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WO2024177720A1 - Phosphoprotéine sécrétée 1 (spp1) utilisée en tant que thérapie neuroprotectrice dans une maladie neurodégénérative - Google Patents

Phosphoprotéine sécrétée 1 (spp1) utilisée en tant que thérapie neuroprotectrice dans une maladie neurodégénérative Download PDF

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WO2024177720A1
WO2024177720A1 PCT/US2023/085619 US2023085619W WO2024177720A1 WO 2024177720 A1 WO2024177720 A1 WO 2024177720A1 US 2023085619 W US2023085619 W US 2023085619W WO 2024177720 A1 WO2024177720 A1 WO 2024177720A1
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spp1
astrocytes
composition
protein
mice
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Tatjana JAKOBS
Song Li
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Schepens Eye Research Institute Inc
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Definitions

  • compositions and methods for treating an ocular neurodegenerative disease in a subject comprising administering to an affected eye of the subject a therapeutically effective amount of a composition comprising a Secreted Phosphoprotein 1 (SPP1) protein or nucleic acid.
  • SPP1 Secreted Phosphoprotein 1
  • compositions comprising SPP1 protein or nucleic acid, e.g., for use in a method for treating an ocular neurodegenerative disease in a subject.
  • the nucleic acid is administered in an expression construct, preferably a viral vector.
  • the viral vector is an adenovirus, adeno-associated virus (AAV), or lentivirus.
  • AAV2 is used.
  • the expression construct comprises a promoter that drives expression of the Spp1 nucleic acid.
  • the promoter is CMV or GFAP.
  • the Spp1 protein is administered in a slow-release formulation, optionally a polymer-based, protein-based, polyphenol-based, lipid- based, or inorganic materials-based formulation.
  • the slow- release formulation is protein-based, optionally wherein the Spp1 protein is attached to polyhedrin, optionally via has a tag that facilitates attachment to the polyhedrin.
  • the tag comprises an H1-tag that comprises an N-terminus H1 helix of a polyhedrin protein or a VP3 tag comprising a region of a capsid protein VP3 of cytoplasmic polyhedrosis virus.
  • the composition is administered by intravitreal or subretinal injection.
  • the subject has glaucoma, diabetic retinopathy/RGC loss in diabetes, Leber’s hereditary optic neuropathy (LHON), toxic optic neuropathy, nonarteritic anterior ischemic optic neuropathy (NAION), ischemic optic neuropathy, or degeneration associated with a pathology involving the Central Nervous System (CNS).
  • LHON hereditary optic neuropathy
  • NAION nonarteritic anterior ischemic optic neuropathy
  • ischemic optic neuropathy or degeneration associated with a pathology involving the Central Nervous System (CNS).
  • CNS Central Nervous System
  • subject has early-stage glaucoma, diabetic retinopathy/RGC loss in diabetes, LHON, toxic optic neuropathy, NAION, ischemic optic neuropathy, or degeneration associated with a pathology involving the CNS.
  • the pathology of the CNS is Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, or demyelinating optic neuritis.
  • the subject is human. Unless otherwise defined, 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 invention belongs.
  • A BRN3A-labeled RGCs in WT (C57BL/6) and Spp1 -/- (Spp1 KO) mice at 3, 10, 16 and 22 months.
  • C BRN3A-labeled RGCs in WT and Spp1 -/- mice after IOP elevation.
  • FIGs.2A-G SPP1 is induced by TGF- ⁇ 1/TGFBR1/RUNX1/E2F1 signaling cascade in astrocytes.
  • A SPP1 immunostaining in the proximal optic nerve at normal conditions (no injury), after high IOP, and after ONC.
  • B SPP1 immunostaining (green) in optic nerve head (ONH) astrocytes counterstained with GFAP (red) after high IOP and on day 3, 7, 14 and 21 after ONC.
  • (D) Quantification of Runx1, E2f1 and Spp1 mRNA in cultured astrocytes when incubated with TGF- ⁇ 1, TGF ⁇ 1 receptor TGFBR1 blocker SB431542, and TGF- ⁇ 1 plus SB431542 (n 5-6).
  • FIGs.3A-L Astrocytic SPP1 deficiency aggravates RGCs loss and vision impairment in aging, glaucoma and traumatic optic nerve injury.
  • A Design of the strain B6.Spp1 fl-EGFP-Stop-tdTomato (Spp1 GFPfl/fl ) before and after cre-mediated recombination.
  • B Validation of Spp1 conditional knockout in ONH astrocytes in Spp1 GFPfl/fl GfapCre mice generated from the Spp1 GFPfl/fl crossed to Gfap-cre mice.
  • C BRN3A-labeled RGCs in young (3 month old) and aged (16 month old) Spp1 GFPfl/fl and Spp1 GFPfl/fl GfapCre mice.
  • J BRN3A-labeled RGCs in Spp1 GFPfl/fl and Spp1 GFPfl/fl GfapCre mice on day 3, 7, 14 and 21 after ONC.
  • FIGs.4A-O Spp1 deletion in astrocytes induces a neurotoxic state and impairs phagocytosis.
  • A Genes involved in neuroinflammatory pathways were up-regulated in the Spp1 KO astrocytes by RNA-sequencing.
  • G tdTomato + astrocyte FACS from the ONH of Gfap-tdTomato and Spp1 GFPfl/fl GfapCre mice.
  • FIGs.5A-J Astrocytic SPP1 promotes mitochondrial function.
  • A Heat map of oxidative phosphorylation genes in cultured Spp1 KO astrocytes by RNA-sequencing analysis.
  • H-I Transmission electron microscopy images of axonal mitochondria (H) and astrocytic mitochondria (I) in the optic nerve in young and aged C57BL/6 and Spp1 KO mice.
  • H-I Transmission electron microscopy images of axonal mitochondria (H) and astrocytic mitochondria (I) in the optic nerve in young and aged C57BL/6 and Spp1 KO mice.
  • J Electron microscopy of the unmyelinated segment of the optic nerves from C57BL/6 and Spp1 KO animals showing mitochondria with few cristae in the axons of RGCs (arrowheads) and accumulations of partially degraded mitochondria in astrocytes (arrows). Astrocytes were false-colored in red (representative TEM images chosen from 4 Spp1 KO and 18 C57BL/6 mice).
  • FIGs.6A-X Astrocytic SPP1 induces mitochondrial VDAC1 expression and promotes mitochondrial function.
  • A VDAC1 immunostaining in cultured C57BL/6 and Spp1 KO astrocytes.
  • R Visualization of cytosolic ATP by transfection of the plasmid encoding the ATP sensor iATPsnFR and mCherry into astrocytes treated with rSPP1 or rSPP1 plus Vdac1 siRNA.
