein incorporated by reference in its entirety. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., International Patent Application Publication Nos: 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941 ; and European Patent No: 0488528, all of which are herein incorporated by reference in their entirety). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus). The AAV recombinants that are produced are then purified by standard techniques.

[0061] In some embodiments, the vector(s) for use in the methods of the invention are encapsidated into a virus particle (e.g. AAV virus particle including, but not limited to, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV12, AAV13, AAV14, AAV15, and AAV16). Accordingly, the invention includes a recombinant virus particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535. In some embodiments, the particle is an AAV5 particle.

METHODS OF USE

[0062] Methods of the present disclosure are directed to increasing the neuroprotective activity of astrocytes in a subject 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.

[0063] Agents of the present disclosure may be any agent that increases nuclear cyclic adenosine monophosphate (cAMP), decreases cytoplasmic cAMP, or increases nuclear cyclic adenosine monophosphate (cAMP) and decreases cytoplasmic cAMP. Agents of the present disclosure may be soluble adenylyl cyclases (sAC) or variants and/or fragments thereof, cAMP sponges, or combinations thereof. In some embodiments, the agent is a sAC or a variant thereof. In some embodiments, the sAC is human sAC. In some embodiments, the human sAC comprises an amino acid sequence having at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, 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%, amino acid sequence identity to SEQ ID NO: 1 . In some embodiments, the sAC is mus musculus sAC. In some embodiments, the mus musculus sAC comprises an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, amino acid sequence identity to SEQ ID NO: 3. . In some embodiments, the sAC is yeast sAC. Yeast sAC may be referred to as yeast CYR. In some embodiments, the yeast sAC comprises an amino acid sequence having at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, 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%, amino acid sequence identity to SEQ ID NO: 33. In some embodiments, the human sAC is truncated human sAC. Truncated sAC is known in the art and has been described by Jaiswal et al. (J Biol Chem. 2001 Aug 24;276(34):31698-708), Kleinboelting et al. (Acta Crystallogr F Struct Biol Commun. 2014 Apr 1 ; 70(Pt 4): 467-469), Kleinboelting et al. (Proc Natl Acad Sci U S A. 2014 Mar 11 ; 11 1 (10): 3727-3732), Chaloupka et al. (Mol Reprod Dev. 2006 Mar; 73(3): 361- 368), and Buck et al. (Proc Natl Acad Sci U S A. 1999 Jan 5; 96(1 ): 79-84), each of which is specifically incorporated by reference herein.

[0064] Truncated sAC comprises the N-terminus of full-length sAC containing the catalytic domain but lacks the C-terminus which contains an autoinhibitory domain. In some embodiments, the truncated sAC comprises the N-terminus of full-length sAC containing the catalytic domain. In some embodiments, the truncated sAC lacks the autoinhibitory domain of full-length sAC. In some embodiments, the truncated sAC is human truncated sAC. In some embodiments, the human truncated sAC comprises an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, amino acid sequence identity to amino acid residues 1 -469 of SEQ ID NO: 1 . In some embodiments, the truncated sAC is mus musculus (i.e., mouse) truncated sAC. In some embodiments, the mouse truncated sAC comprises an amino acid sequence having at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, 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%, amino acid sequence identity to amino acid residues 1 -469 of SEQ ID NO: 3.

[0065] In some embodiments, the sAC is constitutively active sAC. The constitutively active sAC has increased enzymatic activity in the absence of activators. Constitutively active sACs are known in the art and have been described by, for example, Hatley et al. (J Biol Chem. 2000 Dec 8;275(49):38626-32) and Geller et al. Proc Natl Acad Sci U S A 90, 7603-7607 (1993), each of which are specifically incorporated by reference herein. In some embodiments, the sAC is human constitutively active sAC comprises an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, amino acid sequence identity to SEQ ID NO: 5. In some embodiments, the sAC is yeast constitutively active sAC comprises an amino acid sequence having at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, 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%, amino acid sequence identity to SEQ ID NO: 7.

[0066] The sACs of the present disclosure may be operably linked to a nuclear localization signal. The nuclear localization signal allows the sAC to be trafficked to the nucleus where the sAC increases nuclear cAMP levels. The nuclear localization signal may be any of the nuclear localization signals disclosed above.

[0067] In some embodiments, the agent is a cAMP sponge. cAMP “sponge” as used herein refers to any agent that is able to bind and sequester cAMP thereby preventing cAMP from binding to endogenous proteins and activating or participating in signal transduction. In some embodiments, the cAMP sponge is high-affinity cAMP-binding portions of the regulatory subunits of protein kinase A (PKA). In some embodiments, the cAMP sponge comprises full length Protein Kinase A cAMP-Dependent Type I Regulatory Subunit Beta (PRKAR1 B). In some embodiments, the cAMP sponge comprises only the C-terminus of PRKAR1 B. When the cAMP sponge only contains the C-terminus of PRKAR1 B, the cAMP sponge specifically omits the catalytic inhibitory domain of PRKAR1 B located in the N-terminus of PRKAR1 B. In some embodiments, the C-terminus is amino acid residues 133-380 of SEQ ID NO: 26. In some embodiments, the C-terminus is SEQ ID NO: 27. In some embodiments, the cAMP sponge is a phosphodiesterase. In some embodiments, the phosphodiesterase is a partially active phosphodiesterase. In some embodiments, the phosphodiesterase is a constitutively active phosphodiesterase. The phosphodiesterase may be any phosphodiesterase that hydrolyses cAMP thereby reducing cAMP levels. For instance, the phosphodiesterase may be a phosphodiesterase (PDE) 1 , PDE2, PDE3, PDE10, PDE1 1 , PDE4, PDE7, PDE8, etc. In some embodiments, the phosphodiesterase is PDE1 . In some embodiments, the phosphodiesterase is PDE2. In some embodiments, the phosphodiesterase is PDE3. In some embodiments, the phosphodiesterase is PDE10. In some embodiments, the phosphodiesterase is PDE11 . In some embodiments, the phosphodiesterase is PDE4. In some embodiments, the phosphodiesterase is PDE7. In some embodiments, the phosphodiesterase is PDE8. The cAMP sponge may comprise a nuclear export signal or a nuclear exclusion signal such that the cAMP sponge is localized to the cytoplasm thereby decreasing cytoplasmic cAMP levels. In some embodiments, the cAMP sponge comprises a nuclear export signal. The nuclear export or exclusion signal may be any of the nuclear export or exclusion signals described above.

