Compositions and methods for the neuroinflammatory stimulation of microglia against neurodegenerative diseases

Abstract

The present disclosure describes, compositions and methods comprising a recombinant, chimeric poliovirus construct for the neuroinflammatory stimulation of microglia against neurodegenerative diseases. The compositions may include a chimeric poliovirus and a therapeutic agent capable of binding to protein aggregates associated with the neurodegenerative disease. Methods of treating neurodegenerative diseases and methods of activating microglia are also provided.

Description

[0001] COMPOSITIONS AND METHODS FOR THE NEUROINFLAMMATORY STIMULATION OF MICROGLIA AGAINST NEURODEGENERATIVE DISEASES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 660,602 filed on June 17, 2024, the contents of which is incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant no. NS 108773 awarded by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (NIH / NINDS). The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (15555400785.xml; Size: 9, 160 bytes; and Date of Creation: June 16, 2025) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Microglia maintain brain homeostasis by removing neuron-derived components such as myelin and cell debris. The evidence linking microglia to neurodegenerative diseases is growing; however, the precise mechanisms remain poorly understood. It is believed that microglia play a role in neuroprotection by clearing neuron-released a- synuclein. a-synuclein is produced primarily in neurons and constitutes up to 1% of total cytosolic protein in the brain. Neuronal a-synuclein activates microglia, which in turn engulf a-synuclein into autophagosomes for degradation via selective autophagy (termed synucleinphagy). Failure of immune surveillance / tissue homeostasis in the central nervous system (CNS) leads to toxic accumulation of aggregated a-synuclein. At the heart of the problem is an insufficiency of microglia (the tissue resident macrophage population in the brain) of clearing such aggregates. The accumulation of these a-synuclein aggregates is a major component in intraneuronal inclusions known as Lewy bodies (LB) associated with neurodegenerative diseases, such as Parkinson’s disease (PD), dementia with Lewy body (DLB), Alzheimer’s disease and the like. There is a need for new and novel treatments for these neurodegenerative diseases.

[0010] SUMMARY

[0011] The present disclosure describes, compositions and methods comprising a recombinant, chimeric poliovirus construct for the neuroinfl ammatory stimulation of microglia.

[0012] One aspect of the present disclosure provides a method of treating a neurological disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant, chimeric poliovirus construct. In some embodiments, the recombinant, chimeric poliovirus construct comprises a Sabin type I strain of poliovirus with a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the poliovirus' 5' untranslated region between the poliovirus' cloverleaf and the poliovirus' open reading frame of the Sabin type I poliovirus; and the recombinant, chimeric poliovirus construct is administered intracerebrally. In some embodiments, the neurological disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is associated with cerebral protein aggregates. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, dementia with Lewy bodies (DLB), frontotemporal diseases, and multiple system atrophy. In some embodiments, the neurological disease is not a tumor or is not associated with a tumor. In some embodiments, the protein aggregates are amyloid plaques. In some embodiments, the method further comprises administering a therapeutic agent capable of binding to the aggregated proteins and microglia. In some embodiments, the therapeutic agent is an antibody or affinity reagent comprising an Fc region of an antibody and an affinity region capable of binding to the protein aggregates. In some embodiments, the antibody or affinity reagent is an anti-amyloid antibody. In some embodiments, the method decreases cerebral protein aggregates. In some embodiments, the symptoms of the neurological disease are reduced or the progression of the neurological disease is slowed as compared to a subject not treated or prior to treatment.

[0013] Another aspect of the present disclosure provides a composition for the treatment of a neurological disease, the composition comprising a recombinant, chimeric poliovirus construct and a therapeutic agent capable of binding to cerebral protein aggregates and marking these protein aggregates for uptake by microglia. The therapeutic agent may be an affinity reagent with an Fc region and an affinity region capable of binding to the protein aggregates. The antibody or affinity reagent may be an anti-amyloid antibody. In some embodiments, the composition is formulated for intracerebral administration.

[0014] Another aspect of the present disclosure provides a method of activating microglia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant, chimeric poliovirus construct to activate the microglia of the subject wherein the recombinant, chimeric poliovirus construct is administered intracerebrally; and wherein the subject does not have a central nervous system tumor. In some embodiments, the subject in need is diagnosed with or suspected of having a neurological disease. In some embodiments, the method increases microglia with ameboid morphology. In some embodiments, the method increases microglia phagocytosis. In some embodiments, the method increases markers of microglia activation, wherein the markers comprise at least one of RAC 1, CD40, CD86, CD68, or MHCII. In some embodiments, the method further comprises administering to the subject a therapeutic agent capable of binding to cerebral protein aggregates. In some embodiments, the therapeutic agent is an antibody or affinity reagent comprising an Fc region of an antibody and an affinity region capable of binding to the protein aggregates. In some embodiments, the antibody or affinity reagent is an anti-amyloid antibody.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0018] Figure 1. Intratumor (IT) PVSRIPO treatment of murine CT2A glioma shifts resting (“ramified”) microglia shape to activated (“ameboid”) morphology in peritumoral brain, (la-b, e) hCD155-tg mice received stereotactic CT2AhCD155-mCherryimplants (7d) and single IT PVSRIPO infusion [5 x 107plaque forming units (PFU)] before euthanasia (48h post PVSRIPO). Mice were perfused with ice-cold PBS and their brains were removed for immunofluorescence (IF) analysis. The anatomical context of brain areas of interest emerging from our studies is shown: a stereotactic tumor implantation site (central caudate putamen), and the approximate locations of a corresponding contralateral zone and of peritumoral brain are indicated. The anterior commissure is used for orientation; see Yang et al.1for comprehensive description of our stereotactic injection approach, (a, b) Peritumoral brain from mock (a) and PVSRIPO- (b) treated fresh frozen samples were cryo-sectioned (13mm) coronally, formalin-fixed, and stained with IF as shown. The two images shown were used for assessing microglia morphological features in two conditions (mock vs. PVSRIPO). (c) Heatmap of microglial morphological features clustering in microglial subtypes (“rod-like”, “ameboid”, “ramified”, “hypertrophic”) indicated by Z- Score-normalized values across all cells from panels a, b using MicrogliaMorphology ImageJ macro and MicrogliaMorphologyR.2Unsupervised hierarchical clustering of individual microglia based on morphological features extracted from MicrogliaMorphology ImageJ macro and clustering using MicrogliaMorphologyR is shown, (d) Pilot quantification of microglial morphology subtypes in mock vs. PVSRIPO- treated CT2A-bearing brain using the singular examples in panels a, b. (e) To systematically interrogate microglia shape upon PVSRIPO treatment of mouse glioma, we analyzed similar brain areas (corresponding to “peritumoral brain”) from tumor / treatment naive mice (left), CT2A glioma-bearing mice infused with mock (middle), or CT2A glioma-bearing mice infused with PVSRIPO (right). Brains were processed as in panels a, b and stained for TMEM119. (f, g) Quantification of the distribution of microglial morphology subtypes in the brains of mice described for panel e. The brains of two animals from each group were processed, and three adjacent fields from similar brain areas (corresponding to “peritumoral brain”) were analyzed in all of them. Panel f compares subtype distribution in naive vs. (PVSRIPO-treated) tumor-bearing brains, revealing a significant, -55% shift of (resting) ramified to (activated / phagocytic) ameboid shape in the latter. Also, the proportions of rod-like and hypertrophic microglia significantly increased in the latter. Panel g compares PVSRIPO-treated to mock-treated glioma-bearing brains revealing a similarly significant, -50% shift of ramified to ameboid microglia with a minor decrease in the proportion of hypertrophic microglia.