  • T Wild-type (WT) RGCs from C57BL/6 mice were growing in co-culture with C57BL/6 or Spp1 KO astrocytes. Visualization of RGCs and astrocytes by immunostaining of ⁇ -Tubulin and GFAP.
  • FIGs.7A-O SPP1 overexpression prevents RGCs loss and preserves vision in aging, glaucoma and traumatic optic injury.
  • B BRN3A-labeled RGCs in young (4 month old) and aged (16 month old) WT mice treated with AAV2-Spp1 and AAV2-EGFP for 12 month.
  • B-D Quantification of BRN3A + RGCs (B), PERG amplitude (C) and visual acuity (D) in aged WT mice with SPP1 overexpression.
  • E Transmission electron microscopy images of axon in the optic nerve in aged WT mice treated with AAV2-Spp1. Arrows point to enlarged axons with pale axoplasm that are more typically found in aged animals.
  • FIGs.8A-B Functional analysis in mouse model of glaucoma.
  • FIG.9 Traces of IOP in WT and Spp1 -/- mice following microbeads injection into anterior chamber.
  • FIG.9 Representative traces of PERG in WT and Spp1 -/- mice after high IOP.
  • FIGs.10A-F rSPP1 reverses functional impairment in Spp1 KO astrocytes.
  • FIGs.11A-C SPP1 overexpression by AAV2-Spp1 prevents RGCs loss in glaucoma.
  • A Outline of the experimental design in mice with experimental glaucoma (microbead model). AAV2-Spp1 or AAV2-EGFP was intravitreally injected into the WT and Spp1 -/- eyes. Thirty days after AAV2 injection, microbeads were injected unilaterally into the anterior chamber of the eyes to induce elevated IOP.
  • FIGs.12A-D Validation of SPP1 overexpression by AAV2-Spp1 in the retina.
  • RNA-sequencing of purified astrocytes from various regions of the brain demonstrated an up-regulation of inflammatory markers, components of the complement cascade, factors involved in synapse elimination, and others that are similar to those observed in reactive astrocytes 14-16 .
  • astrocytes form the direct cellular environment of the unmyelinated RGC axons 17,18 .
  • SPP1 Secreted Phosphoprotein 1
  • OPN osteopontin
  • Glaucoma is characterized by a progressive apoptotic loss of RGCs and degeneration of the optic nerve 6 .
  • Elevated intraocular pressure (IOP), genetic factors, and age are the most important risk factors. Lowering the IOP is currently the mainstay of glaucoma management, but additional neuroprotective approaches would be welcome.
  • SPP1 is significantly up-regulated in optic nerve astrocytes after injury.
  • SPP1 stimulates mitochondrial activity and ATP production, the secretion of neurotropic factors, and phagocytosis.
  • the expression of neurotoxic and inflammatory mediators is negatively regulated by SPP1.
  • virus-mediated overexpression or direct injection of the protein is in fact highly protective of RGCs in several models of RGC degeneration.
  • SPP1 neuroprotective role for SPP1 that may be used therapeutically in conditions such as glaucoma, diabetic retinopathy/RGC loss in diabetes, Leber’s hereditary optic neuropathy (LHON), toxic optic neuropathy, non- arteritic anterior ischemic optic neuropathy (NAION), ischemic optic neuropathy, or degeneration associated with a pathology involving the Central Nervous System (CNS), e.g., Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, or demyelinating optic neuritis.
  • the subject has early-stage disease.
  • transcriptomic characterizations are, they should not be interpreted as to define fixed cell types 47 .
  • expression profiles of “A1” and “A2” genes overlap in the same population of reactive astrocytes, there is considerable heterogeneity between astrocytes even in the same anatomical region, and astrocyte reactivity itself can be reversible if the pathological stimulus is removed 48-51 .
  • astrocytes take on a more neurotoxic phenotype 15,52 , are less phagocytic, and show declining mitochondrial function 53 .
  • the data herein indicate that supplying a single protein, SPP1, slows the course of age-related or glaucomatous death of RGCs and the concomitant loss of visual function. It does so by mitigating several facets of the decline in astrocyte function.
  • “A2” markers are down-regulated in Spp1 KO astrocytes, suggesting that they show a more neurotoxic profile. Consistent with this observation, in Spp1 KO astrocytes neuroinflammatory markers are up-regulated. Neuroinflammation is observed in human glaucomatous optic nerves and retinas 54 , and it is a common feature of animal models of the disease 55-57 .
  • Spp1 deficiency causes decreased expression of genes involved in oxidative phosphorylation. Functionally, Spp1 deletion leads to a decrease in oxidative phosphorylation and ATP production.
  • phagocytosis is a highly energy-dependent process 59 , the decreased mitochondrial function may partly explain the marked reduction of phagocytic activity we observed in Spp1 KO astrocytes.
  • impaired mitochondrial function and reduced phagocytosis may simultaneously negatively affect axonal integrity from two sides.
  • Astrocytes, especially those of the myelination transition zone – are phagocytic 41 and play an active role in the degradation of axonal mitochondria that have reached the end of their life cycle.
  • transcellular mitophagy In transcellular mitophagy, spent axonal mitochondria are accumulated in bag-like evulsions from the axonal membrane that are engulfed and phagocytosed by surrounding astrocytes 19,60 . This process is not pathological as it is observed even in na ⁇ ve young optic nerves 2 , and in neurons with long axons, transcellular mitophagy may be an energy-saving mechanism that obviates the need to transport mitochondria all the way back to the cell’s soma for degradation 60 .
  • the accumulation of abnormal axonal mitochondria in the axons of Spp1 KO mice may therefore be due to a combination of impaired mitochondrial function and impaired phagocytosis.
  • microglia express SPP1 (or EGFP in the Spp1 fl/fl GfapCre mice) briefly after an optic nerve crush.
  • Spp1 mRNA expression has also been detected in activated retinal microglia 62,63 . All of these cells could secrete SPP1 in response to injury. Whether Müller cells are a source of SPP1 in vivo is less clear. SPP1 is expressed in cultured Müller cells 64 , particularly upon GDNF stimulation 65 , and SPP1 was found in activated Müller cells from surgical specimens of epiretinal membranes 66 , but Spp1 knockout does not affect Müller cell structure 30 . In our study, Attorney Docket No.00633-0381WO1/MEEI 2023-182 we did not observe SPP1 expression in Müller cells either in uninjured young retinas, or after optic nerve crush, an elevation of IOP, or in aging.
  • SPP1 is a secreted protein, from a therapeutic point of view the source may not matter.
  • AAV2 does not specifically target astrocytes and we were able to elicit a neuroprotective effect even with the protein itself.
  • AAV- mediated overexpression of Spp1 preserved RGC function not only in wild-type C57BL/6 retinas but also in Spp1 KO retinas, it did not do so to quite the same extent. This may be due to the difficulty of transfecting astrocytes located deep in the optic nerve, but there is the possibility that endogenous SPP1 – e.g., coming from microglia or alpha RGCs – is also needed for the full neuroprotective effect.