[0068] In some embodiments, the agent is encoded by a polynucleotide. The polynucleotide may encode any of the agents described above. In some embodiments, the polynucleotide encodes 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. The promoter may be any of the promoters described above. In some embodiments, the polynucleotide further comprises a first viral inverted terminal repeat sequence, and a second viral inverted terminal repeat sequence; wherein the nucleic acid sequence is encapsulated by a viral capsid to form a viral particle. The first and second inverted terminal repeat sequence may be any inverted terminal repeat sequence deemed useful. For instance, the inverted terminal repeat sequence may be AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV12, AAV13, AAV14, AAV15, or AAV16 inverted terminal repeat sequence. In some embodiments, the first and second inverted terminal repeat sequence are AAV2 inverted terminal repeat sequences. The viral capsid may be any viral capsid deemed useful. For instance, the viral capsid may be AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAV14, AAV15, or AAV16. In some embodiments, the viral capsid is AAV5.

[0069] As used herein, the term “administration” refers to the administration of an agent or composition (i.e. a composition comprising a nucleic acid sequence encoding the agent) to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be intradermal, intramedullary, intramuscular, intranasal, intrathecal, intravenous, intraventricular, within a specific organ, mucosal, nasal, oral, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), intraocular, intravitreal and subconjunctivally. Intravitreal is preferred. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. The administration may comprise contacting the astrocyte with an effective does of the agent. When the agent is a polynucleotide comprising a first viral inverted terminal repeat sequence, and a second viral inverted terminal repeat sequence wherein the nucleic acid sequence is encapsulated by a viral capsid to form a viral particle, a specific number of viral particles per mL may constitute an effective dose. For instance, the effective dose may be 1 x10⁴, 1 x10⁵, 1 x10⁶, 1x10⁷, 1 x10⁸, 1 x10⁹, 1x10¹°, 1 x10¹¹ , 1 x10¹², 1x10¹³, 1 x10¹⁴, 1 x10¹⁵, 1 x10¹⁶, 1x10¹⁸, 1 x10¹⁹, or 1x1 O²⁰. In some embodiments, the effective does is 1 x10⁸ viral particles per mL.

[0070] In some embodiments, the claimed invention is used to deliver cAMP localizing agents to retinal astrocytes for the treatment of optic neuropathies. In some embodiments, the sequences are expressed in retinal astrocytes and not in microglia, ganglion cells (RGCs) or photoreceptors (PRCs). Optic neuropathies of interest include, but are not limited to, preglaucoma, glaucoma, traumatic optic neuropathy, ischemic optic neuropathy (e.g., arteritic or non-arteritic anterior ischemic neuropathy and posterior ischemic optic neuropathy), compressive optic neuropathy, infiltrative optic neuropathy, mitochondrial optic neuropathy (e.g., Leber's optic neuropathy), nutritional optic neuropathy, toxic optic neuropathy, and hereditary optic neuropathy (e.g., Leber's optic neuropathy, Dominant Optic Atrophy, Behr's syndrome). In some embodiments, the glaucoma is primary open-angle glaucoma, angleclosure glaucoma, normal-tension glaucoma, congenital glaucoma, neovascular glaucoma, steroid-induced glaucoma, or glaucoma related to ocular trauma.

[0071] Non-limiting examples of symptoms associated with the ocular conditions include the loss of retinal ganglion cell viability in the retina of the eye, glaucoma, optic nerve injury, optic neuritis, optic neuropathies, diabetic retinopathy, central retinal artery occlusion, and central retinal vein occlusion.

[0072] Glaucoma is a progressive neuropathy that induces dysfunction and degeneration of the optic nerve and retinal neurons. Of the retinal neurons, retinal ganglion cells (RGCs), which are an essential neuronal subtype that transmit visual information to the brain are the most sensitive to glaucoma. Glaucoma can be triggered when the fluid, called “aqueous humor”, builds up in the front part of the eye. Excess production or reduced draining of the aqueous humor increases the intraocular pressure (IOP), which irreversibly damages the optic nerve and RGCs.

[0073] There are two major classes of glaucoma: primary and secondary glaucoma. Primary glaucoma has no detectable cause of the disease. Secondary glaucoma is caused by other medical conditions. Primary glaucoma is classified into open-angle glaucoma (POAG), normal-tension glaucoma (NTG), angle-closure glaucoma and congenital glaucoma. Secondary glaucoma is classified into neovascular glaucoma, pigmentary glaucoma, exfoliation glaucoma and uveitic glaucoma. POAG is the most common type in the United States and is usually associated with an elevated IOP.

[0074] One of the characteristic features of glaucoma is structural changes in the optic nerve head (ONH). The optic disc (or simply “disc”) in the ONH is the point where RGC axons gather and exit the eye. The gathered RGC axons form the optic nerve. Under the ophthalmoscopy, the disc can be visualized as bright circle in the center of the eye. In the center of the disc, there is brighter spot, termed as “(optic) cup”. Because a size of cup becomes larger in glaucoma patients, the cup-to-disc ratio (often referred to as CDR) is used to assess disease progression. This change is observed in hypertensive glaucoma (POAG) and in NTG. The structural changes of the tissue cause mechanical deformation of the RGC axons running through this region. The ONH is the most vulnerable region and shows the earliest pathological changes.

[0075] Optic nerve atrophy precedes the onset of visual field loss in glaucoma. RGC axons exit the eye through a hole in the sclera that is filled with a mesh-like structure called the lamina cribrosa (LC). Structural change of LC induces ONH cupping. In humans, the LC is enriched in collagens. Because such collagen-rich structures are not observed in rodents, which are often used as glaucoma models, it has been considered that rodents have no LC or ONH cupping. Accumulating evidence has shown that astrocytes-formed LC-like structure can be seen in rodents. The human LC is formed by astrocytes highly expressing glial fibrillary acidic protein (GFAP). The astrocyte-formed mesh-like structure around the optic nerve in rodents is termed glial lamina. Importantly, the axons in the LC are unmyelinated and tightly surrounded by astrocytes. Therefore, changes in astrocytic functions directly affect the optic nerve.