[0019] Figure 2. PVSRIPO elicits sustained viral (v)RNA replication in (hCD155-tg) mouse microglia in vitro and in vivo, triggering type-I IFN signaling responses, (a-c) Enrichment and phenotypic characterization of (CD1 lb+) microglia after CD1 lb+magnetic bead isolation from brain tissue single cell suspensions from mice (a) and humans (b, c). Flow cytometry before and after CDl lb+bead pull down revealed isolation of -93.5% CD45lo / med, CD1 lb+cells (microglia) from mouse brain. Flow cytometry of CD1 lb+bead- isolated microglia from human brain (surplus epilepsy surgery tissue) isolated CD45lo / medcells that were -95% Cx3crl -97.5% CDl lb1and -96% MERTK1(b). Homogeneous positive staining for TMEM119 confirmed microglial identity of CDl lb+bead-isolated microglia from human brain (c). (d) PVSRIPO infection directs vRNA replication in murine and human microglia in vitro and in vivo. Mouse (left panel) or human (right panel) CD l lb bead-isolated microglia were cultured and infected with mock / PVSRIPO (MOI 10) for the indicated intervals / hrs post infection (hpi); tumor-naive brains injected with mock / PVSRIPO as described in Figure 1 were used for CD1 lb+bead isolation of microglia at the indicated hpis. Cultured microglia or microglia isolated from ipsi- / contralateral brain hemispheres were processed for total RNA isolation and RT-qPCR of (+) strand vRNA in PVSRIPO-treated samples vs. mock, (e) PVSRIPO infection elicits the formation of (-) / (+) (s)strand vRNA+viral RNA replication organelles in human / mouse microglia. Hybridization chain reaction (HCR)-fluorescent in situ hybridization (FISH) reactions conducted on human CD1 lb+bead-isolated microglia show (-) and (+)strand vRNA at the indicated intervals. Note the transition of detection of (-)strand vRNA (the replication intermediate generated first) to (+)s vRNA progeny [generated by copying (+)strand vRNA templates], (f) HCR RNA-FISH and IF conducted on (tumor) naive mouse brains infected with IT PVSRIPO (5.0 x 107pfu) (96hpi) and processed as described in Figure 1 (top row). Tissue in the area of the stereotactic injection site [corresponding to “approx, tumor location” (Fig. 1)] was analyzed. Note the formation of characteristic (-)strand vRNA+replication organelles in TMEM119+microglia, (g) Widespread, global type-I IFN signaling in TMEM119+microglia upon PVSRIPO infection of (tumor) naive mouse brains (middle row; in the same area as in the top row samples). We observed the formation of abundant microglial ISG15+clusters / nodules (bottom row), which were disseminated throughout the brain, in areas distant from the virus inoculation site.

[0020] Figure 3. Mouse and human microglia show elevated activation phenotypes and phagocytic activity after PVSRIPO infection, (a) Mouse microglia isolated and cultured as shown above were infected with PVSRIPO (MOI 10) or mock (96h) prior to co-culture with UV-irradiated, Cell Trace Violet (CTV)-labeled CT2A mouse glioma cells for the intervals shown. Cells were harvested and analyzed by flow cytometry. Microglia were gated as CD45lo / medCDl lb+; the percentages of CTV+microglia are shown, (b) HCRRNA- FISH and IF conducted on mouse microglia reveal (+)strand vRNA+microglia engulfing (24hpi) and phagocytosing (48hpi) CTV1glioma cells leading to the appearance of break up CTV+material >72hpi. (c) Human microglia were interrogated similar to mouse microglia described in panels a, b (human CTV+M059J glioma cells were used as bait), (d) Total RNA isolated from human microglia infected with mock / PVSRIPO (96h) and co-cultured with CT2A cells for the indicated intervals was analyzed by RT-qPCR for the poliovirus pattern recognition receptor (MDA5), the type-I IFN signaling response (IRF7) and for the microglia activation / phagocytosis marker TREM2.

[0021] Figure 4. Induction of microglia phagocytic activity in the glioma-bearing brain and tumor antigen cross presentation by PVSRIPO infection, (a-d) Mice were implanted with CT2AmCherrygliomas (7d) as described in Figure 1, treated with single IT PVSRIPO (5 x 107pfu) (n=2) or mock (n=l), euthanized after treatment (48h), and brains were processed for flow cytometry and IF as described above. Microglia phagocytosis (CD45lo / medCdl lb+CT2AmCherry+) 48h after IT PVSRIPO vs. mock was assessed by flow cytometry (a). Almost 50% of microglia isolated by CDl lb+bead pull down stained positive for the CTV tracer (left panel) in the PVSRIPO-treated mice (a, b) with isolation of CTV+microglia decreasing thereafter (b); this was associated with robust induction of antigen presenting cell activation markers MHC II (middle panel), CD86 (right panel) and MERTK (c). (d) In vivo phagocytosis of CT2Amcl'cr'v' mouse glioma by microglia after IT PVSRIPO was visually evident by IF. (e) To test cross presentation of tumor antigens after PVSRIPO-induced phagocytic uptake, microglia were infected in vitro with mock or PVSRIPO (MOI 10) and co-cultured with UV-irradiated B16OVAmelanoma cells for the intervals shown. At the indicated time points cells were harvested and analyzed by flow cytometry with antibody specific to the H-2Kb(MHC I):SIINFEKL (OVA epitope) complex (right panels). PVSRIPO-induced phagocytic uptake / tumor antigen cross presentation (e; left top panel) was associated with TREM2 induction (e; left bottom panel).

[0022] Figure 5. PVSRIPO promotes microglia tumor antigen cross presentation at levels superior to poly(I:C) and IFNg; in vivo evidence for microglia: CD8+T cell interactions induced by PVSRIPO. (a, b) Mouse microglia were treated in vitro with mock, transfected poly(I:C) (lOng / mL), IFNg (200u / mL), PVSRIPO (MOI 10) (96h) at the same time of initiating co-culture with UV-irradiated CTV-labeled B16OVAcells (for the intervals shown). Cells were harvested and analyzed by flow cytometry for phagocytic uptake of CTV (a) and SIINFEKL cross presentation (b; the proportion of CTV microglia cross presenting H-2Kb: SIINFEKL is shown over time); flow cytometric plots (bottom row) show CTV+microglia cross presenting H-2Kb: SIINFEKL at 72h after the various treatments, (c) Mice were implanted with CT2AmChe,Ty(7d), treated with single IT PVSRIPO (5 x 107PFU), euthanized (48h), and their brains were processed for IF as described above. TMEM119+microglia, infiltrating CD8+T cells and microglia:CD8+T cell engagement (yellow arrows) is shown in an overview (top row) and in detail (insert; bottom row).