  • SPP1 can be used as a neuroprotective treatment.
  • SPP1 administration could be combined with IOP-lowering drugs, including those that currently are the first-line treatment of glaucoma 67 .
  • SPP1 protein could be directly injected, e.g., into the vitreous for delivery to the eye. Intraocular injection is commonly used in anti-VEGF therapy for age-related macular degeneration.
  • Gene therapy e.g., using viral (e.g., AAV)- mediated overexpression of Spp1, can also be used.
  • AAV is generally considered safe 70-72 , and several clinical trials for monogenic eye diseases in humans are under way or completed 73-76 .
  • compositions and methods for delivering Spp1 protein or nucleic acids to a subject e.g., to a tissue in a subject.
  • Exemplary sequences of human Spp1 are provided in GenBank as shown in Table 1. TABLE 1.
  • the resulting isoform (OPN-a) has a shorter and distinct N-terminus compared to isoform e.
  • Variant 2 differs in the 5' UTR and coding sequence and lacks an alternate Attorney Docket No.00633-0381WO1/MEEI 2023-182 in-frame exon compared to variant 5.
  • the resulting isoform (OPN-b) has a shorter and distinct N-terminus and lacks an alternate internal segment compared to isoform e.
  • Variant 3 differs in the 5' UTR and coding sequence and lacks an alternate in-frame exon compared to variant 5.
  • the resulting isoform (OPN-c) has a shorter and distinct N-terminus and lacks an alternate internal segment compared to isoform e.
  • Variant 4 differs in the 5' UTR and coding sequence and lacks two alternate in-frame exons compared to variant 5.
  • the resulting isoform (isoform 4 or d) has a shorter and distinct N-terminus and lacks an alternate internal segment compared to isoform e.
  • Variant 5 represents the longest transcript and encodes the longest isoform (isoform 5 or e). See, e.g., Saitoh et al., Lab Invest.1995 Jan;72(1):55-63; Silva et al., Mol Biol Rep.2020 Oct;47(10):8339-8345; Hao et al., Int J Mol Med.2017 Jun;39(6):1327- 1337.
  • OPN-a is used.
  • the sequence of a protein or nucleic acid used in a composition or method described herein is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a sequence set forth herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol.48:444-453 ) algorithm which has been Attorney Docket No.00633-0381WO1/MEEI 2023-182 incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the sequence of a protein (or nucleic acid encoding a protein) used in a composition or method described herein has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions as compared to a sequence set forth herein. In some embodiments, the substitutions are conservative substitutions.
  • Spp1 Gene Therapy Constructs Spp1 nucleic acids can include naked mRNA or DNA, as well as expression constructs comprising sequences encoding Spp1, e.g., as shown in Table 1.
  • Expression constructs comprising sequences encoding Spp1 can include viral vectors, including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids.
  • Suitable expression constructs can include: a coding region; a promoter sequence, e.g., a promoter sequence that restricts expression to a selected cell type, a conditional promoter, or a strong general promoter; an enhancer sequence; untranslated regulatory sequences, e.g., a 5'untranslated region (UTR), a 3'UTR; a polyadenylation site; and/or an insulator sequence.
  • a promoter sequence e.g., a promoter sequence that restricts expression to a selected cell type, a conditional promoter, or a strong general promoter
  • an enhancer sequence untranslated regulatory sequences, e.g.,
  • the expression construct is capable of directing expression of the Spp1 nucleic acid preferentially in a particular cell type (e.g., astrocytes, retinal ganglion cells, or microglia, or Muller cells).
  • a particular cell type e.g., astrocytes, retinal ganglion cells, or microglia, or Muller cells.
  • Spp1 expression can be driven by a promoter known in the art.
  • expression is driven by a ubiquitous promoter, such as cytomegalovirus (CMV); a hybrid CMV enhance/chicken ⁇ -actin (CBA) promoter; a promoter comprising the CMV early enhancer element, the first exon and first intron of the chicken ⁇ -actin gene, and the splice acceptor of the rabbit ⁇ -globin gene (commonly call the “CAG promoter”); beta glucuronidase (GUSB); ubiquitin; UBC, Rous sarcoma virus (RSV) promoter; or a 1.6-kb hybrid promoter composed of a CMV Attorney Docket No.00633-0381WO1/MEEI 2023-182 immediate-early enhancer and CBA intron 1/exon 1 (commonly called the CAGGS promoter; Niwa et al.
  • CMV cytomegalovirus
  • CBA hybrid CMV enhance/chicken ⁇
  • a promoter for a gene that is specifically expressed in astrocytes can be used, e.g., GFAP or GfaABC1D. Modifications of these sequences may be possible or desirable in certain applications, and such modifications are within the scope of this disclosure.
  • the woodchuck hepatitis virus posttranscriptional response element (WPRE) can also be used. For targeting astrocytes, see, e.g., Merienne et al., Front Cell Neurosci.2013 Jul 5:7:106 Spp1 expression constructs that can be administered to a subject in need thereof.
  • the constructs can include, e.g., a viral delivery vector, e.g., preferably an adeno-associated virus (AAV) vector that comprises sequences encoding the Spp1.
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • AAV vectors efficiently transduce various cell types and can produce long-term expression of transgenes in vivo.
  • AAV vectors have been extensively used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models as well as in the clinic; see, e.g., Mingozzi and High, Nat Rev Genet, 2011.12(5): p.341-55; Deyle and Russell, Curr Opin Mol Ther, 2009.11(4): p.442-7; Asokan et al., Mol Ther, 2012.20(4): p.699- 708).
  • AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression.
  • AAV2, AAV5, AAV2/5, AAV2/8 and AAV2/7 vectors have been used to introduce DNA into photoreceptor cells (see, e.g., Pang et al., Vision Res, 2008.48(3): p.377-85; Khani et al., Invest Ophthalmol Vis Sci, 2007.48(9): p.3954-61; Allocca et al., J Virol, 2007. 81(20): p.11372-80).
  • the AAV vector can include (or include a sequence encoding) an AAV capsid polypeptide described in WO 2015/054653; for example, a virus particle comprising an AAV capsid polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 of WO 2015/054653, and a sequence encoding Spp1 as described herein.
  • the AAV capsid polypeptide is an Anc80 polypeptide, e.g., Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; or Anc80L44.
  • the AAV incorporates inverted terminal repeats (ITRs), e.g., derived from the AAV2 serotype.
  • ITRs inverted terminal repeats
  • numerous modified versions of the AAV2 ITRs are used in the field. Modifications of Attorney Docket No.00633-0381WO1/MEEI 2023-182 these sequences are known in the art, or will be evident to skilled artisans, and are thus included in the scope of this disclosure.
