Brincidofovir to suppress beta amyloid aggregation

Abstract

A method of using brincidofovir (BCV) or a pharmaceutical composition thereof to suppress amyloid β aggregation, neuronal cell degeneration, or phosphorylation of Tau is described.

Description

BRINCIDOFOVIR TO SUPPRESS BETA AMYLOID AGGREGATIONSEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 15, 2025, is named 14884WO1 and is 12,076 bytes in size.STATEMENT AS TO FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant number P41EB027062 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The technical field of the present invention, for example, relates to the suppression of amyloid P aggregation, neuronal degeneration, or phosphorylation of Tau, or the treatment of neurodegenerative diseases.BACKGROUND

[0004] Alzheimer’s disease (AD) is a progressive neurodegenerative condition that is the most common cause of dementia in aged adults and the sixth leading cause of death in the United States. An estimated 6.2 million Americans aged 65 and older have Alzheimer’s dementia (an underestimate of the total population with AD), which is expected to reach 12.7 million by 2050. With the possible exception of the controversial aducanumab treatment, no disease-modifying preventive or therapeutic strategies for AD currently exist. Barenholtz Levy, H., Ann Pharmacother, 2022. 56(6): p. 736-739.

[0005] The pathological hallmarks of AD are the aggregation and deposition of P-amyloid (AP) peptide into senile plaques and of hyperphosphorylated tau into neurofibrillary tangles (NFTs). Ap is derived from the integral transmembrane protein amyloid precursor protein (APP) via sequential processing by P-secretase (BACE1) and y-secretase and can vary in size from 37 to 43 peptides in length. Liu, P.P., et al., Signal Transduct Target Ther, 2019. 4: p. 29. The most common isoforms are Api-40 and Api-42, the latter of which is more prone to aggregate into neurotoxic oligomersthat are considered to be an initiator of AD pathology. Watts, J.C. and S.B. Prusiner, Cold Spring Harb Perspect Med, 2018. 8(5). Early onset AD (EOAD) is caused by mutations in APP or y- secretase subunits PSEN1 or PSEN2, resulting in increased overall Ap production or an increased AP42 / AP40 ratio. Jarrett, J.T., E.P. Berger, and P.T. Lansbury, Jr., Biochemistry, 1993. 32(18): p. 4693-7. However, EOAD only accounts for approximately 5.5% of AD cases, therefore models utilizing EOAD mutations can only provide limited insight into the complex mechanisms and risk factors for the far more prevalent sporadic form of AD (sAD).

[0006] Evidence is growing for the pathogen hypothesis in sAD, which states that pathogens act as triggers for the amyloid cascade, tau hyperphosphorylation, and neuroinflammation for some subsets of patients. Harris, S.A. and E.A. Harris, J Alzheimers Dis, 2015. 48(2): p. 319-53.SUMMARY OF THE INVENTION

[0007] The inventors have conducted intensive research of the antiviral effects of brincidofovir (BCV) on a 3D human cortical brain tissue model of herpes-induced AD, which provides some of the first evidence for direct causality of HSV-1 in AD in human brain tissues. As a result, the inventors have discovered that BCV has an excellent effect in reducing HSV-1 infection and resulting AD-like phenotypes. Additionally, the inventors have studied the kinetics of antiviral activity of BCV and demonstrate that early BCV treatment post-infection prevents herpes-induced AD-like phenotypes in 3D human brain-like tissues.

[0008] In one aspect of the invention provides a method of suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau in a subject in need thereof, the method comprising administering an effective amount of brincidofovir (BCV) to the subject. Utilizing this method, neurodegenerative diseases such as Alzheimer's disease can be treated or prevented.BRIEF DESCRIPTION OF THE FIGURES

[0009] The present invention may be more readily understood by reference to the following drawings, wherein:

[0010] Figure 1 provides a schematic representation of the experimental plan for assaying antiviral activity of BCV in herpes-induced AD.

[0011] Figures 2A-C provide graphs and images showing BCV prevents HSV-1 infection in monolayer cultures of hiNSCs. (A) Immuno staining results showing expression of HSV marker(top part: HSV (red text)) across various concentrations of BCV, scale = 100 mm. The bottom part is DAPI (blue text). (B) Quantification of HSV immunostaining. (C) At BCV concentrations > 10 mM, substantial cytotoxicity is observed. The top part is HSV marker (red text). The bottom part is DAPI (blue text).