[0023] Figure 6. Microglia exhibit robustly enhanced phagocytosis of Ab(l-42) amyloid plaques upon intracerebral PVSRIPO treatment, (a-c) Pre-aggregated Hi Lyte 488 Ab(l- 42) peptide was injected into the dorsal hippocampus of hCD155-tg mice followed by 7d of plaque aggregation in 9 mice, each, treated with control (vehicle) or PVSRIPO using the same stereotactic coordinates. Mice were euthanized at the indicated intervals (n=3 control s / PVSRIPO at 24, 48, and 72hpi, respectively), the brains were collected and analyzed by flow cytometry and sectioned for IF verification of Ab plaque formation (a). CD45lo / medCDl lb+microglia were analyzed by flow cytometry for determining the proportions of CD86+cells (top panels) and Cx3crl+Hi Lyte 488 Ab(l-42)+cells (bottom panels) in one mock / PVSRIPO-treated animal each (48hpi; b). (c) Mice were euthanized at the indicated intervals (n=3 control s / PVSRIPO for each interval) and the Hi Lyte 488 Ab(l-42) Normalized Mean Fluorescence Activity (MFI) in the microglia compartment was calculated by subtracting mock control from PVSRIPO MFI values for microglia at each time interval up to 72h. To assess the effects of intracerebral PVSRIPO in a model with more substantial disease burden, pre-aggregated Hi Lyte 488 Ab(l-42) peptide was injected into the caudal putamen (see Figure 1) of hCD155-tg mice followed by 28d of plaque aggregation and mock vs. PVSRIPO treatment in cohorts of four mice, each (d-i). The proportion of Hi Lyte 488 Ab(l-42)+microglia from animals treated with PVSRIPO (n=4; d; bottom) was significantly increased compared to the mock controls (n=4; d; top) (e). This was associated with significant induction of the phagocytosis / microglia shape determinant RAC1 in the PVSRIPO-treated cohort (f),3and induction of the activation / maturation markers CD40, CD86 and CD68 (g; representative findings from 1 animal in each cohort are shown), (h, i) Flow cytometry with gating of Cx3crlhland CD1 lb microglia isolated from the mock and PVSRIPO cohorts (n=4 each) revealed ~6- fold expansion of a microglia subset (11.7% of cells in PVSRIPO-treated brains; h). This microglia subset exhibited MHC II induction (i). j) data showing cytokine secretion of PVSRIPO-infected microglia (fold-induction PVSRIPO vs. mock). This cytokine secretion panel demonstrates proinflammatory engagement of microglia after PVSRIPO infection, underpinning the effects shown in Figures A-I.

[0024] DETAILED DESCRIPTION

[0025] The present disclosure is based, in part, on the discovery by the inventors of a unique innate immune stimulatory phenotype in myeloid cells upon stimulation with a profoundly attenuated, chimeric viral construct. Without wishing to be bound by any one theory, the inventors have demonstrated that administration of a recombinant viral construct can replicate inside of the microglia without production of progeny virus to activate microglia cells which increases their phagocytic activity.

[0026] One aspect of the present disclosure provides a method of treating a neurological disease, disorder or physiological condition in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an attenuated chimeric poliovirus construct to the subject such that the neurodegenerative disease, disorder, or physiological condition is treated or prevented in the subject. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the neurological disease. In some embodiments, the subject is responsive to methods and or compositions disclosed herein and include use in combination with one or more additional therapeutic agents. The term "treat" further includes the reduction in one or more symptom associated with neurological diseases or neurodegenerative disorders.

[0027] The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. As used herein, “subject” or "patient" refers to both mammals and non-mammals. “Mammals” include any member of the class Mammalia, such as humans, non-human primates (e.g., chimpanzees, other apes and monkey species), farm animals (e.g., cattle, horses, sheep, goats, and swine), domestic animals (e.g., rabbits, dogs, and cats), and laboratory animals (e.g., rats, mice, and guinea pigs). The term “subject” does not denote a particular age or sex. In one embodiment, the subject is a human.

[0028] In some embodiments, an attenuated chimeric poliovirus construct is used. In some embodiments the attenuated chimeric poliovirus construct comprises a Sabin type I strain of poliovirus with a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the poliovirus' 5' untranslated region between the poliovirus' cloverleaf and the poliovirus' open reading frame of the Sabin type I poliovirus. In some embodiments, the chimeric vial construct is a highly attenuated, non-cytopathogenic polio:rhinovirus chimera, known as PVSRIPO.

[0029] PVSRIPO is a recombinant, profoundly attenuated poliovirus (also referred to herein as “chimeric poliovirus”). PVSRIPO consists of the live attenuated type 1 (Sabin) PV vaccine containing a foreign internal ribosomal entry site (IRES) of human rhinovirus type 2 (HRV2) (see Gromeier et al., PNAS 93: 2370-2375 (1996); Dobrikova et al., J Virol 86:2750-2759 (2012); Dobrikov et al., mBio 13:e0085422 (2022); Dobrikov et al., mBio 14:e:019523 (2023) and United States Patent No. 6,264,940, both of which are incorporated by reference in their entirety; SEQ ID NO: 1). The IRES is a cis-acting genetic element located in the 5’ untranslated region of the poliovirus genome, mediating viral, m7G-cap-independent translation. The anti-tumor effects of PVSRIPO comprise direct, virus-mediated cytopathogenic or inflammatory events in cancerous cells; and — more importantly — profound proinflammatory and immunogenic events in myeloid cells (either in myeloid cells associated with a tumor, or tissue-resident myeloid cells in bystander normal organs). PVSRIPO takes advantage of its marked tropism for infection and proinflammatory engagement of solid cancers mediated by natural ectopic overexpression of the human poliovirus receptor, CD 155 (also known as Nectin-like molecule 5 (Necl-5)) on the surface of tumor cells (see U.S. Patent No. 11, 506,666, which is incorporated by reference in its entirety); and natural expression of CD155 in all cells of monocytic lineage, eg. monocytes, macrophages, dendritic cells, and microglia. PVSRIPO is administered directly to a tumor site and the virus is likely not capable of spreading to tumors distant to the site of intratumoral administration, U.S. PatentNo. 10,398,743, which is incorporated by reference in its entirety, describes use of PVSRIPO in treatment of primary tumor by intratumoral administration to the primary tumor.

[0030] Inducers of innate immunity may also be used in the present disclosure with or in place of PVSRIPO. These include without limitation imiquimod, polyinosinic- polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC), Stimulator of interferon genes (STING) agonist, Toll-like receptors (TLR) agonist, and cytokine-inducible SH2-containing protein (CISH).

[0031] This disclosure demonstrates that PVSRIPO targets microglia for infection (by virtue of the poliovirus receptor CD 155 expressed in them) and induces activation upon sensing of viral RNA by the pattern recognition receptor MDA5. As shown herein PVSRIPO is able to cause innate inflammatory activation in microglia with increased phagocytosis activity and TREM2 upregulation. Moreover, the innate response to PVSRIPO is sustained, lasting for weeks or months (at a minimum), due to ongoing, low- level, non-cytopathogenic viral RNA replication. Thus, PVSRIPO, represented as SEQ ID NO: 1 or highly homologous variants thereof capable of replicating the viral RNA in microglia cells are encompassed. The PVSRIPO construct may be SEQ ID NO: 1 or sequences with at least 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 1.

[0032] In some embodiments, the recombinant viral construct is administered intracranially. Intracranial administration includes intracerebral administration. Intracerebral administration refers to the direct delivery of therapeutic agents into the brain. This approach aims to bypass the blood-brain barrier. Intracerebral administration can be used to administer therapeutics to specific sites or areas of the brain and may allow for increased therapeutic concentration and or reduced systemic toxicity. Intracerebral administration includes Intracerebroventricular (ICV) Injection, intracerebral implants, Convection-Enhanced Delivery (CED), intracistemal administration, intraparenchymal administration, transmeningeal administration and intracarotid delivery. In some embodiments the recombinant, chimeric poliovirus construct may be administered one or more times as determined by one of skill in the art.

[0033] As used herein the term “effective amount” refers to the amount or dose of the compound that provides the desired effect. In some embodiments, the effective amount is the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. Suitably the desired effect may be reducing the symptoms or progression of a neurologic disease.