  • the AAV e.g., packaged in AAV capsids, can be included in compositions (such as pharmaceutical compositions) and/or administered to subjects.
  • An exemplary pharmaceutical composition comprising an AAV according to this disclosure can include a pharmaceutically acceptable carrier such as balanced saline solution (BSS) and one or more surfactants (e.g., Tween 20) and/or a thermosensitive or reverse- thermosensitive polymer (e.g., pluronic).
  • BSS balanced saline solution
  • surfactants e.g., Tween 20
  • thermosensitive or reverse- thermosensitive polymer e.g., pluronic
  • Other pharmaceutical formulation elements known in the art may also be suitable for use in the compositions described here.
  • Slow Release Spp1 Formulations Provided herein are slow-release compositions for administration of Spp1 protein to a tissue over time. Time-release, delayed release, or sustained release compositions are known in the art and can include polymer-based, protein-based, polyphenol-based, lipid-based, and inorganic materials-based systems.
  • Exemplary polymer-based systems can include poly(lactideglycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides, encapsulating the Spp1 protein.
  • Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent No.5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. See, e.g., Vaishya et al., Expert Opin Drug Deliv.2015 Mar; 12(3): 415–440; Nie et al., Biomacromolecules 2021, 22, 6, 2299–2324; Markwalter et al., Journal of Controlled Release, 334:11-20 (2021).
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems such as lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and
  • compositions include complexes of a cargo protein comprising Spp1 attached to a micron-sized proteinaceous particle, known as polyhedra, for zero-order sustained-release of the Spp1.
  • the polyhedra comprise or consist of polyhedrin protein derived from polyhedrosis virus.
  • the Spp1 have a tag that facilitates attaching to the polyhedrin, e.g., comprising a region of a capsid protein VP3 of cytoplasmic polyhedrosis virus, more specifically, a region which is either a region from the N-terminus to the 40th amino acid residue or a region from the 41st amino acid residue to the 79th amino acid residue as an Attorney Docket No.00633-0381WO1/MEEI 2023-182 embedding signal for polyhedron (see US7619060 and 7432347).
  • a tag that facilitates attaching to the polyhedrin e.g., comprising a region of a capsid protein VP3 of cytoplasmic polyhedrosis virus, more specifically, a region which is either a region from the N-terminus to the 40th amino acid residue or a region from the 41st amino acid residue to the 79th amino acid residue as an Attorney Docket No.00633-0381WO1/MEEI 2023-182 embedd
  • the Spp1 has a tag, e.g., an N-terminal tag (e.g., an H1-tag that comprises an N-terminus H1 helix of a polyhedrin protein or functional equivalent thereof, preferably comprising Met Ala Asp Val Ala Gly Thr Ser Asn Arg Asp Phe Arg Gly Arg Glu Gln Arg Asn Ser Glu Gln Tyr Asn Tyr Asn Ser Ser (SEQ ID NO:1); see, e.g., US8554493).
  • an N-terminal tag e.g., an H1-tag that comprises an N-terminus H1 helix of a polyhedrin protein or functional equivalent thereof, preferably comprising Met Ala Asp Val Ala Gly Thr Ser Asn Arg Asp Phe Arg Gly Arg Glu Gln Arg Asn Ser Glu Gln Tyr Asn Tyr Asn Ser Ser (SEQ ID NO:1); see, e.g., US8554493).
  • Polyhedrin proteins useful in the present compositions are known in the art, and include those described in US7619060, US7432347, and US8554493; US2020/0277570; and Mori et al., J. Gen. Virol., (1989), 70 (Pt 7):1885- 1888; Mori et al., J. Gen. Virol., (1993) 74:99-102; Ikeda et al., J. Virol., (2001), 75:988-995.
  • PODS® the POlyhedrin Delivery System
  • Spp1-polyhedrin protein complexes can be made using methods known in the art; see, e.g., Mori et al., J. Gen. Virol.74 (1), 99-102 (1993); US7619060, US7432347, and US8554493.
  • Spp1 protein expression, folding and incorporation into polyhedrin crystals are achieved within insect cells, e.g., Spodoptera frugiperda IPLB-SF21-AE cells (Sf21) or Sf9 cells (a clonal isolate of Spodoptera frugiperda Sf21 cells), but other cells (e.g., insect cells S2, Tni and others) can also be used.
  • the methods can be used to treat optic neuropathies associated with neurodegeneration in the eye and loss of RGCs, including glaucoma, diabetic retinopathy/RGC loss in diabetes, Leber’s hereditary optic neuropathy (LHON), dominant optic atrophy (DOA), nonarteritic anterior ischemic optic neuropathy (NAION), ischemic optic neuropathy, and degeneration associated with pathologies involving the Central Nervous System (e.g., Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and demyelinating optic neuritis). See, e.g., Carelli et al., Hum Mol Genet.2017 Oct 1; 26(R2): R139–R150.
  • Subjects who have loss of RGCs and can be treated using the present methods can be identified by a skilled health care provider, e.g., using ophthalmoscopic examination of the optic nerve head, Visual Field Test, optic nerve head photographs, confocal scanning laser ophthalmoscopy, Attorney Docket No.00633-0381WO1/MEEI 2023-182 scanning laser polarimetry, and Retinal Nerve Fiber Layer (RNFL) thickness assessed by optical coherence tomography (OCT). See, e.g., Weinreb et al., JAMA.2014 May 14; 311(18): 1901–1911.
  • OCT optical coherence tomography
  • the methods include administering a composition comprising a therapeutically effective amount of a nucleic acid encoding Spp1 or Spp1 protein, e.g., Spp1 linked to PODS, e.g., as described herein, to an eye of a subject who is in need of, or who has been determined to be in need of, such treatment.
  • the methods include administering the composition directly to the eye of the subject, e.g., by intravitreal or subretinal injection.
  • to “treat” means to ameliorate at least one symptom of the disorder associated with loss of RGCs.
  • a treatment comprising or consisting of administration of a therapeutically effective amount of a composition described herein can result in a reduction in rate or extent of RGC loss and a return or approach to normal vision (e.g., with an associated improvement in neuronal structure and/or function), or a preservation of existing vision (e.g., with associated preservation of neuronal structure and/or function).
  • the subject has an early stage of the disease, and the present methods slow or reduce risk of progression.
  • mice Animals Spp1 KO (B6.129S6(Cg)-Spp1 tm1Blh /J) mice were obtained from the Jackson Laboratories (stock number 004936) and maintained in our facility as homozygous breeding stock. This strain is deficient for SPP1 expression in all tissues. C57BL/6 mice (Jackson Laboratories, stock number 000664) were used as controls. The strain, B6.Spp1 fl-EGFP-stop-tdTomato , for conditional deletion of Spp1 was produced by Cyagen. The cre donor strain for creating the astrocyte specific deletion was B6.Cg-Tg(Gfap- cre)77.6Mvs/2J (Jackson Laboratory, stock number 024098).