[0012] Figure 3 provides an image showing BCV prevents induction of herpes-induced amyloid beta fibril expression in monolayer cultures of hiNSCs. Immuno staining results showing expression of Abeta marker (middle part: AB (red text)) and pan-neuronal marker beta III tubulin (top part: TUJ1 (green text)) across various concentrations of BCV, scale = 100 m. The bottom part is DAPI (blue text).

[0013] Figures 4 A-C provide a scheme, an image, and a graph showing Pilot studies of HSV-1 infection and BCV treatment using 3D human brain-like tissue constructs. (A) Schematic representation of the generation of 3D silk scaffold-based human brain like tissue constructs. (B) Image of 3D donuts in culture. (C) LDH leakage assay using conditioned media from 3D human brain-like tissue constructs in the presence or absence of HSV-1 and BCV.

[0014] Figures 5 A-C provide graphs and images showing BCV prevents infection and induction of herpes-induced amyloid beta fibril expression in 3D human brain-like tissues. (A) Immunostaining results showing expression of HSV marker (top part: HSV (green text)) and Abeta (middle part: AB (red text)) across various concentrations of BCV, scale = 100 pm. The bottom part is HSV marker (red text), AB (red text), and DAPI (blue text). (B) Quantification of Abeta immuno staining. (C) Quantification of cell number across treatments.

[0015] Figures 6 A-H provide graphs showing BCV suppresses herpes-induced expression of AD markers, active gliosis and neuro-inflammation in 3D human brain-like tissues. qRT-PCR results showing HSV-1 expression (A), known mediators of AD, APP (B), BACE1 (C) and PSEN2 (D) as well as gliosis marker GFAP (E) and inflammatory markers ILlb (F), IFNy (G) and TNFa (H).

[0016] Figure 7 provides a graph showing the experimental plan for assaying antiviral activity of BCV in herpes induced AD.

[0017] Figures 8 A-C provides graphs and images showing BCV prevents herpes-induced amyloid beta expression in a time-dependent manner in 3D human brain-like tissues. (A) Immunostaining results showing expression of pan-neuronal marker beta III tubulin (top part: TUJ1 (green text)) and Abeta (middle part: AB (red text)) across various time points of BCV addition, scale = 100pm. The bottom part is TUJ1 (green text), AB (red text), and DAPI (blue text). (B) Quantification of Abeta immuno staining. (C) Quantification of cell number across treatments.

[0018] Figures 9A-B provides a graph showing the experimental plan for assaying activity of BCV in herpes induced phosphorylation of Tau (pTau) and images showing BCV suppresses HSV-1- induced pTau in a time-dependent manner in 3D human brain-like tissues. (A) Experimental plan for assaying activity of BCV in herpes induced phosphorylation of Tau (pTau). (B) Immuno staining results showing expression of pan-neuronal marker beta III tubulin (top part: TUJ1 (green text)) and pTau (middle part: PTAU (red text)) across various timepoints of BCV addition, scale = 100 m. The bottom part is TUJ1 (green text), PTAU (red text), and DAPI (blue text).

[0019] Figure 10 provides schematics showing Lentiviral constructs expressing shAPOE and relevant controls. Short hairpin RNA construct, shAPOE, as well as relevant control constructs (GFP and non-target shRNA) were used to generate hiNSC lines.

[0020] Figures 11 A-D provide a schematic and images showing Generation of stable shAPOE expressing hiNSC lines. (A) Schematic of shAPOE construct which contains both a GFP and puromycin resistance cassette. (B) Initial shAPOE lentiviral infection using increasing MOI. (C) Titration of puromycin in untransduced hiNSCs showing substantial cell death at 1 pg / ml puromycin. (D) Titration of puromycin in shAPOE- transduced hiNSCs showing higher selection for transduced cells at higher concentration as indicated by the presence of increased GFP. Scale = 100 pm.