[0034] An effective amount can be readily determined by those of skill in the art, including an attending diagnostician, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. Appropriate dosages may be determined, for example, by extrapolation from animal studies or in clinical trials taking into account body weight of the patient, absorption rate, half-life, disease severity and the like. Suitable booster schedules may be determined by a skilled artisan. For example, the compositions may be given once, twice or yearly, or may be given in a series of booster schedule, for example, once a month, every other month, every 4 months, every 6 months, once a year, once every two years, and any range of time in between. Neurological diseases are conditions that affect the brain, spinal cord, and nerves, impacting physical, cognitive, emotional, and behavioral functions. In some embodiments, the neurological disease, disorder or physiological condition comprises a neurodegenerative disease. Neurodegenerative diseases are characterized by the progressive degeneration of central nervous system cells, including nerve cells, leading to the impairment of various functions including physical, cognitive, emotional, and behavioral functions. In some embodiments, the neurodegenerative disease is associated with cerebral protein aggregates. Protein aggregation, the clumping of proteins, is a hallmark of many neurodegenerative diseases. These aggregates, often misfolded proteins, can disrupt cellular function and lead to neurodegeneration. Specific proteins involved in these aggregates vary depending on the disease, but they often include amyloid-beta and tau in Alzheimer's disease, alpha-synuclein in Parkinson's disease, and polyQ expansions in Huntington's disease. Specific protein aggregates may also be referred to as plaques. Protein aggregation in neurodegenerative diseases may be the result of mis-expressed protein, mis-folded proteins, or lack of clearing of proteins. One embodiment of the present disclosure provides methods and compositions to increase the clearance of protein aggregates through phagocytosis by microglia. Another embodiment of the present disclosure provides methods and compositions to increase the clearance of any type of extracellular proteinaceous debris that fails to be removed spontaneously through intrinsic mechanisms, eg. those that involve glymphatic flow “washing” such materials out of brain tissue and / or uptake into microglia and other phagocytic cells in the CNS.

[0035] As is known in the art, a neurological diseases, disorders and / or physiological conditions is generally considered diseases, disorders and / or physiological conditions that affect the nervous system and require clinical care by a physician or other healthcare professional. The methods of the present disclosure can be used to prevent and / or treat a neurological disease, disorder, and / or condition. Suitable neurological diseases, disorders and / or physiological conditions include, but are not limited to, Absence of the Septum Pellucidum, Acid Lipase Disease, Acute Disseminated, Encephalomyelitis, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Goutieres Syndrome Disorder, Aicardi Syndrome, Alexander Disease, Alpers Disease, ALS Amyotrophic Lateral Sclerosis, Alternating Hemiplegia, Alzheimer’s Disease, Amyotrophic Lateral Sclerosis ALS, Anencephaly, Angelman Syndrome, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arteriovenous Malformation, Asperger Syndrome, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit Hyperactivity Disorder, Autism, Autism Spectrum Disorder, Back Pain, Barth Syndrome, Batten Disease, Behcet s Disease, Bell s Palsy, Benign Essential Blepharospasm, Binswanger s Disease, Brachial Plexus Injuries, Brain and Spinal Tumors, Brown Sequard Syndrome, CADASIL, Canavan Disease, Carpal Tunnel Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral, Cavernous Malformation, Cerebral Hypoxia, Cerebral Palsy, Cerebro Oculo Facio Skeletal Syndrome COFS, Charcot Marie Tooth Disease, Chiari Malformation, Chorea, Chronic Inflammatory Demyelinating Polyneuropathy CIDP, Chronic Pain, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Myasthenia, Congenital Myopathy, Corticobasal Degeneration, Craniosynostosis, Creutzfeldt Jakob Disease, Cushing s Syndrome, Dandy Walker Syndrome, Deep Brain Stimulation for Parkinson s Disease, Dementia, Dementia With Lewy Bodies, Dermatomyositis, Developmental Dyspraxia, Diabetic Neuropathy, Dravet Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dyssynergia Cerebellaris Myoclonica, Dystonias, Empty Sella Syndrome, Encephalitis Lethargica, Encephaloceles, Encephalopathy, Epilepsy, Erb Duchenne and Dejerine Klumpke Palsies, Essential Tremor, Fabry Disease, Fahr s Syndrome, Familial Periodic Paralyses, Farber s Disease, Febrile Seizures, Fibromuscular, Dysplasia, Foot Drop, Friedreich s Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses, Gerstmann s Syndrome, Gerstmann Straussler Scheinker Disease, Giant Axonal Neuropathy, Glossopharyngeal Neuralgia, Guillain Barre Syndrome, Headache, Hemicrania Continua, Hemifacial Spasm, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Herpes Zoster Oticus, Holmes Adie syndrome, Holoprosencephaly, Huntington s Disease, Hydranencephaly, Hydrocephalus, Hydromyelia, Hypersomnia,, Hypertonia, Hypotonia, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Neuroaxonal Dystrophy, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Isaac s Syndrome, Isaacs Syndrome, Joubert Syndrome, Kearns Sayre Syndrome, Kennedy s Disease, Kleine Levin Syndrome, Klippel Feil Syndrome, Klippel Trenaunay Syndrome KTS, Kliiver Bucy Syndrome, Krabbe Disease, Kuru, Lambert Eaton Myasthenic Syndrome, Landau Kleffner Syndrome, Learning Disabilities, Leigh s Disease, Lennox Gastaut Syndrome, Lesch Nyhan Syndrome, Leukodystrophy, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked In Syndrome, Machado Joseph Disease, Megalencephaly, Melkersson Rosenthal Syndrome, Meningitis and Encephalitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mitochondrial Myopathies, Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia Gravis, Myoclonus, Myopathy, Myotonia, Myotonia Congenita, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, Neurological Consequences of Cytomegalovirus Infection, Neurological Sequelae Of Lupus, Neuromyelitis Optica, Neuronal Migration Disorders, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Niemann Pick Disease, Normal Pressure Hydrocephalus, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Paraneoplastic Syndromes, Paresthesia, Parkinson s Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry Romberg, Pelizaeus Merzbacher Disease, Peripheral Neuropathy, Periventricular Leukomalacia, Pervasive Developmental Disorders, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post Polio Syndrome, Postural Tachycardia Syndrome, Primary Lateral Sclerosis, Progressive Multifocal Leukoencephalopathy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudotumor Cerebri, Psychogenic Movement, Rasmussen s Encephalitis, Refsum Disease, Repetitive Motion Disorders, Restless Legs Syndrome, Rett Syndrome, Reye s Syndrome, Sandhoff Disease, Schilder s Disease, Schizencephaly, Septo Optic Dysplasia, Shaken Baby Syndrome, Shingles, Sjogren s Syndrome, Sleep Apnea, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Muscular Atrophy, Stiff Person Syndrome, Striatonigral Degeneration, Stroke, Sturge Weber Syndrome, Subacute Sclerosing Panencephalitis, SUNCT Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syringomyelia, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay Sachs Disease, Tethered Spinal Cord Syndrome, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Todd s Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vasculitis Syndromes of the Central and Peripheral Nervous Systems, Von Hippel Lindau Disease VHL, Wallenberg s Syndrome, Wernicke Korsakoff Syndrome, Whiplash, Whipple s Disease, Williams Syndrome, Wilson Disease, Zellweger Syndrome and the like. In some embodiments, the neurological disease, disorder and / or physiological condition comprises a neurodegenerative disease. Neurodegenerative diseases are those neurological condition where nerve cells are damaged or die. Such disease include, but are not limited to, Parkinson’s Disease, Alzheimer’s Disease, and dementia with Lewy body (DLB).

[0036] In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, dementia with Lewy bodies (DLB), frontotemporal diseases, and multiple system atrophy. In some embodiments, the neurological disease is associated with protein aggregates. In some embodiments, the neurological disease and the neurodegenerative disease is not a tumor or is not associated with a tumor. For example, in some embodiments, the subject in need does not have a brain tumor.

[0037] In some embodiments, the aggregated proteins are amyloid plaques. Amyloid plaques are extracellular deposits of amyloid beta (A ) protein that present in the brain. Amyloid plaques are associated with Alzheimer's disease, Lewy body dementia, hereditary cerebral amyloid angiopathy, and some cases of amyloidosis.

[0038] In some embodiments, the methods may further comprise administering a therapeutic agent capable of binding to the aggregated proteins and microglia. For example, the method may comprise administering a therapeutically effective amount of a recombinant, chimeric poliovirus construct intracerebrally and a therapeutic agent capable of binding to the aggregated proteins and microglia.