  • the reporter strain Ai14 to create Spp1 + astrocytes expressing red fluorescent protein was B6.Cg- Attorney Docket No.00633-0381WO1/MEEI 2023-182 Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J (Jackson Laboratory, stock number 007914). Male and female mice were used in equal numbers, and all control mice were strictly age- and sex- matched. Experiments included young adult mice (3-5 months) and aged mice (16 months). Mice were kept at a 12 hour dark/light cycle and received water and standard food at libitum. Mice were anesthetized by i.p.
  • mice received an s.c. injection of 0.1 mg/kg buprenorphine.
  • mice were euthanized by CO2 inhalation according to the Guide for the Care and Use of Laboratory Animals of the AAALAC. All animal procedures were done in accordance with the guidelines of the Association for Research in Vision and Ophthalmology and approved by the Institutional Animal Care and Use Committee at Schepens Eye Research Institute.
  • astrocytes Primary cell culture (astrocytes) Retinas and optic nerves from either C57BL/6 wild-type or Spp1 KO neonatal mice of 1–3 days old were dissociated by incubation with 0.25% trypsin and 0.01% DNase in DMEM, followed by mechanical trituration as described previously 77,78 . Dissociated cells were filtered through nylon mesh screening filters with a 52- ⁇ m pore size to efficiently remove debris. Cells were plated on polylysine-coated coverslips in DMEM/F12 with 10% FBS and then cultured at 37 °C in a 95% air and 5% CO2 incubator with a change of medium twice a week.
  • RGCs and astrocytes Primary cell co-culture (RGCs and astrocytes) Confluent astrocytes were trypsinized and re-seeded in 24-well plates and further incubated for 7 days for astrocyte–neuron co-cultures as described previously 79,80 . Primary cultures of RGCs were prepared from P1-P3 C57BL/6 mice.
  • Eyes were enucleated and retinas were dissected and placed in cold (4°C) DMEM with 100 U/ml Penicillin-Streptomycin. Retinas were cut into pieces and digested in 20 U/ml pre- warmed papain solution with 100 U/ml DNase I at 37°C for 15 min. An equal volume of 5 mg/ml ovomucoid was added to stop the reaction. Lysates were triturated several times and centrifuged at 3000 rpm for 5 min at 4°C. Cells were resuspended in 500 ⁇ l Attorney Docket No.00633-0381WO1/MEEI 2023-182 washing buffer (0.5% BSA, 2 mM EDTA in 1 ⁇ PBS).
  • micro-magnetic beads conjugated Thy1.2 (CD90.2) antibody (Invitrogen, 11443D) were added into cells and incubated at 4°C for 20 min.
  • a MACS magnetic separation system was used to isolate RGCs according to the manufacturer’s instructions.
  • the purified cells were centrifuged at 3000 rpm for 5 min at 4°C and re-suspended in culture medium (DMEM/F12 with 10%FBS, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml Penicillin-Streptomycin).
  • astrocytes were grown to confluency and washed with phosphate-buffered saline (PBS, pH 7.4). Resuspended RGCs were seeded onto the astrocyte layer. ß-III tubulin, BRN3Aand SMI32 antibodies were used to verify the purification of RGCs.
  • RGCs on the astrocyte layer were fixed with 4% paraformaldehyde for 10 min and then incubated with ß- tubulin. Images were taken on a SP8 confocal microscope and the length of neurites was measured using the FIJI distribution of ImageJ 81 .
  • Fluorescence ⁇ activated cell sorting Optic nerve head was collected from Gfap-tdTomato and Spp1 GFPfl/fl GfapCre mice and digested with 0.6 mg protein/ml papain (Worthington, LS003126), 0.012 mg/ml L-cysteine(Sigma, C7352) in HBSS (Ca 2+ and Ma 2+ free) (Gibco, 14185-052) for 15 min at 37 °C. After the incubation, tissues were centrifuged at 2000 rpm for 5 min and then removed papain/HBSS solution.
  • Tissues were resuspended in 10% horse serum (Gibco, 26050-088) and 60 U/ml DNase I (Sigma, D-5025) in HBSS and were triturated carefully with a heat polished Pasteur pipet (World Precision Instruments, TW150-4) until all visible chunks of tissue were completely dissociated. Dissociated cells were centrifuged and resuspended in HBSS. Cell suspensions were passed through a 35- ⁇ m cell strainer into 5 ml round bottom polystyrene test tube (Falcon, 352235) for further cell sorting.
  • Live, singlet tdTomato + cells were identified as astrocytes and sorted on a FACS Aria III cell sorter (BD Biosciences) directly into collection medium. Cells sorted by FACS were centrifuged at 1000g for 10 min and cell pellets were used for further RNA extraction. Fundus photography and optical coherence tomography Mice were anesthetized and their pupils were dilated. Fundus images were captured using a Micron III retinal imaging system (Phoenix Research Laboratories, Pleasanton, CA). Optical coherence tomography (OCT) was performed directly after fundus photography.
  • FACS Aria III cell sorter BD Biosciences
  • OCT optical coherence tomography
  • Anterior and posterior segment OCT images were obtained with Attorney Docket No.00633-0381WO1/MEEI 2023-182 a spectral domain OCT system (Bioptigen, Morrisville, NC). Averaged single B scans and volume scans were obtained with images centered on optic nerve head or the cornea, respectively. Electroretinography and optomotor reflex testing Pattern electroretinograms (PERG) were recorded from both eyes of light- adapted mice on a Celeris small animal testing system (Diagnosys LLC). PERG amplitudes were defined as the difference between the P1 peak and N2. Conventional, light-adapted ERG was recorded directly afterwards to ensure that reductions of PERG amplitudes were not due to unrelated ocular disease.
  • PERG Electroretinography and optomotor reflex testing Pattern electroretinograms
  • the optomotor reflex was tested in awake mice that were placed on a platform in the middle of an arena made from 4 computer monitors 82 .
  • Retro-orbital optic nerve crush and microbead occlusion model of glaucoma Mice were anesthetized and the intraorbital optic nerve was exposed by dissection of the conjunctiva 83 .
  • the nerve was clamped for 10 s in the myelinated region approximately 200 ⁇ m behind the sclera using self-closing forceps.
  • Polystyrene microbeads (15 ⁇ m diameter, Invitrogen, Carlsbad, CA, USA) were injected into the anterior chamber of the right eye through the cornea 31,84 . Control groups received an injection of sterile saline solution.
  • Intraocular pressure was measured in both eyes with a rebound tonometer (TonoLab, iCare, Espoo, Finland) under isoflurane anesthesia. All measurements were taken in the morning to minimize circadian variation of IOP.