COMPOSITIONS

[0076] Also compositions for practicing the methods are described in the present disclosure. In general, subject compositions may have an agent as described above in addition to a pharmaceutically acceptable excipient. In some embodiments, the subject compositions contain a polynucleotide comprising polynucleotide encodes 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. In some embodiments, the polynucleotide further comprises a first viral inverted terminal repeat sequence, and a second viral inverted terminal repeat sequence; wherein the nucleic acid sequence is encapsulated by a viral capsid to form a viral particle.

[0077] Compositions of the present disclosure can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, Intravitreal, or others as well as oral, nasal, ophthalmic, rectal, or topical. Intravitreal is preferred. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.

[0078] As noted above, the agent can be formulated with a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

[0079] The composition may be administered in a unit dosage form and may be prepared by any methods well known in the art. Such methods include combining agent with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients. A pharmaceutically acceptable carrier is selected on the basis of the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used.

[0080] In some embodiments, the composition is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from 5 mM to 100 mM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80. Optionally the composition may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the composition is stored at about 4²C. Pharmaceutical compositions may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.

[0081] Compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997. The compositions of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

EXPERIMENTAL

[0082] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Example 1

A Molecular Switch for Neuroprotective Astrocyte Reactivity

[0083] Here, a new gene therapy approach is described to specifically modulate compartmented cAMP in optic nerve head astrocytes to promote RGC survival after optic nerve injury. Specifically, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cAMP in reactive astrocytes are shown to act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data provide methods of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

RESULTS

[0084] Soluble adenylyl cyclase is required for astrocyte proliferation. To test the hypothesis that proliferation may be a key defining element of neuroprotective reactive astrocytes, possible molecular mechanisms regulating astrocyte proliferation and reactivity were explored, in vitro. Cyclic-adenosine monophosphate (cAMP) has been shown to differentially regulate cell cycle progression across multiple cell-types, but its role in reactive astrocyte proliferation is unknown. There are two main classes of adenylate cyclases, 9 transmembrane (tmACs) and 1 soluble (sAC), previously shown to be expressed in astrocytes. We treated cultured primary human astrocytes with the pan-tmAC inhibitor, dDADO, and two sAC-specific inhibitors, KH7 and 2HE, and assayed their effects on astrocyte proliferation by 5-ethynyl-2’- deoxyuridine (EdU) incorporation. Surprisingly, it was found that both KH7 and 2HE, but not dDADO, significantly suppressed EdU incorporation in a concentration-dependent manner, implicating sAC as a required source of cAMP in astrocyte proliferation (Fig. 1 A-B). This observed decrease in proliferation was not the result of sAC inhibition-mediated apoptosis as KH7 and 2HE did not induce significant astrocyte cell death at the effective concentrations (Fig. 1 C). To confirm the putative role of sAC in astrocyte proliferation, purified sAC^fl/fl mouse astrocytes were transduced with AAV-cre (Cre; KO) or deactivated-cre (dCre, CTRL); cre- mediated excision of the sAC^,l/fl allele deletes the second catalytic domain (C2) found in all sAC isoforms (Fig. 1 D) resulting in a complete sAC KO. Cre-mediated sAC KO was confirmed by quantitative PCR (qPCR) using primers that specifically recognize the first catalytic domain (C1 ) present in all sAC isoforms (sAC^a") and an extended C- terminal tail present only in full- length sAC (sAC^,ul1) (Fig. 1 D-E). Cre-mediated sAC KO was also confirmed by western blot (Fig. 1 F-G). Consistent with the KH7 and 2HE assays (Fig. 1 A-B), it was found that genetic deletion of sAC significantly inhibited astrocyte proliferation (Fig. 1 H-l). Interestingly, at least two sAC isoforms were observed, sAC^,LJ" and sAC*, that are expressed in proliferating mouse astrocytes after 15 days in vitro (15DIVs), however, with extended time in culture (30DIV) we found a significant decrease in sAC^fu" mRNA and protein (Fig. 1 E-F). Notably, this decrease in full-length sAC was associated with a significant reduction in astrocyte proliferation (Fig. 1 H-l). Taken together, these data demonstrate sAC activity is required for astrocyte proliferation and multiple sAC isoforms are expressed in proliferating astrocytes.

[0085] Soluble adenylyl cyclase is required for reactive astrocyte proliferation and suppresses microglial- induced neurotoxic astrocyte differentiation after optic nerve injury. To test whether sAC-derived cAMP is necessary for reactive astrocyte proliferation in vivo, a transgenic mouse line was generated in which GFAP-positive reactive astrocytes undergo cre-mediated deletion of sAC and express a cre-dependent GFP reporter (GFAP-cre/ZEG/sAC^fl/") and assayed the effect of sAC KO on reactive astrocyte proliferation following optic nerve crush (ONC) injury (Fig. 2A). To label proliferating cells, mice were injected with EdU intraperitoneally 3-, 4-, and 5-days post-crush. Seven days’ post-crush mice were perfused, and optic nerves dissected, sectioned, and immunostained for GFP, Sox9, and EdU. Optic nerve crush induced broad ere recombination in reactive optic nerve astrocytes identified by dual expression of GFP and the astrocyte-specific transcription factor Sox9 (Fig. 2B). Consistent with the in vitro data, genetic loss of sAC in reactive optic nerve astrocytes significantly inhibited reactive proliferation in recombinant (cre-positive; GFP+) astrocytes compared to controls (GFAP-cre/ZEG), but not non-recombinant (cre-negative; GFP-) astrocytes (Fig. 2C). This effect was likely underestimated due to the short half-life of EdU in vivo as well as the opportunity for incorporation of EdU in cells before sAC KO-induced G2/M cell cycle arrest. Similar to that reported following spinal cord injury, reactive astrocyte proliferation was highest closest to the site of injury and decreased with distance towards the chiasm consistent with the heterogeneous nature of reactive astrogliosis. Thus, astrocyte-expressed sAC is necessary for reactive astrocyte proliferation, in vivo.