[0021] Figures 12 A-D provide images showing HSV-1 does not cause AD-like morphological changes in APOE Knockdown hiNSCs. (A) Immunostaining results showing expression of panneuronal beta III tubulin marker (top part: TUJ 1 (red text)) in response to mock or HSV-1 infection in (A) untransduced as well as cells transduced with control GFP (B), non-mammalian control shRNA (C) and shAPOE (D). The middle part is DAPI (blue text), and the bottom part is TUJ1 (green text) and DAPI (blue text). hiNSCs exhibiting the typical morphological response to HSV- 1 infection are boxed in red. shAPOE hiNSCs infected with HSV-1 were morphologically indistinguishable from mock- infected cells. Importantly, BCV treatment (0.1 pM) rescued herpes- induced morphological differences across all cell lines. Scale = 100 pm.

[0022] Figures 13 A-B provide images showing shAPOE hiNSCs do not express Abeta in response to HSV-1 infection. A) Immunostaining results showing expression of amyloid beta (toppart: AB (red text)) in response to mock or HSV-1 in both control and shAPOE hiNSCs. The bottom part is AB (red text) and DAPI (blue text). B) Immuno staining results showing expression of APOE4 (top part: E4 (red text)) in response to mock or HSV-1 in both control and shAPOE hiNSCs. Scale =100 m. The bottom part is E4 (red text) and DAPI (blue text).DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0023] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0024] The invention is inclusive of the compounds described herein in any of their pharmaceutically acceptable forms, including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, and the like. In particular, if a compound is optically active, the invention specifically includes each of the compound's enantiomers as well as racemic mixtures of the enantiomers. It should be understood that the term "compound" includes any or all of such forms, whether explicitly stated or not (although at times, "salts" are explicitly stated).

[0025] A “subject,” as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a mammal, such as a research animal (e.g., a monkey, rabbit, mouse or rat) or a domesticated farm animal (e.g., cow, goat, horse, pig) or pet (e.g., dog, cat). In some embodiments, the subject is a human. The subject may be, for example, a subject in need of treatment for a disease associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, a subject with a disease associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, a subject diagnosed as having developed a disease associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, a subject suspected of developing a disease associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, a subject with reactivated herpes virus, a subject with an indicator of reactivated herpes virus (e.g., an antibody to herpes virus antigen), or a subject with an ApoE allele (e.g., ApoE4).

[0026] "Treat", "treating", and "treatment", etc., as used herein, refer to providing a benefit to a subject having a disease, including improvement in the condition through lessening or suppressionof at least one symptom, delay in progression of the disease, etc. Treatment, as used herein, can also include reducing the viral load within cells infected with herpes virus. The lessening or suppression may include, for example, inhibition of 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more, or 100%.

[0027] As used herein, the term “prevention” includes either preventing or decreasing the risk of developing a disease or disorder. This includes prophylactic treatment of those having an enhanced risk of developing a disease or disorder. An elevated risk represents an above-average risk that a subject will develop a particular disease or disorder, which can be determined, for example, through family history or the detection of genes causing a predisposition to develop a particular disease or disorder.

[0028] “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject for the methods described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0029] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualify and / or quantify the amount of each agent which will achieve the goal of decreasing disease severity. The therapeutically effective amount may be administered in one or more doses. An effective amount, on the other hand, is an amount sufficient to provide a significant chemical effect, which may or may not affect disease severity.