  • Tissue preparation After euthanasia, the skull was opened, the brain was removed, and eyes and optic nerves were dissected from the surrounding tissue 85 and immediately fixed in 4% paraformaldehyde overnight. Brains were fixed separately. Eyes were hemisected along the ora serrata and the retinas were gently removed from the posterior eyecup. Four relieving cuts were made to facilitate whole-mounting of the tissue. Optic nerves were left intact and processed for sectioning.
  • tissue was designed for electron microscopy, perfusion fixation was performed directly after euthanasia with 5 ml heparinized saline solution, followed by 4% paraformaldehyde solution.
  • the tissue was dissected as described above and immediately post-fixed with 4% paraformaldehyde/0.8% glutaraldehyde in 0.5x sodium cacodylate buffer 2,32 .
  • Attorney Docket No.00633-0381WO1/MEEI 2023-182 Immunohistochemistry Retinas were mounted ganglion cell side-up on nitrocellulose membranes and stained for 3 days with primary antibodies (Key Resources Table).
  • Optic nerves or whole brains were cryoprotected, embedded in OCT compound and sectioned at 12 ⁇ m and 30 ⁇ m, respectively, in a Leica cryostat. Sections were incubated with primary antibodies overnight at 4°C. Primary antibodies were visualized with appropriate secondary antibodies conjugated with Alexa fluorophores (Jackson ImmunoResearch and Molecular Probes). DAPI were used to counterstain nuclei. Labeled sections were mounted with Vectashield (Vector Laboratories) and imaged by confocal microscopy. Confocal microscopy and transmission electron microscopy Confocal image stacks were taken on a Leica SP8 confocal microscope (Leica, Wetzlar, Germany).
  • the axons number per optic nerve was calculated by axonal density and optic nerve area. Axon counts were performed by an individual blinded to the genotype of the animals and the injury induced. cDNA synthesis and quantitative PCR Due to the small amount of tissue from the optic nerve head, three optic nerve heads were pooled for one sample of crushed and uncrushed eyes. One well of cultured astrocytes growing in 6-well plates was one sample. After total RNA extraction using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA), the purity of RNA were assessed using the NanoDrop 2000 (Thermo Fisher Scientific) and the RNA integrity was determined on the BioAnalyzer (Agilent Technologies, Santa Clara, CA).
  • RNA samples that had a 260/280 ratio > 1.8 and a RNA integrity number (RIN) higher than 5 (mean RIN of 7.37 ⁇ 0.67) were used for cDNA synthesis.
  • Ten ng of RNA from optic nerve heads were reverse-transcribed using the Ovation qPCR System (NuGen, San Carlos, CA), and the cDNAs were diluted 1:50 as templates for quantitative PCR.
  • GAPDH was used as a reference gene as the expression level in the optic nerve head is stable after optic nerve crush 26 .
  • RNA-sequencing Total RNA was extracted from cultured astrocytes isolated from either C57BL/6 wild-type or Spp1 KO neonatal mice using the RNeasy Plus Micro Kit (Qiagen; 74034). RNA purity and integrity was examined using the NanoPhotometer spectrophotometer (IMPLEN) and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies).
  • RNA concentration was measured with Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies). Libraries were prepared and sequenced by Novogene Sequencing was performed on an Illumina NovaSeq 6000 System, and 150-bp paired-end reads were generated.
  • Attorney Docket No.00633-0381WO1/MEEI 2023-182 Data analysis of RNA-seq Reads were aligned to the mouse reference genome (GRCm38) on the computer cluster of the Harvard Chan Bioinformatics Core (HBC) using the STAR software package. Gene-expression levels were calculated using the transcripts per kilobase of exon per million fragments mapped (FPKM). All statistical analyses were conducted using R version 4.0.5. Differential expression analysis were performed using the R package DESeq2.
  • astrocytes were plated onto 24-well culture plates for 3 days and incubated with pHrodo Green E. coli BioParticles (Invitrogen, P35366) for 4 hours. Phagocytic activity was determined based on the fluorescence intensity of the cells by confocal microscopy. Phagocytic activity was also measured by spectrophotometry. Plate astrocytes into a 96-well plate as experimental wells. Leave two wells empty of cells for every control well, so that a no-cell control background subtraction may be performed. Astrocytes were cultured for 24 h, and the culture medium was then replaced with 200 ⁇ l of pHrodo Green E.
  • coli BioParticles conjugate solution in experimental and no- cell control wells Cover and transfer the microplate to an incubator warmed to 37°C for 4 hours to allow phagocytosis and acidification to reach its maximum. Scan all experimental and no-cell control wells of the microplate in the fluorescence plate reader (BioTek Synergy H1 instrument). Phagocytic activity was determined based on relative fluorescence intensity of pHrodo particles in astrocytes.
  • oligomycin an ATP synthase inhibitor
  • FCCP carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
  • BCA bicinchoninic acid
  • Plasmid GfaABC1D-cyto-Ruby3-iATPSnFR1.0 was developed by the laboratory of Baljit Khakh and expresses cytoplasmic ATP sensor in astrocytes with red reference protein 44 .
  • Plasmid #102554 was purchased from Addgene. Bacteria containing the plasmid was amplified in LB medium with ampicillin and the plasmid was extracted using the EndoFree Plasmid Maxi Kit (QIAGEN, #12362). Astrocytes at 70–90% confluency were transfected with the plasmid and Lipofectamine 3000.
  • the transfection of a single well of a 24-well dish was as following: 1 ⁇ g of DNA and 2 ⁇ l P3000 were added to one tube containing 50 ⁇ l of Opti-MEM and 3 ⁇ l of Lipofectamine 3000 was added to the other tube containing 50 ⁇ l of Opti-MEM.
  • the content of the two tubes was mixed and incubated for 20 min at room temperature, and 400 ⁇ l of fresh complete medium were added into the DNA-lipid complexes mixture before adding the mixture to astrocytes. The medium was changed the following day. Cells were incubated for 2–4 days at 37°C before fixation with 4% PFA. Images were taken on SP8 confocal microscope.
  • siRNA knockdown CD44 siRNA (165814), Integrin alpha 5 siRNA (67718), Vdac1 siRNA (187532) and Silencer® Negative Control #1 siRNA (AM4611) were obtained from Thermo Fisher Scientific. siRNA were resuspended at 50 ⁇ M. Lipofectamine 3000 transfection reagent (ThermoFisher Scientific) was used to transfect siRNA into astrocytes according to the manufacturer’s instructions. Astrocytes were grown in DMEM/F-12 complete media with siRNA (50 nM) and incubated for 24-72 h after treatment. Reagents are summarized in the Key Resources Table.
  • astrocytes were plated onto 24-well culture plates for 3 days, and incubated with pHrodo Green E. coli BioParticles (Invitrogen, P35366) for 4 hours. Phagocytic activity was determined based on the fluorescence intensity of the cells by using a BioTek instrument.