[0086] Perturbing astrocyte reactivity and associated glial scar formation has been described in some cases to increase and in others to decrease neuronal health and CNS recovery, for example following traumatic spinal cord injury. To determine if sAC KO in reactive optic nerve astrocytes affects neuronal health, retinal ganglion cell (RGC) survival was quantified in retinal flatmounts by RNA binding factor mRNA processing factor (RBPMS) immunostaining after optic nerve crush injury, where it was found that sAC KO in reactive astrocytes led to significantly more RGC death compared to controls (Fig. 2E-F). I ntriguingly, when the variance between animals was analyzed, the extent of RGC survival directly correlated with the amount of reactive astrocyte proliferation in the optic nerve (Fig. 2G), supporting a new model in which sAC signaling in reactive astrocytes promotes their neuroprotective functions.

[0087] Because sAC KO led to an increase in RGC death after optic nerve injury, it was sought to determine whether loss of sAC in reactive astrocytes also affected microglial and macrophage activation. Using Iba1 as a marker of activated microglia and infiltrating macrophages, it was found thatsAC KO optic nerves had significantly larger lesion cores relative to controls (Fig. 2H-I). This increase was, at least in part, due to an increase in microglial and macrophage proliferation within the lesion core, which was also significantly higher than controls (Fig. 2J). Similar increases in microglia/macrophage cell density and proliferation were also found outside the lesion core in sAC KO optic nerves (Fig. 2K-L). As with astrocyte proliferation, microglia/macrophage density inversely correlated with RGC survival (Fig. 2M), suggesting sAC-derived cAMP in reactive astrocytes promotes proliferation and neuroprotective reactivity to inhibit deleterious immune cell activation and downstream RGC death.

[0088] Given sAC KO in reactive optic nerve astrocytes led to increased microglial infiltration and RGC death, it was hypothesized that sAC-regulated astrocyte proliferation in vivo may be specific to C3-positive versus C3-negative reactive astrocyte. To test this, control and sAC KO optic nerves were immuno-stained for complement component C3500 pm from the lesion core (LC) where reactive astrocyte recombination was the highest (Fig. 2N). After sAC KO, it was found that a significant increase in C3 immunoreactivity in GFP+ astrocytes measured as average C3 intensity (Fig. 20) or as a percent of C3-positive A1 astrocytes (Fig. 2P) relative to controls, which with the decrease in astrocyte proliferation this points to an even more dramatic increase in the ratio of C3-positive to C3-negative astrocytes. Thus, sAC KO and blockade of astrocyte proliferation is associated with a downstream increase in neurotoxic 03- positive astrocytes, consistent with the increased immune cell infiltration and RGC death observed in these experiments.

[0089] Nuclear and cytoplasmic pools of cAMP differentially regulate astrocyte proliferation, in vitro. Cyclic-AMP is highly compartmentalized in cells and sAC reportedly localizes in multiple subcellular compartments including the nucleus and cytoplasm. These findings and the profound effects of sAC activity in regulating neuroprotective astrocyte proliferation led us to hypothesize that nuclear sAC activity may be critical to this process. To test this, the effects of compartment-specific cAMP depletion were assayed on astrocyte proliferation in vitro using a series of genetically encoded cAMP-binding “sponges” derived from the PKA-R1 |3 subunit fused to different subcellular targeting sequences and a mCherry reporter (Fig. 3A).

[0090] Proper localization of each construct was confirmed by mCherry fluorescence (Fig. 3B- C) and effects on proliferation determined by EdU incorporation relative to mCherry alone (Fig. 3D; dotted line) and inactive mutant controls (Fig. 3D; grey bars). Inactive mutants served as critical controls for non-specific effects on proliferation caused by elevated transgene expression in each compartment. Consistent with the hypothesis, it was found that buffering cAMP exclusively in the nucleus (NLS) significantly inhibited astrocyte proliferation, whereas buffering cAMP in the cytoplasm (NES) surprisingly induced a significant increase in astrocyte proliferation. Depleting cAMP in all compartments with a construct lacking a targeting sequence (Ubiq) only slightly but significantly inhibited astrocyte proliferation, perhaps reflecting a dominance of nuclear cAMP signaling over cytoplasmic cAMP in the regulation of astrocyte proliferation. Lastly, buffering cAMP exclusively at the plasma membrane (PM) had no effect on astrocyte proliferation. Thus, nuclear and cytoplasmic pools of cAMP oppose one another in the regulation of astrocyte proliferation, whereby, nuclear cAMP promotes and cytoplasmic cAMP inhibits proliferation.

[0091] Due to the opposing effects of nuclear and cytosolic cAMP on astrocyte proliferation their effects on cell cycle progression was assayed by the propidium iodide assay (Fig. 3E).