[0039] A therapeutic agent is any substance administered to influence the outcome of a disease. In some embodiments, the therapeutic agent is an antibody or affinity reagent comprising an Fc region of an antibody and an affinity region which is capable of binding to the protein aggregates. By way of example and not limitation, the therapeutic agent may comprise an affinity region that is able to bind to an aggregated protein, and an Fc portion that is able to bind to a microglial cell, e.g., via the Fc receptor on the microglia cell.

[0040] In some embodiments, the antibody or affinity reagent is an anti -amyloid antibody. Anti-amyloid antibodies are antibodies designed to target and remove amyloid plaques in the brain. Anti-amyloid antibodies bind to A plaques which labels them for removal by immune cells, particularly microglia. Examples of anti-amyloid antibodies presently in clinical use include Aducanumab, Lecanemab, Donanemab. In some embodiments, the antibody or affinity reagent is an anti-tau antibody. Examples of anti-tau antibody include JNJ-6373365, BMS-986446, Bepranemab, ABBV-8E12, E2814, and Semorinemab. Other agents that directly target debris for phagocytic uptake (via Fc receptor) may also be used. Further, any therapeutic agent that aims for removal of extracellular debris, given that such debris can only be removed effectively through uptake into phagocytic cells, most pertinently, microglia may be included.

[0041] Other therapeutic agent suitable for use in the treatment of a neurological disease, disorder and / or physiological condition can be used and can be readily determined by one skilled in the art. Suitable examples of additional therapeutics include, but are not limited to, acetylcholinesterase inhibitors (e.g., Donepezil (Aricept1M’ Adlarity1M), rivastigmine (Exelon™), galatamine (Reminyl™, Razadyne ER™); Memantine (Namenda™); lecanemab (Leqembi™), risperidone, haloperidol, antidepressants, sleep aids, and the like. Other agents that indirectly target debris for phagocytic uptake, including the ACI-24 peptide-based vaccine which stimulates the production of antibodies that target Ap plaques may also be included.

[0042] Also included are non-pharmaceutical therapies / treatment regimens suitable for the treatment of a neurological disease, disorder and / or physiological condition. Treatment regimens may include, in addition to delivery of the chimeric poliovirus construct and therapeutic agent(s) include, but are not limited to, cognitive stimulation therapy, occupational therapy, cognitive rehabilitation, and the like.

[0043] As used herein, the term "administering" an agent, such as a therapeutic agent or composition described herein to a subject, is intended to refer to dispensing, delivering or applying the substance to the subject. In some embodiments, the therapeutic agent of the present disclosure may be administered systemically. The therapeutic agent may also be administered in the same manner as the recombinant, chimeric poliovirus (e.g. intracranially). Administration methods are well known to those skilled in the art and include, but are not limited to, transdermal administration, administration by inhalation, nasal administration, and parenteral administration, including injectable such as intravenously (IV), intramuscular administration, intradermal administration, and subcutaneous administration. The therapeutic agent of the present disclosure may be administered one or more times, separated by a period of time. For example, the antibody or affinity reagent may be administered once every week, once every two weeks, once a month for a time period as determined by one skilled in the art.

[0044] In some embodiments, the therapeutic agent of the present disclosure may be administered at the same time as the recombinant, chimeric poliovirus construct. Alternatively, the therapeutic agent of the present disclosure may be administered within days of the chimeric poliovirus construct. By way of example and not limitation, the chimeric poliovirus construct may be administered followed by an antibody or affinity reagent 30, 28, 21, 14, 10, 9, 8, 7, 6, 5, 4, 3 , 2, or 1 day(s) after administration of the chimeric poliovirus construct. The therapeutic agent may be administered via intracranial administration or via any other means of administration.

[0045] In some embodiments, the present disclosure provides a method of treating a neurological disease in a subject in need thereof, wherein the method decreases cerebral protein aggregates. As used herein, the term “decrease” or the related terms “decreased,” “reduce” or “reduced” refers to a statistically significant decrease. For the avoidance of doubt, the terms generally refer to at least a 10% decrease in a given parameter, and can encompass at least a 20% decrease, 30% decrease, 40% decrease, 50% decrease, 60% decrease, 70% decrease, 80% decrease, 90% decrease, 95% decrease, 97% decrease, 99% or even a 100% decrease (i.e., the measured parameter is at zero). In some embodiments, the protein aggregates are decreased in size. In some embodiments, the protein aggregates are decreased in number. In some embodiments, the protein aggregates are decreased in a specific location of the brain.

[0046] In some embodiments, the present disclosure provides a method of treating a neurological disease in a subject in need thereof, wherein the symptoms of the neurological disease are reduced or the progression of the neurological disease is slowed as compared to a subject not treated or prior to treatment. In some embodiments, one or more symptoms of the neurological disease are reduced including but not limited to memory loss, impaired thinking and judgement, language problems, disorientation, or executive dysfunction. Other symptoms include muscle and movement symptoms such as tremors, muscle rigidity, balance and coordination muscle fatigue, difficulty swallowing and breathing, changes in vision or hearing, mood changes, personality changes, behavioral changes, and changes in sleep. Other symptoms may include cellular or biomarker changes. For example, a decrease or loss of neurons, neuronal damage, an increase in the number or size of plaques, inflammation, brain atrophy and changes in autophagy or mitophagy. In some embodiments, the progression of the neurological disease is slowed as compared to a subject not treated or prior to treatment. Neurological symptoms and progression can be evaluated by one of skill in the art and through physical and neurological exams, lab test and brain imaging.

[0047] Another aspect of the present disclosure provides a composition for the treatment of a neurological disease. In some embodiments, the composition comprises a recombinant, chimeric poliovirus construct and a therapeutic agent capable of binding to cerebral protein aggregates. In some embodiments, the composition is formulated for intracranial administration. In some embodiments, the neurological disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is not a tumor or associated with a tumor. In some embodiments, the protein aggregates are amyloid plaques. In some embodiments, the therapeutic agent is an antibody or affinity reagent. In some embodiments the antibody or affinity reagent comprises an Fc region of an antibody and an affinity region capable of binding to the protein aggregates. In some embodiments, the antibody or affinity reagent is an anti-amyloid antibody. Another aspect of the present disclosure provides a method of activating microglia in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a recombinant, chimeric poliovirus construct to activate the microglia of the subject. In some embodiments, the subject does not have a central nervous system tumor. A central nervous system (CNS) tumor is also known as a brain tumor or a spinal cord tumor. In some embodiments, the subject in need is diagnosed with or suspected of having a neurological disease. In some embodiments, the neurological disease is associated with cerebral protein aggregates. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, dementia with Lewy bodies (DLB), frontotemporal diseases, and multiple system atrophy.

[0048] Microglia are a class of brain mononuclear phagocytes and have functions similar to those of other tissue macrophages, including phagocytosis, antigen presentation, and production of cytokines, chemokines, eicosanoids, complement components, matrix metalloproteinases (MMPs), oxidative radicals, and nitric oxide. Microglia also regulate brain development, maintenance of neuronal networks, and injury repair. Microglia are located throughout the brain and spinal cord of the CNS. Microglia can be activated in response to various stimuli, including infection, injury, and neurodegenerative diseases.