  • AAV2-mediated gene transfer The coding region of Spp1, followed by a cleavable F2A linker region and the EGFP gene was synthetized (Integrated DNA Technologies, Coralville, IA), inserted into the AAV2 backbone plasmid, sequenced, and used to generate the virus (1.41E12 GC/ml, Viral Vector Core, MEEI).
  • AAV2 expressing only EGFP served as a control. In both constructs, gene expression was driven by the CMV promoter.
  • One ⁇ l of AAV2-Spp1-EGFP or control virus was injected intravitreally, and transfection efficiency was monitored by fundus photography and immunohistochemistry.
  • SPP1 PODS slow-release SPP1 protein
  • SPP1 was up-regulated in most datasets, and participated in a network containing TGF- ⁇ 1 and at least the transcription factors RUNX1 and E2F1, which regulated SPP1 expression, and were also up-regulated in glaucoma and optic nerve injury 27-29 .
  • BRN3A is a common marker of RGCs in healthy and glaucomatous retinas 32,33 , there is the possibility that it is down-regulated in disease 34 , and that a lack of BRN3A staining indicates dysfunctional cells rather than the complete disappearance of the cells after apoptosis 35,36 . Therefore, we counted ganglion cell axons in the optic nerve after PPD staining. As expected for young animals, there was no difference at baseline between wild-type C57BL/6 and Spp1 KO mice, and both groups lost axons after induction of elevated IOP (Figure 1G). There was a significant difference between the wild-type and the Spp1 KO mice, which lost more axons ( Figures 1G).
  • B6.GFAP-cre a strain expressing cre recombinase under the control of the astrocyte-specific GFAP promoter, with a tdTomato reporter strain (Ai14) and collected tdTomato-labeled astrocytes by FACS.
  • Tgf- ⁇ 1, Runx1, E2f1, and Spp1 were up-regulated in astrocytes in vivo after elevation of IOP and optic nerve crush compared to na ⁇ ve control nerves.
  • Spp1 GFPfl/fl mice were used as controls; and they lost about 32% of their RGCs over a period of 16 months, the expected rate in mice on the B6 background, but the cell loss in the Spp1 cKO strain Spp1 GFPfl/fl GfapCre was significantly worse over the same time period (44% in Spp1 cKO vs 32% in control, Figures 3C-3E). Similarly, after induction of elevated IOP, the Spp1 cKO mice did worse in terms of ganglion cell survival and visual function ( Figures 3F-3I). We also followed the timeline of RGC loss after optic nerve crush in the cKO and the control strain.
  • Astrocytes are the major glial component of the unmyelinated optic nerve head (ONH), but microglial cells are also present in the nerve and the retina, and Müller glia are abundant in the retina. In some injuries, Müller glia are known to up- regulate GFAP, thus Gfap-driven cre may be active in Müller cells.
  • ONH unmyelinated optic nerve head
  • Müller glia are known to up- regulate GFAP, thus Gfap-driven cre may be active in Müller cells.
  • Spp1 KO astrocytes filled the gap faster than wild-type astrocytes, however, this may be due primarily to the increased cell proliferation of the KO cells.
  • a BrdU incorporation assay showed that 10% of Spp1 KO astrocytes incorporated the BrdU label versus 5% of wild-type cells. Since these experiments relied on cultured astrocytes, we verified the results with an analysis of freshly isolated optic nerve head SPP1 + astrocytes from Gfap- tdTomato and SPP1- astrocytes from Spp1 GFPfl/fl GfapCre (cKO) mice (Figure 4G).
  • Spp1 KO astrocytes showed an “A1” type with up-regulation of A1 specific genes and down-regulation of most A2 specific genes compared to wild-type nerves.
  • IOP elevation or crush injury there was no clear pattern indicative of either an “A1” or “A2” type in Spp1 + astrocytes or Spp1 KO astrocytes in vivo.
  • Spp1 deletion induces a neurotoxic phenotype in astrocytes in vitro and in vivo, mainly including activation of neuroinflammation and neurotoxic factors, and inhibition of synaptogenic, phagocytic, and neurotrophic processes.
  • Example 5 Spp1 depletion leads to impaired phagocytosis in astrocytes.
  • Astrocytes have been implicated in the degradation of axonal mitochondria, a process that involves the phagocytosis of axonal evulsions that contain damaged mitochondria by neighboring astrocytes 19 .
  • This process relies at least in part on LGALS3 (Galectin 3) 41 .
  • LGALS3 Galectin 3
  • Lgals3 mRNA is detectable at similar levels in uninjured nerves from wild-type and Spp1 KO mice, and it is up-regulated by an increase in IOP and an optic nerve crush injury in optic nerves of wild-type animals, only to a very limited extent in the knock-out (Figure 4M), suggesting Spp1 deficiency prevented the injury-induced increase in Lgals3 expression.
  • Spp1 deletion causes mitochondrial dysfunction Since oxidative phosphorylation was one of the major down-regulated pathways in Spp1 KO astrocytes ( Figure 5A) that affected genes complexes I, III, IV, and V of the electron transport chain, we used metabolic analysis to measure mitochondrial respiration in Spp1 KO versus wild-type astrocytes. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) ( Figures 5B and 5C) and basal and maximal respiration and ATP production were significantly lower in the Spp1 KO astrocytes ( Figures 5D-5F), indicating an impaired mitochondrial respiration in Spp1 KO astrocytes.
  • OCR Oxygen consumption rate
  • ECAR extracellular acidification rate
  • basal and maximal respiration and ATP production were significantly lower in the Spp1 KO astrocytes ( Figures 5D-5F), indicating an impaired mitochondrial respiration in Spp1 KO astrocytes.
  • TGF- ⁇ 1 positively regulated SPP1 and TGF- ⁇ 1 induced increased OCR, ECAR, basal and maximal respiration, and ATP production in cultured astrocytes, an effect that was blocked by the TGF- ⁇ 1 receptor blocker SB431542, the RUNX1 antagonist Ro5-3335, and the E2F1 antagonist HLM006474.
  • the TGF- ⁇ 1 receptor blocker SB431542 In electron microscopy of the unmyelinated portion of the optic nerve, Spp1 KO mice showed a high prevalence of axonal mitochondria with few or no cristae even at young ages (Figure 5H). Degenerating mitochondria were also present in the astrocytes, especially in aged animals ( Figures 5I and 5J).
  • Example 7 Exogenous recombinant SPP1 restores astrocytic gene expression.
  • rSPP1 Recombinant SPP1 protein significantly inhibited the expression of C3 in cultured C57BL/6 astrocytes ( Figure 10A). Exogenous rSPP1 was also found to stimulate the expression of the astrocytes’ own Spp1 gene, an effect that was Attorney Docket No.00633-0381WO1/MEEI 2023-182 dependent on the SPP1 receptor Itg ⁇ 5, but not on the other known SPP1 receptor CD44 ( Figure 10B). We also found rSPP1 reversed the decreased expression of “A2” genes to almost normal level, but had little effects on the expression of “A1” genes in Spp1 KO astrocytes ( Figure 10C).