[0092] Compartmented cAMP in optic nerve head (ONH) astrocytes differentially regulates microglial/macrophage activation and RGC survival after optic nerve injury, in vivo. Several adeno-associated viral (AAV) vectors have been reported to transduce astrocytes in the brain and spinal cord including AAV2, AAV5, and AAV8 typically in combination with an astrocytespecific promoter, however, use of these vectors to target inner retinal or optic nerve head astrocytes has not been reported. To test this, injected 2 pL of ~5x10¹³vp/mL of AAV5 or AAV8 expressing tdTomato under control of a CMV, GFAP, or gfaABC(1 )D promoter was injected intravitreally and quantified astrocyte transduction in retinal flat-mounts and cross sections (Fig 4A). As expected, neither AAV with the CMV promoter specifically or efficiently transduced retinal astrocytes. AAV8 also failed to efficiently transduce retinal astrocytes when combined with either GFAP promoter. Remarkably, however, it was found that intravitreal injection of recombinant AAV5 combined with the truncated GFAP promoter, gfaABC(1 )D, was highly specific to optic nerve head (ONH) astrocytes (Fig. 4B-C). Retinal astrocytes were broadly identified by dual GFAP and Sox9 immunoreactivity throughout the retina and ONH region (defined by the area between the glial lamina and optic disc rim -200 pm from the center of the optic disk) (Fig. 4D-F). Notably, this vector also transduced a small subset of nerve fiber layer (NFL) astrocytes, and 1 -2% percent of Muller glia (MG) (Fig. 4G-H), but was predominantly ONH astrocyte specific. Intravitreal injection of AAV5.gfaABC(1 )D was very specific to the inner retina as it did not transduce any other neuronal cell-type including photoreceptor cells, as evidenced by the absence of tdTomato expression in the inner and outer nuclear layers (INL, ONL). Importantly, AAV5.gfaABC(1 )D did not transduce RGCs as measured in retinal flat-mounts (Fig. 4I), nor did it transduce retinal microglia around the ONH (Fig. 4J-K); two celltypes directly affected by reactive astrocyte signaling. Thus, intravitreal injection of AAV5.gfaABC(1 )D transduces ONH astrocytes with >94% efficiency and high specificity, only transducing ~8% of NFL astrocyte, less than 2% of MG, and no other retinal cell-types (Fig. 41), supporting its use to deliver molecular constructs to ONH astrocytes, a tissue location hypothesized to be critical in the pathogenesis of glaucoma and other optic neuropathies.

[0093] Given the findings on sAC and the opposing effects of nuclear and cytoplasmic cAMP in astrocyte proliferation (Fig. 3C-D), this new ONH astrocyte targeting approach was used to ask whether similar compartment-specific cAMP modulation is relevant to reactive astrogliosis, microglial infiltration, and neuronal survival after optic nerve crush injury, in vivo. To do so, nuclear- and cytoplasmic-targeted cAMP sponges were packaged (NLS-sp and NES-sp) as well as a nuclear-targeted sAC* (NLS-AC) fused to tdTomato into our AAV5.gfaABC(1 )D viral vector (Fig. 5A). 2 Lils of ~5x10^A13 vp/mL of each construct was intravitreally injected into adult C57BL/6 mice followed by ONC injury seven days’ post injection, and immune cell infiltration and retinal ganglion cell (RGC) survival assayed after 2- weeks (Fig. 5B). Robust expression and proper localization of each construct was detected almost exclusively at the optic nerve head region (Fig. 5C-D). Initially, we quantified total Iba1 immunoreactivity in whole retinal flat mounts where we found buffering nuclear cAMP in ONH astrocytes (NLS-sp) significantly increased retinal immune cell infiltration, whereas, buffering cytoplasmic or elevating nuclear cAMP had no significant effects (Fig. 5C, G). When Iba1 + density was quantified in the ONH region only, however, it was found that local immune cell infiltration was significantly inhibited by both elevating nuclear (NLS-sAC) and buffering cytoplasmic cAMP (NES-sp) (Fig. 5E-H). Buffering nuclear cAMP in reactive ONH astrocytes also induced more significant local immune cell infiltration. These results demonstrate that cytoplasmic and nuclear pools of cAMP in reactive ONH astrocytes regulate opposing immune-modulatory functions after ONC injury.

[0094] Finally, it was found that sAC-dependent astrocyte-mediated immune suppression and neuronal health to be inversely related in our transgenic sAC KO mice (Fig. 2), prompting whether compartmented cAMP modulation in ONH astrocytes similarly affects RGC survival. To focus on astrocyte regulation of adjacent (proximal, P) RGCs, RGC survival was quantified relative to treatment with a tdTomato control vector, and found that depleting nuclear cAMP in reactive ONH astrocytes significantly exacerbated RGC death by 23.9%, whereas elevating nuclear cAMP or depleting cytoplasmic cAMP in ONH astrocytes led to greater than 10% and 21 % increases in RGC survival, respectively (Fig. 5F, I). Significant differences in RGC survival were not detected across all conditions when quantified distally from the ONH. This relationship between local astrocyte cAMP- regulated reactivity and RGC survival was further supported by the finding that the extent of proximal RGC survival across all conditions inversely correlated with immune cell density in the ONH region (Fig. 5J). Taken together these data support a novel model by which sAC-derived nuclear and cytoplasmic cAMP differentially regulate neuroprotective reactive astrocyte-mediated suppression of local microglial/macrophage activation and downstream neurotoxic reactivity and RGC survival. Thus, proliferating neuroprotective astrocytes act upstream of microglial-induced C3-positive neurotoxic astrocyte differentiation to regulate neuronal survival that can be targeted to therapeutic effect.

DISCUSSION

[0095] Here it was shown that a causal linkage between astrocytic sAC expression and differentially compartmented cAMP to reactive astrocyte proliferation, the balance of neurotoxic versus neuroprotective astrocyte reactivity, immune cell activation, and neuronal survival. First, it was found that compartmented signaling is a crucial element of this signaling pathway, specifically that differentially compartmented astrocyte cAMP similarly plays opposing roles in vivo in astrocyte-mediated microglial recruitment and RGC survival. Indeed, several recent reports have highlighted the importance of second messenger compartmentation in conferring specificity to cellular functions including RGC survival and growth after injury, axon guidance, synaptic plasticity, apoptosis, and cardiac contractility. Multiple catalytically active isoforms of sAC (sAC^MI and sAC’) are expressed in proliferating astrocytes, and genetic deletion of all sAC isoforms inhibits astrocyte proliferation while developmental loss of sAC^fu" is associated with a decrease in proliferative index. These data indicate isoform-specific expression and/or localization of sAC may account for the differential effects that nuclear and cytoplasmic cAMP exert on proliferation reported here

[0096] Optic nerve head astrocytes have been implicated in pathogenesis of optic neuropathies from glaucoma to trauma and others. Therapeutic approaches to target this cell population for pre-clinical studies of pathophysiology or clinical investigation have been lacking. Targeting specific astrocyte populations, e.g. retinal versus optic nerve astrocytes, can provide greater therapeutic benefits in different disease contexts, e.g. in glaucoma where optic nerve head astrocytes have been more specifically implicated. The novel AAV-ONH targeting approach is the first description of such a strategy and allows specifically studying this population and targeting genetic manipulations toward gliotherapeutics in future work.