[0049] Activated microglia can acquire different phenotypes and morphological characteristics. Resting microglia are ramified, while activated microglia adopt an amoeboid shape and can become phagocytic. In some embodiments, a method described herein increases microglia with ameboid morphology. Phagocytosis is the process by which a cell uses its plasma membrane to engulf a large particle, giving rise to an internal compartment called the phagosome. A cell that performs phagocytosis is called a phagocyte. In some embodiments, a method described herein increases microglia phagocytosis. Activated microglia can also be identified by several markers. Markers of microglia activation include, but are not limited to increases in CD1 lb, CD68, CD45, Ibal, RAC1, TREM2 and CD206. Additionally, markers related to antigen presentation, like MHC class II, CD80, and CD86, are upregulated during activation. Additional markers include APOE, CDl lc, AXL, Clec7A, Tyrobp CST7, LPL, CD40, NLRP3, Salll, LAMP 1 / 2, MERTK. In some embodiments, the method further comprises administering to the subject a therapeutic agent capable of binding to cerebral protein aggregates. In some embodiments the therapeutic agent is an antibody or affinity reagent comprising an Fc region of an antibody and an affinity region capable of binding to the protein aggregates. In some embodiments, the antibody or affinity reagent is an anti-amyloid antibody.

[0050] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps.

[0051] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0052] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0053] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0054] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0055] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g, “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0056] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0057] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0058] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0059] Examples

[0060] Microglia, the ubiquitous, by far most abundant, phagocytic cells in the CNS, assume crucial roles in maintaining CNS homeostasis, for example by i) sensing challenges (eg. infectious, destructive, or injurious); ii) remove extracellular debris that inappropriately accumulates within CNS parenchyma; iii) coordinate inflammatory or immunological reactions, eg. by controlling T cell influx, traffic within CNS, or effector phenotypes.

[0061] While highly desirable therapy targets, microglia are notoriously difficult to “guide”, “instruct” or “engage” safely and effectively. This is mostly because the overarching physiological role of microglia is protecting the exceedingly delicate, often irreplaceable, architecture of the CNS. Thus, compared to tissue-resident macrophages in other tissues that are more capable of withstanding inflammation / damage (such as liver, lung), microglia are far less pliant in initiating inflammatory activation programs that may damage the CNS.

[0062] This invention uses the unique properties of a recombinant, chimeric poliovirus (PVSRIPO), that 1) naturally infects microglia (which express the poliovirus receptor CD 155); 2) executes a peculiar viral program of extended viral (v)RNA replication without any cytopathogenic damage / particle propagation; 3) triggers a highly specific innate inflammatory program limited to sustained type-I / III release; 4) durably enhances the ingrained roles of microglia in CNS protection — most pertinently phagocytosis / debris removal — by reinforcing their primordial physiological functions. Thus, in short, PVSRIPO infection provides ^positive, non-damaging, pro-activation, pro-protection, prosurvival program in microglia. This is most evident as the extension of survival in explanted microglia: PVSRIPO-infected microglia survive for at least 2 months (while exhibiting vRNA replication) ex vivo (they essentially appear normal on microscopy) while their non-infected controls succumb to senescence / death when cultured ex vivo within approximately 10-14 days (evident as floating cells, cell fragmentation, clumping). For these reasons, PVSRIPO provides a unique resource to safely and effectively engage microglia in CNS-protective functions, such as phagocytic uptake of protein aggregates. Mice

[0063] Wt C57B1 / 6 mice were obtained from Jakson Laboratory. Homozygous hCD155- tg mice were provided generously by S. Koike (Tokyo Metropolitan Insitute of Medical Sciene, Tokyo, Japan) and maintained as a breeding colony at the Duke University Laboratory Animal resource. Male and female mice were used at 8-12 weeks of age and 6 months of age. Mice were housed in the Duke University Cancer Center Isolation Facility under Biosafety Level 2 (BSL2) conditions with 12-hour light / dark cycles, relative humidity of 50 ± 20%, and temperature of 21 ± 3 °C.

[0064] Virus, Cells. Amyloid Beta Aggregates

[0065] PVSRIPO was propagated in HeLa R19 cells, purified, and quantified by plaque assay as described earlier. Isolation of supernatant 48 h after infection. Virus-containing supernatants were purified through a 0.1 pm syringe filter (Pall) followed by a lOOKda cutoff spin-filter (Millipore-Sigma) to remove small debris. Murine CT2A Wild Type (plain) glioma cells, B16 OVA, and CT2A mCherry, CT2AhCD155, and M059J glioma cells (Duke CCF) were used. CT2ACellTraceGreencells were used for in vitro phagocytosis experiments. All cell lines were confirmed to be mycoplasma negative (Duke Cell Culture Facility). For hCD155-transduction the cells were infected with lentiviral hCD155 expression vector and selected through fluorescence-activated cell sorting (FACS) with an anti-CD155-PE antibody (BioLegend). Murine CT2A Wild Type (plain), CT2A mCherry and CT2AhCD135cells were used for implantation into hCD155 tg C57B1 / 6 mice. For tumor implantation, cells were propagated in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) with 10% fetal bovine serum (FBS; Sigma, #F0926) to 60-70% confluency for harvest. Beta Amyloid (1-42), HiLyte Fluor 488 and 647-labeled peptide (Anaspec) was resuspended in 50 uL NH40H and stored in aliquots at 0.2 mg / mL of PBS at -80 until use. For pre-aggregated HiLyte 488 A ? (1-42) (fA / ?42) was incubated in PBS at 37°C. Tumor and Amyloid Beta Models

[0066] All animal procedures were performed under a Duke University School of Medicine lACUC-approved vertebrate animal use protocol. Where indicated, antipoliovirus immunization occurred 30 and 45 days prior to tumor administration with two IM injections of PVSRIPO diluted in PBS to 2 xlO7pfu / mL and mixed 1 : 1 with Alhydrogel (Invitrogen) with one (50 uL) injection in each hind quad. Stereotactic implantation of mouse glioma along with stereotactic virus / mock are described in detail elsewhere. PVSRIPO / mock IT treatment in the brain was performed on day 7 after tumor implantation. All stereotactic infusions delivered a volume of 5 uL. Amyloid beta HiLyte 488 A ? (1-42) (fA ?42) was injected as described above as well as injection -1.0 ± 0.06 mm from bregma and 1.8 ± 0.1 mm sagittally when noted. In all cases of amyloid beta injection 5 ug of semi-aggregated peptide incubated for 7 days (37 degrees Celsius) in PBS was injected. Alternate injections when noted used a 50 pl micro syringe (Hamilton, #80901) prefdled with tumor cells or HiLyte 488 A / ? (1-42) (fA / ?42) homogenously suspended in 2.4% methylcellulose, and a keyhole was created at (x = 2.0 mm and z = -0.5 mm) by microdrill, with a depth of 3.6 mm measuring from where the bevel tip intersects with the brain surface. Intratumoral injection of PVSRIPO (5 x 107pfu) was given the same manner as tumor implantation. Intracerebral injection of PVSRIPO (5 x 107pfu) at the same concentration was given in the same manner as amyloid beta injection in accordance with injection coordinates.

[0067] RNA Analyses

[0068] Mice were perfused with IX PBS and whole brain was collected or brains split at the midline using a razor blade for experiments comparing ipsilateral and contralateral vRNA burden. Microglia were isolated using a CD1 Ib-antibody based procedure, according to published methods. Total cellular / tissue RNA was isolated form cell pellets by resuspension in Trizol Reagant (Invitrogen). Chloroform (250uL) was added and mixed before centrifugation at 12,000 rpm for 15 min. at 4 degrees Celsius to induce phase separation. The aqueous layer was removed and transferred to a fresh centrifuge tube. After being mixed with isopropanol (500uL) and spun once again at 12,000 rpm for 10 min. at 4 degrees Celsius the supernatant was removed. The RNA pellet was washed with 70% EtOH twice and dried at RT and resuspended in nuclease-free water. Column purification (ThermoFisher) was then performed to concentrate the RNA and remove impurities. The Monarch Total RNA Miniprep Kit (NEB) was also used to isolate RNA. RNA concentration was checked using a nanodrop. For RT-qPCR analysis, the RNA was treated with PrimeScript™ FAST RT Reagent Kit (Takara Bio) for DNase removal and reverse transcribed into cDNA following the manufacturer’s instructions. One-step qPCR was performed with custom primer pairs for PVSRIPO and standard primer pairs for MDA5 (Hs00223420_ml FAM), IRF7 (Hs01014809_gl FAM) ,TREM2 (Hs00219132_ml FAM), and RAC1 (MmO12O1653_mH FAM) using the TaqMan Fast Advanced Master Mix (Applied Biosystems).