  • SPP1 also restored the expression of phagocytosis-related genes, neurotrophic factors, and genes related to oxidative phosphorylation and inhibited toxic factors C1q and C3 expression in Spp1 KO astrocytes ( Figures 10D and 10E). There was no further effect of rSPP1 on the expression of oxidative phosphorylation-related genes in wild-type astrocytes ( Figure 10F).
  • SPP1 up-regulates VDAC1
  • the Vdac1 (voltage-dependent anion channel 1) gene was significantly down- regulated in Spp1 KO astrocytes in our RNA-sequencing dataset, by qPCR, and in immunohistochemistry ( Figures 5Aand 6A-6C).
  • SPP1 and VDAC1 were co- expressed in cultured astrocytes.
  • Recombinant SPP1 led to a significant increase in the VDAC1 labeling intensity in cultured wild-type astrocytes and to a 50-fold increase in Vdac1 mRNA expression.
  • This effect was inhibited by knock-down of the SPP1 receptors CD44 and Itg ⁇ 5, and by inhibition of AP1, which is downstream in the SPP1 signaling pathway 43 ( Figures 6D-6F, qPCR validation for the siRNAs was also performed).
  • TGF- ⁇ 1 increased the expression of Vdac1 mRNA and the effect was prevented by RUNX1 and E2F1 inhibition ( Figure 6G).
  • SPP1 increases intracellular ATP concentration via VDAC1. Since VDAC1 allows the diffusion of ATP from the mitochondria into the cytoplasm, we asked if SPP1 administration leads to an increase in cytosolic ATP concentration via VDAC1.
  • cytosolic ATP in astrocytes by transfection with a plasmid encoding the ATP sensor iATPsnFR and mCherry tag 44 .
  • the ATP sensor increased in fluorescence upon binding ATP (Figure 6R).
  • Exogenous rSPP1 led to an increase in cytosolic fluorescence, indicating that SPP1 not only increased mitochondrial ATP production but also promoted its delivery into the cytosol.
  • Attorney Docket No.00633-0381WO1/MEEI 2023-182 Increased fluorescence of the ATP sensor induced by rSPP1 was blocked by Vdac1 knockdown, suggesting that SPP1 promoted ATP delivery into the cytosol through up-regulation of VDAC1 ( Figures 6R and 6S).
  • Example 10 Astrocytic SPP1 enhances neurite growth and branching in retinal ganglion cells in a VDAC1-dependent manner.
  • AAV2-mediated overexpression of SPP1 would have an anti-aging effect.
  • AAV2 is not specific to astrocytes, but we reasoned that as a secretory protein, SPP1 from any source may be beneficial.
  • AAV2-Spp1-EGFP AAV2-Spp1 for short.
  • Age-, strain-, and sex-matched control animals received AAV2-EGFP.
  • ERG and visual acuity of the mice were measured, and RGCs were counted ( Figure 7A).
  • SPP1 PODS SPP1-polyhedrin complexes
  • TGFbeta and BMP-2 activation of the OPN promoter roles of smad- and hox- binding elements.
  • the microbead occlusion model a paradigm for induced ocular hypertension in rats and mice.
  • a small peptide antagonist of the Fas receptor inhibits neuroinflammation and prevents axon degeneration and retinal ganglion cell death in an inducible mouse model of glaucoma.
  • Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481-487.10.1038/nature21029. 41.
  • BDNF Contributes to Spinal Long- Term Potentiation and Mechanical Hypersensitivity Via Fyn-Mediated Phosphorylation of NMDA Receptor GluN2B Subunit at Tyrosine 1472 in Rats Following Spinal Nerve Ligation.
  • Hernansanz-Agustin P., Choya-Foces, C., Carregal-Romero, S., Ramos, E., Oliva, T., Villa-Pina, T., Moreno, L., Izquierdo-Alvarez, A., Cabrera- Garcia, J.D., Cortes, A., et al. (2020). Na(+) controls hypoxic signalling by the mitochondrial respiratory chain. Nature 586, 287-291.10.1038/s41586-020-2551-y. 89.
  • SLC25A51 is a mammalian mitochondrial NAD(+) transporter. Nature 588, 174-179. 10.1038/s41586-020-2741-7. 90. Li, D., Liu, N., Zhao, H.H., Zhang, X., Kawano, H., Liu, L., Zhao, L., and Li, H.P. (2017).

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Abstract

L'invention concerne des compositions et des méthodes de traitement d'une maladie neurodégénérative oculaire chez un sujet, comprenant l'administration à un oeil affecté du sujet d'une quantité thérapeutiquement efficace d'une composition comprenant une protéine phosphoprotéine 1 sécrétée (SPP1) ou un acide nucléique.
PCT/US2023/085619 2023-02-26 2023-12-22 Phosphoprotéine sécrétée 1 (spp1) utilisée en tant que thérapie neuroprotectrice dans une maladie neurodégénérative Pending WO2024177720A1 (fr)

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* Cited by examiner, † Cited by third party
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JP2025172257A (ja) * 2024-05-11 2025-11-21 首都医科大学宣武医院 抗老化におけるｒｏ ５－３３３５の応用

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US20100216651A1 (en) * 2007-02-28 2010-08-26 National University Corporation Kyoto Institute Of Technology Viral polyhedra complexes and methods of use
US20200277570A1 (en) * 2017-04-13 2020-09-03 Yuka MATSUZAKI Delivery method
WO2021148653A1 (fr) * 2020-01-22 2021-07-29 Ucl Business Ltd Procédé de diagnostic

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Publication number Priority date Publication date Assignee Title
US20100216651A1 (en) * 2007-02-28 2010-08-26 National University Corporation Kyoto Institute Of Technology Viral polyhedra complexes and methods of use
US20200277570A1 (en) * 2017-04-13 2020-09-03 Yuka MATSUZAKI Delivery method
WO2021148653A1 (fr) * 2020-01-22 2021-07-29 Ucl Business Ltd Procédé de diagnostic

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LI SONG, JAKOBS TATJANA C.: "Secreted phosphoprotein 1 slows neurodegeneration and rescues visual function in mouse models of aging and glaucoma", CELL REPORTS, ELSEVIER INC, US, vol. 41, no. 13, 1 December 2022 (2022-12-01), US , pages 111880, XP093206897, ISSN: 2211-1247, DOI: 10.1016/j.celrep.2022.111880 *

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JP2025172257A (ja) * 2024-05-11 2025-11-21 首都医科大学宣武医院 抗老化におけるｒｏ ５－３３３５の応用

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