METHODS

[0097] Animals. Timed pregnant (E17) C57BL/6 mice were purchased from Charles-River for primary astrocyte cultures. GEAR- ere (B6.Cg-Tg(GFAP-cre)77.6Mevs/J) and ZEG reporter mice (Tg(CAG-Bgeo/GFP)21 Lbe/J) were purchased from Jackson Laboratories. Conditional sAC knockout mice (Adcy10^fl/fl) were generously provided by Jochen Buck and Lonny Levin (Dept, of Pharmacology, Weill Cornell Medical College). sAC^fl/fl, GFAP-cre/ZEG, and GFAP- cre/sAC^fl/,l/ZEG mice were generated in-house. Mice of both sexes aged P30-P60 were used for in vivo experiments. [0098] Astrocyte purification and cell culture. Mouse cortical astrocytes were isolated from embryonic day 17 (E17) C57BL/6 and sAC^fl/fl mice as described in Schildge et al. (J. Vis. Exp. 2013). Briefly, E17 time pregnant mice were sacrificed, pups removed, and fetal brains quickly isolated in ice cold DPBS (Gibco, 14287-080). Cortices were carefully dissected away from the olfactory bulbs, hippocampus, and subcortical structures, and meninges removed to prevent contamination by meningeal cells and fibroblasts. Dissected cortices were sliced into 4 pieces per cortex and incubated in 0.25% Trypsin diluted in DPBS (Gibco, 14287-080) at 37^aC for 30 mins, with gentle shaking every 10 mins. Tissue was then pelleted at 200 x g for 5 mins at room temperature, trypsin aspirated, and cell pellet vigorously pipetted about 25 times in 10 mL mouse astrocyte media (mAM), consisting of DMEM (Gibco, 10569-010) with 10% fetal bovine serum (Hyclone, sh30071.03) and 1 % penicillin/streptomycin (Sigma-Aldrich, P4333-100mL). Dissociated cells were plated at 15 million cells per T75 flask (Thermo Fisher Scientific, 430641 U), placed in the incubator at 37 ^SC, and media was changed the following day and every third day thereafter. After 7 days, the culture was placed directly on an orbital shaker (Barnstead Thermolyne RotoMix Type 50800) for at least 4 hours at maximum speed at 37 ²C, then washed twice with 5 mL DPBS lacking Ca²⁺ and Mg²⁺ ions (Gibco, 14190- 144), followed by three rounds of vigorous manual shaking for 30 seconds in 10 mLs of DPBS (-Ca, -Mg) to remove any contaminating microglia and oligodendrocyte precursor cells (OPCs). Adherent astrocytes were released by incubating 5mL of 0.05% Trypsin-EDTA diluted in DPBS (-Ca, -Mg) for 4 mins at 37 ²C and were plated accordingly in mouse astrocyte media. Each culture of astrocytes was passaged a maximum of two times.

[0099] Human cortical astrocytes were purchased from ScienCell (#1800) and expanded twice before freezing in human astrocyte media (ScienCell, #1801 ) and 10% dimethyl sulfoxide (Sigma-Aldrich, D2650-100mL). Cells were plated in the PDL-coated T75 flasks and media was replaced every three days until the astrocytes were -90% confluent. Cells were passaged using 5 mL 0.05% Trypsin-EDTA (Thermo Fisher Scientific, 25300062) diluted 1 :1 with DPBS (-Ca, -Mg), incubated for 4 mins at 37 ^aC, and plated accordingly in human astrocyte media. All human astrocyte experiments were performed between passages 3 and 5.

[00100] All astrocytes were grown at 37^aC in 5% CO2, in tissue culture dishes (Falcon, 353046 or 353047) pretreated with poly-D-lysine (PDL). PDL was dissolved in water at a concentration of 0.01 mg/mL and allowed to cover the culture surface for one hour at room temperature. Afterwards, the solution was aspirated and washed with water three times, before letting dry at room temperature.

[00101] In vitro proliferation assays. Pharmacological sAC inhibition: Human astrocytes were plated at a density of 50,000 cells per well in PDL-coated 24-well dishes (Falcon, 353047) and incubated overnight before pharmacological treatment. The following day, astrocytes were treated with KH7 (Sigma-Aldrich, K3394), 2-hydroxyestradiol (2HE, Sigma-Aldrich, H3131 ), 2’5’-dideoxyadenosine (dDADO, Sigma-Aldrich, D7408), aphidicolin (Tocris, 5736), nocodazole (Tocris, 1228), and DMSO (vehicle; Sigma, D8418) diluted in human astrocyte media containing 10 pM 5-ethynyl-2’deoxyuridine (Edll (Invitrogen, c10640 or c10637) and incubated for 20hrs at 37^aC to label proliferating cells. After 20hrs, astrocytes were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences 15710), diluted in 1X PBS, for 15 mins and washed with 1X PBS three times prior to EdU visualization using the Click-iT Plus EdU Alexa Fluor 647 or 488 Imaging Kit (Invitrogen c10640 or c10637). Cells were incubated in DAPI (Molecular Probes, D3571 ) diluted 1 :2,000 in 1 X PBS for 5 minutes at room temperature to label all cell nuclei. Automated quantification of proliferating (EDU positive) astrocytes was performed using the ThermoFisher Celllnsight CX-5 platform.

[00102] Genetic sAC knockout: Mouse astrocytes were plated at a density of 50,000 cells per well in PDL-coated 24-well dishes (Falcon, 353047) or 250,000 per 6-well plate (Falcon, 353047) and allowed to expand for two days before transduction. For in vitro proliferation assays, mouse astrocytes were transduced with crude lentivirus, LV-eGFP-cre and LV-eGFP- deleted-cre, produced by the Stanford Neuroscience Gene Vector and Viral Core. Polybrene (Millapore Sigma, TR-1003-G) was diluted 1 :1000 (10mM) directly in virus to increase transduction efficiency before transduction. Crude virus added directly to each well and cells incubated for 30 mins at 37²C and subsequently spun at 800g for 30 mins. Following the spin, LV was aspirated and replaced with fresh mouse astrocyte media and cells placed back in the incubator. After 3 days in culture, media was replaced with fresh mouse astrocyte media containing EdU diluted 1 :1000 (10mM) and incubated for 20 hours before PFA fixation and EdU staining. For in vitro cell cycle assays and western blots, mouse astrocytes were transduced with AAV2-GFP (Addgene, 105530- AAV2) and AAV2-Cre-GFP (Addgene, 105525-AAV2) diluted in mouse astrocyte media at 20,000 multiples of infection (MOI) and incubated overnight, followed by a media change the following day. After 4-5 days in culture, cells were EtOH fixed for propidium iodide staining or harvested for western blot as described below.