[0069] Microglial and Tissue Staining

[0070] For immunohistochemistry experiments mice were perfused with ice-cold PBS. The brain tissue was subsequently embedded in OCT and flash frozen over liquid nitrogen and stored at -80 until cut into 15-micron cryosections and fixed in 4% PFA for 15 minutes. For immunofluorescence staining of Rabbit / Mouse anti-TMEM119 (Cell Signaling E3E10 / E4B9S), IBA1, ISG15 (Invitrogen #703132), and CD8 (Invitrogen MAI-81180) the sections were permeabilized (0.05% Triton X-100 in PBST), incubated in blocking buffer (Donkey or Goat Serum, 10%), incubated in primary antibody (1:200 dilution; 24h, at 4 degrees Celsius), rinsed (TBS, 3x), incubated with correlating secondary anti-species antibody. The secondary antibodies used were H+L 647 Goat Anti-Rabbit+ (Invitrogen A32733), 594 Donkey Anti-Mouse (Invitrogen A21203), and H+L 488 Goat Anti- Rabbit+(Invitrogen A32731) for 2 hours at RT. Sections were then counterstained with Hoechst 33342 (ThermoFisher 62249) and mounted using vectashield. Where indicated HCR-FISH was performed on the stained slides for imaging of PVSRIPO (-) strand vRNA with specific probes (Molecular Instruments Reagents). Microglia were cultured on poly- d-lysine 1.5 mm coated coverslips. Slides were then rinsed with PBS-T and incubated with probe hybridization buffer (Molecular Instruments Reagents) for pre-hybridization without probes (30min; 37°C). The probe solution was prepared by adding 6pM of each probe set to lOOpl probe hybridization buffer (preheated to 37°C) and added directly onto the sample and incubated (24h, 37°C). After probe hybridization, the slides were washed with probe wash buffer 3x (Molecular Instruments Reagents) to remove the probe solution. Hairpin amplification occurred by incubating the cells with snap-cooled hl and h2 hairpins in amplification buffer (Molecular Instruments Reagents) (24h in the dark, 20°C). The hairpin solution was removed, the slides were rinsed five times with SSC buffer (150mM sodium chloride, 15mM sodium citrate) and counterstained with Hoechst 33342 and mounted for imaging.

[0071] Microglia Imaging and Morphology Analysis

[0072] Tissue sections stained with ISG15 and CD8 as well of microglia stained with TMEM1 19 were imaged on a Leica Stellaris 8 confocal microscope at 20x magnification at a resolution of 1024 x 1024, 2048 x 2048, and 4096 x 4096 in the Light Microscopy Core Facility, Duke University School of Medicine. Using Imaged .tiff images were created of each fluorescent channel and used as input for downstream morphological analysis in MicrogliaMorphology and MicrogliaMorphologyR as described in (The ImageJ macro MicrogliaMorpholgy was used to determine dataset specific parameters to account for variability in image preparation and acquisition. All supporting code for MicrogliaMorphology is available on GitHub at github.com / ciernialab / MicrogliaMorphology. Optimal local thresholding methods and radius values were selected to accurately segment individual microglial cell fragments or overlapping cells. These parameters are then applied within the macro to guide all downstream morphological analyses. Images are then binarized and converted to greyscale. The final images are used as input for creation of individual ROI manager functions to create and save images of every individual microglial cell. This allows for measurement of morphology measures. The cells are skeletonized to generate measures of different morphology features. Fractal analysis is then performed to measure additional morphology features. MicrogliaMorpholgyR is an R package that wraps several packages including tidyverse, Hmisc, pheatmap, factoextra, ImerTest, Ime4, Matrix, SciViews, ggpubr, glmmTMB, DHARMa, rstatix, and gridExtra. All source code for MicrogliaMorphologyR and descriptions of functions can be found on GitHub at github.com / ciernialab / MicrogliaMorphologyR. Statistical analyses were conducted on GraphPad Prism.

[0073] Phagocytosis and Cross Presentation assays

[0074] For in vitro phagocytosis studies microglia were isolated and cultured according to established methods, (insert the method) CT2A WT cells were stained with Cell Trace Violet (Thermo Fisher) by adding 20 pL of DMSO to one vial of CellTrace Violet. Making a stock solution of 5 mM. 2 uL of CTV was added to 10 mb of pre warmed PBS. Place 1 mL of the loading solution (IpM) was added into a conical with 25 x 105cells and incubated (20 min. 37 degrees Celsius). The cells were washed and resuspended with 10 mL of media (DMEM + 10% FBS) for 10 min. They were then irradiated and verified to be dead by trypan blue staining. Cells were stored at 4 degrees Celsius until use. Microglia were plated at 70% confluency and infected with PVSRIPO (MOI 10) or mock, IFN- gamma (200 U / mL), or Poly I:C (10 ug / mL) and incubated with 200,000 CT2A mCherry or B16-OVA cells (up to 96 hpi). For cross presentation assays H-2Kb: SIINFEKL-PE was used to assess cross-presentation. At the specified time points, cells were washed to remove non-adherent cells or spun down if microglia were not adherent. For in vitro phagocytosis of A ? pre-aggregated HiLyte 488 A ? (1-42) (fA ?42) was added to microglia at a concentration of luM (96 hours) and infected with PVSRIPO (MOI 10). For in vivo phagocytosis assays with amyloid beta (1-42) and CT2AmCHerry, microglia were isolated into a single cell suspension and analyzed for phagocytosis. Cells were stained with antibodies and analyzed by spectral flow cytometry (Cytek Aurora) for phagocytosis (HiLyte 488 or mCherry). Controls included single stained cells and beads, as well as unstained controls to account for autofluorescence. Multiple antibody panels and combinations were used including combinations of PE-Cy7-TMEM119, PE-H-2Kb bound to SIINFEKL, PE / APC-CD155, BV605-CD40, BV421 / AF488-Ki-67, FITC / BV785-IAIE, BV711 / BV570-CD1 lb, BV421 / PerCP-Cy5.5-Cx3crl, APC-Cy7 / APC / BUV737-CD45.2, APC / Fire-750-CD68, BUV496-CD86, FITC-TREM2, PE-CY7-H-2Kb, APC-CD11c, PE- MERTK, BUV805-F4 / 80, and 7-AAD. (All Bio Legend, All 1 :250 dilution) Data were analyzed with FlowJo software.

[0075] Cytokine Analysis

[0076] Microglia isolated by above methods were cultured in microglia medium and infected with PVSRIPO (MOI 10) up to 96 hours along with M059J cells when noted. Supernatant was collected and were analyzed using the LEGENDplex Mouse and Human Anti-Virus Response Panel. Samples and standards were plated in biological and technical replicated, and the assay was executed per the manufacturer’s protocol. Data was collected using the LSR Fortessa (BD) as well as the CytoFLEX Flow Cytometer (Beckman Coulter). Minimum threshold values were shown if cytokine concentrations were below the sensitivity of detection (automatically calculated by analysis software); in rare cases where analyte concentration exceeded maximum threshold values, the maximum value was shown. Data was analyzed using the LEGENDplex software available on the manufacturer’s website.