[0030] “AP” and “Abeta” as used herein mean amyloid p.Methods of Treatment

[0031] One aspect of the invention provides a method of suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau in a subject in need thereof, the method comprising administering an effective amount of brincidofovir (BCV) to the subject. Utilizing this method, neurodegenerative diseases such as Alzheimer's disease can be treated. In some embodiments, the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau occurs in response to viral infection. In some embodiments, the virus is a herpes virus. In some embodiments, the herpes virus is Herpes simplex virus type I (HSV-1), Herpes simplex virus type 2 (HSV-2), or Varicella-zoster virus (VZV). In some embodiments, the method is a method of suppressing amyloid P aggregation. In some embodiments, the method is a method of suppressing neuronal cell degeneration. In some embodiments, the method is a method of suppressing phosphorylation of Tau. In some embodiments, the subject is in a state of reactivation of herpesvirus. In some embodiments, the method comprises detecting reactivation of herpes virus in the subject. In some embodiments, the detecting comprises detecting an antibody against herpes virus antigen. In some embodiments, the suppression is carried out in a treatment of a neurodegenerative disease, and the method comprises identifying a subject in a state of reactivation of a herpes virus as a treatment target for a neurodegenerative disease. In some embodiments, the subject has an ApoE4 allele. In some embodiments, the method comprises detecting an ApoE4 allele in the subject. In some embodiments, the suppression is carried out in a treatment of a neurodegenerative disease, and the method comprises identifying a subject with an ApoE4 allele as a treatment target for a neurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease. In some embodiments, the method comprises diagnosing an onset of a neurodegenerative disease in the subject and identifying the subject with the onset of the neurodegenerative disease as a candidate for treatment, and the identified subject is tested for the presence or absence of reactivation of herpes virus. In some embodiments, the subject has a neurodegenerative disease. In some embodiments, the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau is accompanied by a neurodegenerative disease. In some embodiments, the suppression is carried out in a treatment of a neurodegenerative disease. In some embodiments, the suppression of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau results in a treatment of a neurodegenerative disease. In some embodiments, the neurodegenerative disease occurs as a result of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. In some embodiments, the neurodegenerative disease is Alzheimer's disease or dementia. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the suppression is carried out in a treatment of encephalitis. In some embodiments, the method does not comprise testing for edema after administering BCV to the subject. In some embodiments, the subject is not suitable for receiving or continuing treatment with administration of an antibody against amyloid-p. In some embodiments, the method of suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau is a method of treating neurodegenerative diseases. In some embodiments, the extent of suppression of amyloid P aggregation may include, for example, 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more, or 100%. In some embodiments, the extent of suppression of neuronal cell degeneration, for example, 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more, or 100%. In some embodiments, the extent of suppression of phosphorylation of Tau may include, for example, 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more, or 100%. In some embodiments, the method of suppressing amyloid P aggregation may be, for example, a method of suppressing the accumulation of amyloid P aggregates or a method of suppressing the accumulation of amyloid P plaques.

[0032] An embodiment of the present invention provides a method of treating a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau in a subject in need thereof, comprising administration of an effective amount of BCV to the subject. Utilizing this method, a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, such as Alzheimer's disease, can be treated. In some embodiments, the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau occurs in response to viral infection. In some embodiments, the virus is a herpes virus. In some embodiments, the herpes virus is HSV-1, HSV-2, or VZV. In some embodiments, the subject is in a state of reactivation of herpes virus. In some embodiments, the method comprises detecting reactivation of herpes virus in the subject. In some embodiments, the detecting comprises detecting an antibody against herpes virus antigen. In some embodiments, the method comprises identifying a subject in a state of reactivation of a herpes virus as a treatment target for a neurodegenerative disease. In some embodiments, the subject has an ApoE4 allele. In some embodiments, the method comprises detecting an ApoE4 allele in the subject. In some embodiments, the method comprises identifying a subject with an ApoE4 allele as a treatment target for a neurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease. In some embodiments, the method comprises diagnosing an onset of a neurodegenerative disease in the subject and identifying the subject with the onset of the neurodegenerative disease as a candidate for treatment, and the identified subject is tested for the presence or absence of reactivation of herpes virus. In some embodiments, the subject has a neurodegenerative disease. In some embodiments, the suppression is carried out in a treatment of a neurodegenerative disease. In some embodiments, the suppression of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau results in a treatment of a neurodegenerative disease. In some embodiments, neurodegenerative diseases result from amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. In some embodiments, the neurodegenerative disease is Alzheimer's disease or dementia. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the method results in a treatment of encephalitis by suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. In some embodiments, the disease may not include encephalitis. In some embodiments, the method does not comprise testing for edema after administering BCV to the subject. In some embodiments, the subject is not suitable for receiving or continuing treatment with administration of an antibody against amyloid-p.

[0033] An embodiment of the present invention provides a method of using BCV or a pharmaceutical composition to suppress amyloid P aggregation, neuronal cell degeneration, orphosphorylation of Tau. In some embodiments, the aggregation, degeneration, or phosphorylation occurs via pathogens including herpes virus. In some embodiments, the aggregation, degeneration, or phosphorylation is associated with neurological diseases including cognitive impairment. In some embodiments, the aggregation, degeneration, or phosphorylation occurs in a subject that has a particular ApoE allele, especially ApoE4. In some embodiments, the aggregation, degeneration, or phosphorylation is accompanied by neuro-inflammation, active gliosis, or phosphorylation of Tau. In further embodiments, the subject has brain amyloid P pathology or reactivation of herpes virus.