[00103] cAMP sponges: Cyclic-AMP sponges were designed and synthesized commercially by Vector Builder (vectorbuilder.com). All transfections were performed using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, L3000015) as per the manufacturer’s protocol. Briefly, cells were seeded at 50,000 cells per well in a PDL-coated 24-well plate and transfected with 500ng of DNA, 0.75ul lipofectamine, and 1 ul P3000 per well. After 3 days in culture, media was replaced with fresh mouse astrocyte media containing EdU diluted 1 :1000 (10mM) and incubated for 20 hours before PFA fixation and EdU staining. Cells were incubated in DAPI (Molecular Probes, D3571 ) diluted 1 :2,000 in 1 X PBS for 5 minutes at room temperature to label all cell nuclei. Cells were imaged on a Zeiss Axio Observer Inverted microscope and proliferation (EdU) of sponge expressing cells quantified using the “cell counter” function in FIJI Imaged. For in vitro cell cycle assays, cells were methanol fixed 4 days after transfection before propidium iodide staining as described below.

[00104] In vitro cell cycle assays. For all cell cycle assays, astrocytes were fixed with 70% ice cold methanol for 5 mins and washed once with 1X PBS before staining with propidium iodide (PI). Astrocytes were then incubated in FxCycle PI Far Red stain (Invitrogen, F10348) or FxCycle PI Violet stain (Invitrogen, F10347) diluted in 1 X PBS for at least 4 hours at room temperature. Automated imaging and quantification were performed using the ThermoFisher Cell Insight CX-5 platform.

[00105] In vitro survival/cytotoxicity assay. To measure cytotoxicity in vitro, KH7, 2HE, and dDADO treated HAs were stained SYTOX® Orange Nucleic Acid Stain (Invitrogen, s34859) diluted 1 :5,000 and DAPI (1 :2, 000), for 5 mins at 37 ^SC before imaging and quantification using the ThermoFisher Celllnsight CX-5 platform.

[00106] Western blots. Five days after AAV-Cre transduction, cells were washed with 1 mL ice cold 1 X PBS then scraped down in —1 OOul RIPA buffer (Pierce, 89901 ) that included 1 X Halt Protease and Phosphatase inhibitor cocktail (ThermoFischer, 78446). Cells were briefly sonicated, cell lysate spun down at 13,000g for 5 mins at 4³C and supernatant transferred to freshly prepared tubes. Protein concentration was determined using Pierce BCA Protein Assay Kit (Pierce, 23227). Samples were diluted in LDS sample buffer (ThermoFischer, NP0007) and heated to 69°C for 10 min, cooled on ice, spun down at 13,000g for 5 mins and 20ug/well of total protein loaded onto a NuPage 3-8% Tris-Acetate gel (Invitrogen, EA0375). Protein was transferred to polyvinyldiene fluoride (PVDF) membranes (Bio Rad, 1704156) using the Trans-Blot® Turbo Transfer Starter System (BioRad, 17001918). Membranes were blocked with 5% non-fat milk (Labscientific, M0841 ) diluted in TBST (0.2% tween) and probed with anti-sAC (Abeam, ab82854) diluted 1 :500 or anti-GAPDH (Cell Signaling, 2118) diluted 1 :500 in TBST overnight at 4^SC on a rocker. The following day, membranes were washed 5X with TBST for at least 10 mins on a rocker and subsequently probed with anti-rabbit IgG horseradish peroxidase conjugated secondary antibody (GE Healthcare, NA9340) diluted 1 :500 in TBST for 4 hours at room temperature. Proteins were visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific, 32106) and imaged on an Ambersham Imager 600. Relative protein expression normalized to Ponceau or GAPDH and determined by densitometry using FIJI ImageJ.

[00107] Intravitreal injections and optic nerve injury. I ntravitreal injections and optic nerve crush were performed as previously described. For intravitreal injections, 2pls of >5x10^Al3vps/mL of AAV5.gfaABC(1 )D was injected into the posterior chamber of each eye using a Hamilton syringe. For optic nerve injury, the optic nerve was exposed from the lateral canthus and crushed for 3 secs with Dumont #5 forceps (FST, 91 150-20) ~1 .5 mm behind the globe. Care was taken to avoid damaging the blood supply to the retina. Mice with any significant post- operative complications (e.g., retinal ischemia, cataract) were excluded from further analysis. On days 3,4, and 5 post-crush, EdU (10mg/mL; ThermoFisher, A10044) diluted in 1X PBS was injected intraperitoneally (100ug/g bodyweight) to label proliferating cells.

[00108] Mice were sacrificed by intracardial perfusion with 4% PFA and optic nerves and retinal flat-mounts were prepared as below. Briefly, eyes were enucleated, and optic nerves carefully dissected and then post-fixed in 4% PFA for two hours at room temperature. Optic nerves were sequentially cryoprotected in 15% and 30% sucrose overnight, mounted in OCT (Fisher Scientific, 23-730-571 ) and sectioned in 12pm sections on Superfrost Plus glass slides (VWR, 4831 1 -703) for immunostaining. Retinas were carefully dissected from the eye cup, lens removed, and partitioned into 4 quadrants and post-fixed in ice cold methanol before immunostaining.