[0077] Mouse and Human Flow Cytometric Analyses

[0078] Cell and protein analyses were performed by flow cytometry. Brain was harvested and microglia were isolated using a CD1 Ib-antibody based procedure, according to published methods and stored on ice cold PBS. Isolation accuracy of microglia isolation was verified by flow cytometry. 7-AAD viability dye (ThermoFisher) was used to stain dead cells in samples. All samples were resuspended in FACS buffer. (2% FBS in PBS) Cells were washed with FACS buffer and incubated with 1% Mouse or Human TruStain FcX FC receptor blocking solution (BioLegend) and incubated at 20 degrees Celsius. (30 min) Flow antibodies were then added to each tube; antibodies used in flow cytometry analysis are listed. Cells were incubated with the antibodies (Ih, 20 degrees Celsius), with shaking every 15 min in the dark. Cells were then rinsed and washed with 1 mb FACS buffer and spun down at 1500G for 5 minutes and a pellet was formed. After pelleting, cells were resuspended in 300 uL FACS buffer and analyzed by flow cytometry using SpectroFlo using the Cytek Aurora (Cytek Biosciences) or LSR Fortessa (BD) instruments. Microglia were stained for CD45.2-APC, Cx3crl-PerCp-Cy5.5, Cdl lb-BV570 (All Biolegend) for identification as well as various other antibodies for further analysis. For intracellular vRNA staining, HCR-FISH was combined with flow cytometry. This procedure consisted of the cell suspensions being fixed in 4% paraformaldehyde. (Ih. 20 degrees Celsius) Fixed cells were pelleted by centrifugation for 3 min. at 500g , washed 4 times with PBS-T (PBS, Tween-200.5%), and resuspended in 70% EtOH and stored (24h, 4 degrees Celsius). Cells were washed with PBST and resuspended with pre-warmed probe-hybridization buffer for HCR FISH (Molecular Instruments Reagents). Hairpin amplification was then performed by incubating the cells with snap-cooled hl and h2 hairpins in amplification buffer (Molecular Instruments Reagents) (24h in the dark, 20°C). The hairpin solution was removed; the cells were then washed five times with SSC buffer (150mM sodium chloride, 15mM sodium citrate) an analyzed with the Cytek Aurora using Spectro Flo. Negative cell populations, single stain controls and isotype controls were used to define positive vs negative staining using beads and isolated unstained microglial cells. Appropriate compensation / spectral unmixing, and specificity of antibodies was confirmed. VersaComp beads (Beckmann-Coulter) and staining of fresh cells were used to standardize compensation. All flow cytometry data were analyzed using FlowJo vlO.10 (BD Biosciences).

[0079] Citations from Brief Description of the Drawings

[0080] 1 Yang, Y. et al. Polio virotherapy targets the malignant glioma myeloid infiltrate with diffuse microglia activation engulfing the CNS. Neuro Oncol 25, 1631-1643, doi : 10.1093 / neuonc / noad052 (2023). Kim, J., Pavlidis, P. & Ciernia, A. V. Development of a High-Throughput Pipeline to Characterize Microglia Morphological States at a Single-Cell Resolution. eNeuro 11, doi: 10.1523 / ENEUR0.0014-24.2024 (2024). Wang, S. et al. TREM2 drives microglia response to amyloid-beta via SYK- dependent and -independent pathways. Cell 185, 4153-4169 e4119, doi : 10.1016 / j . cell .2022.09.033 (2022).

Claims

CLAIMSWhat is claimed:

1. A method of treating a neurological disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant, chimeric poliovirus construct, wherein the recombinant, chimeric poliovirus construct comprises a Sabin type I strain of poliovirus with a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the poliovirus' 5' untranslated region between the poliovirus' cloverleaf and the poliovirus' open reading frame of the Sabin type I poliovirus; and wherein the recombinant, chimeric poliovirus construct is administered intracerebrally.

2. The method of claim 1, wherein the neurological disease is a neurodegenerative disease.

3. The method of claim 1 or 2, wherein the neurodegenerative disease is associated with cerebral protein aggregates.

4. The method of claim 2 or 3, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, dementia with Lewy bodies (DLB), frontotemporal diseases, and multiple system atrophy.

5. The method of any one of the preceding claims, wherein the neurological disease is not a tumor or is not associated with a tumor.

6. The method of any one of claims 3-5, wherein the protein aggregates are amyloid plaques.

7. The method of any one of claims 3-6, further comprising administering a therapeutic agent capable of binding to the aggregated proteins and microglia.

8. The method of claim 7, wherein the therapeutic agent is an antibody or affinity reagent comprising an Fc region of an antibody and an affinity region capable of binding to the protein aggregates.

9. The method of claim 8, wherein the antibody or affinity reagent is an anti-amyloid antibody.

10. The method of any one of the preceding claims, wherein the recombinant, chimeric poliovirus construct is PVSRIPO.

11. The method of any one of the preceding claims, wherein the method decreases cerebral protein aggregates.

12. The method of any one of the preceding claims, wherein the symptoms of the neurological disease are reduced or the progression of the neurological disease is slowed as compared to a subject not treated or prior to treatment.

13. A composition for the treatment of a neurological disease, the composition comprising a recombinant, chimeric poliovirus construct and a therapeutic agent capable of binding to cerebral protein aggregates, wherein the recombinant, chimeric poliovirus construct comprises a Sabin type I strain of poliovirus with a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the poliovirus' 5' untranslated region between the poliovirus' cloverleaf and the poliovirus' open reading frame of the Sabin type I poliovirus.

14. The composition of claim 13, wherein the composition is formulated for intracerebral administration.

15. The composition of claim 13 or 14, wherein the neurological disease is a neurodegen erative disease.

16. The composition of any one of claims 13-15, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, dementia with Lewy bodies (DLB), frontotemporal diseases, and multiple system atrophy.

17. The composition of any one of claims 13-16, wherein the neurological disease is not a tumor or is not associated with a tumor.

18. The composition of any one of claims 13-17, wherein the protein aggregates are amyloid plaques.

19. The composition of any one of claims 13-18, wherein the therapeutic agent is an antibody or affinity reagent comprising an Fc region of an antibody and an affinity region capable of binding to the protein aggregates.

20. The method of claim 19, wherein the antibody or affinity reagent is an anti-amyloid antibody.

21. A method of activating microglia in a subj ect in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant, chimeric poliovirus construct to activate the microglia of the subject, wherein the recombinant, chimeric poliovirus construct comprises a Sabin type I strain of poliovirus with a human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in the poliovirus' 5' untranslated region between the poliovirus' cloverleaf and the poliovirus' open reading frame of the Sabin type I poliovirus, wherein the recombinant, chimeric poliovirus construct is administered intracerebrally; and wherein the subject does not have a central nervous system tumor.

20. The method of claim 19, wherein the subject in need is diagnosed with or suspected of having a neurological disease.

21. The method of claim 20, wherein the neurological disease is associated with cerebral protein aggregates.

22. The method of claim 20 or 21, wherein the neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, dementia with Lewy bodies (DLB), frontotemporal diseases, and multiple system atrophy.

23. The method of any one of claims 19-22, wherein the method increases microglia with ameboid morphology.

24. The method of any one of claims 19-22, wherein the method increases microglia phagocytosis.

25. The method of any one of claims 19-24, wherein the method increases markers of microglia activation, wherein the markers comprise at least one of RAC 1, CD40, CD86, CD68, or MHCII.

26. The method of any one of claims 19-25, further comprising administering to the subject a therapeutic agent capable of binding to cerebral protein aggregates.

27. The method of claim 26, wherein the therapeutic agent is an antibody or affinity reagent comprising an Fc region of an antibody and an affinity region capable of binding to the protein aggregates.

28. The method of claim 27, wherein the antibody or affinity reagent is an anti-amyloid antibody.

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