[00109] Immunohistochemistry. Cell culture. Cells were fixed with 4% PFA for 15 minutes and then washed three times with PBS. Cells were blocked and permeabilized with 0.5% Trition- X100 (Sigma-Aldrich, x100-100mL) and 5% normal goat serum (Thermo Fisher Scientific, 16- 210-064) in PBS for one hour at room temperature and then incubated with primary antibodies at 4^SC overnight in blocking solution: anti-GFAP (Abeam, ab4674), anti-mCherry (Abeam, ab125096), and anti-l ba1 (Fujifilm, 019-19741 ). The following day, cells were washed 3 times in 1 X PBS for 5 mins each and then incubated in Alexafluor secondary antibodies diluted 1 :500 in blocking buffer for 4 hours at room temperature. Cells were then washed 3 times with 1X PBS and stained with DAPI before imaging.

[00110] Optic nerves. Sectioned optic nerves were thawed at room temperature for 10 mins and a hydrophobic barrier was drawn around each section using a pap-pen. EdU visualization was performed using the Click-iT Plus EdU Alexa Fluor 647 or 488 Imaging Kit (Invitrogen, c10640 or c10637) as per the manufactures protocol. Following EdU staining, optic nerve sections were blocked and permeabilized in approx. 200ul with 0.5% Trition-X100 (Sigma- Aldrich, x100-1 OOmL) and 5% normal goat serum (Thermo Fisher Scientific, 16-210-064) in 1 X PBS for one hour at room temperature. Blocking buffer was then replaced with anti-GFP (Abeam, ab13970), anti-Sox9 (Abeam, ab185966), anti-lba1 (Fujifilm, 019-19741 ), and/or anti- C3 (Abeam, ab97462) diluted in blocking buffer and incubated on a flat surface overnight. The following day, slides were washed 3 time in 1X PBS for 10 mins each and then incubated in Alexafluor secondary antibodies diluted 1 :500 in blocking buffer overnight at 4^SC. Slides were washed 3 times in 1 X PBS and cover slipped with mounting media plus DAPI. Images were acquired with an inverted Ziess microscope or confocal laser scanning microscope (Zeiss 880) at 20X and 63X magnification.

[00111] Flat mounts. Dissected retinas were transferred to a 48 well dish and incubated for 1 hour at room temperature on a rocker in approx. 500ul of flat mount blocking buffer consisting of 1X PBS, 3% Triton-X 100, 0.5% Tween-20, 1% BSA, and 0.1 % sodium azide. RGCs were stained with RNA binding protein with multiple splicing (RBPMS) antibody (PhosphoSolutions, 1832-RBPMS) diluted 1 :250 in flat mount blocking buffer and placed on a rocker at 4^aC for 48 hours. Retinas were washed three times with 1 X PBS for 10 mins each and then incubated in Alexafluor secondary antibodies diluted 1 :250 in flat mount blocking buffer for 24 hours at 4^aC. The following day, retinas were washed three times with 1 X PBS for 10 mins each and then mounted RGC side up on Superfrost Plus glass slides (VWR, 4831 1 -703). Images were acquired with a confocal laser scanning microscope (Zeiss 880) at 20X magnification. Imaging and quantification were performed in a masked fashion as previously described.

[00112] Statistical analysis. Data was analyzed using Fiji Imaged, R, and GraphPad Prism. All results are shown as the means ± SEM of at least three independent replicates. Number of replicates and statistical details for each experiment can be found in the corresponding figures and figure legends.

Table 1 . Sequences used in Example 1 .

Example 2

[00113] As disclosed in Example 1 , soluble adenylyl cyclase (sAC) signaling in reactive astrocytes is critical for neuroprotective astrocyte proliferation and retinal ganglion cell (RGC) survival after optic nerve injury. These effects are mediated through compartment-specific cAMP, and buffering cAMP in the nuclear compartment decreases neuroprotective astrocyte proliferation and RGC survival after optic nerve injury.

[00114] Here, it was determined if increasing cAMP in the nucleus can increase neuroprotective astrocyte proliferation and promote RGC survival after injury. A constitutively- active AC (AC^ra) derived from yeast has been shown to cause long-lasting elevation in cAMP levels when expressed in cells. It was shown that nuclear-targeted constitutively active AC (NLS-AC^ca) increases proliferation in primary cultured astrocytes almost four-fold compared to control plasmid (FIG. 6). Next, the optic nerve head-targeting AAV5.gfaABC(1 )D viral vector was used to deliver nuclear-targeted AC^ca constructs to ONH astrocytes (FIG. 7A, B). After allowing 7 days for adequate expression, optic nerve crush (ONC) was performed to induce optic nerve injury, and then compared the effect of NLS-AC^ca versus control virus on immune infiltration at the ONH and RGC survival 2 weeks after ONC. It was found that nuclear-targeted AC^ca decreases immune cell activation at the ONH and promotes RGC survival after injury (FIG. 7C, D). These data show that nuclear-targeted AC^ca can be a therapeutic agent for the treatment of optic neuropathies.

Table 1. Sequences used in Example 2.

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Claims

WHAT IS CLAIMED IS:

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. 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. The method of claim 2, wherein the AC is operably linked to a nuclear localization signal.

4. The method of claim 2 or claim 3, wherein the AC is soluble AC (sAC) or yeast CYR.

5. The method of claim 4, wherein the sAC is truncated sAC.

6. The method of any of claims 1 -5, wherein the AC is constitutively active, or activated by an exogenous signal.

7. The method of claim 1 , wherein the agent comprises a cytoplasmic localized cAMP sponge, which decreases cytoplasmic cAMP levels.

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. 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. The method of any of claims 6-9, wherein the cAMP sponge is operably linked to a nuclear export signal.

1 1 . The method of any of claims 1 -10, wherein the subject has or is predicted to have an optic neuropathy.

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. The method of any of claims 1 -12, wherein the astrocytes are optic nerve head (ONH) astrocytes.

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. 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. The method of claim 15, wherein the viral inverted terminal repeat sequences are AAV2 inverted terminal repeats.

17. The method of any of claims 14-16, wherein the promoter is gfaABCi D or a variant thereof.

18. The method of any of claims 15-17, wherein the viral capsid is AAV5.

19. The method of any of claims 15-18, wherein the effective dose comprises at least 1 x10⁸ viral particles per ml.

20. The method of any of claims 1 -19, wherein the agent is administered via intravitreal injection.