[0034] In some embodiments, the detecting step comprises detecting an antibody against a herpes virus antigen, detecting a herpes virus genome DNA, or detecting an ApoE4 allele. Detection of herpes virus reactivation can be performed, for example, by detecting an antibody to a herpes virus antigen or a herpes virus genome DNA in a sample (e.g., blood or cerebrospinal fluid) from the subject. The sample includes, for example, blood sample (e.g. plasma or serum) or cerebrospinal fluid sample. For example, when antibodies to herpes virus antigen or herpes virus genome DNA are detected in the blood sample or cerebrospinal fluid sample, BCV may be administered to the patient who provided the sample. In this way, it is possible to selectively treat a patient who is highly likely to respond well to BCV treatment among patients with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. The antibodies to herpes virus to be, for example, detected may be an antibody that recognizes specifically herpes virus antigens as described in Front Aging Neurosci. 2014 Oct 15;6:285, or PLoS ONE 2008, 3(11): e3637. The antibody may be, for example, IgA or IgM, or it may be IgG. The antibody may be detected, for example, by an ELISA assay (enzyme-linked immunosorbent assay) in which the antigen recognized by the antibody is immobilized on a solid phase surface. A method for detecting the antibody in the sample may include, for example, a step of contacting a sample containing the antibody with a solid phase having the antigen, or a step of detecting the antibody bound to the antigen. The antibody bound to the antigen may be, for example, detected by combining it with a labeled secondary antibody. The detecting step may include, for example, a step of contacting a labeled secondary antibody to the antibody bound to the antigen, or a step of detecting enzyme activity. After the contact step, there may be, for example, a washing step. The detecting step may include, for example, a step of collecting a sample from the patient. The detecting step may include, for example, a step of measuring absorbance (e.g., at a wavelength of 450 nm). The detecting step may include, for example, a step of measuring the titer of herpes virus antigen- specific antibodies. The solid phase may be, for example, a plate (e.g., a microplate). The label may be, for example, an enzyme label. For the detection of enzyme activity, a substrate maybe used, and the substrate may be a substrate whose absorbance spectrum changes by reaction. The enzyme may include, for example, peroxidase, alkaline phosphatase, or P-galactosidase. The substrate may include, for example, DAB (3,3'-Diaminobenzidine), BCIP / NBT (5-Bromo-4- chloro-3'-indolylphos-phatase / Nitroblue tetrazolium), or X-Gal (5-Bromo-4-Chloro-3-Indolyl-P- D-Galactoside). HSV-specific antibodies may be detected using a commercially available kit (BEIA HSV- 1 IgG or IgM, Technogenetics, Milano, Italy). For example, the patient with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau has a sample (e.g., plasma), and an absorbance value when the sample is used in the above method may be 0.13 or more compared to an absorbance value when the negative control is used in the above method. The 0.1 or more may be, for example, 0.1, 0.15, 0.2, 0.3, 0.4, or 0.5 or more, or may be within the range of any two of those values. For example, when an absorbance value obtained using the sample from the patient with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau is 0.1 or more compared to an absorbance value obtained using a negative control, it may be assessed that herpes virus is reactivated. The herpes virus genomic DNA to be detected may be, for example, detected by a PCR method (e.g., quantitative PCR) using DNA extracted from the sample (e.g., plasma). Detection of herpes virus genomic DNA may be performed by detecting herpes virus-derived genes. Detecting herpes virus genomic DNA includes detecting a portion of the herpes virus genomic DNA. The extraction may be, for example, performed using NucliSens Extractor (Organon Teknika, Boxtel, The Netherlands) using a silica-based nucleic acid isolation method from 0.5 to 1.5 mL of plasma. The 0.5 to 1.5 mL may be, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5 mL, or may be within the range of any two of those values. The PCR may be performed using, for example, specific primers for a sequence in the gene for the viral DNA polymerase (pol) (5- GGTGAACGTCTTTTCGCACT-3Z(SEQ ID NO: 4) and 5-GTGTTGTGCCGCGGTCTCA C- 3Z(SEQ ID NO: 5)) as described in J Virol. 2002, 76(23): 12394-12398. The copy number of herpes virus genomes in plasma can be calculated, for example, from the calibration curve obtained from DNA extracted from purified HSV-1 propagated in Vero cells (copy number of viral genome is calculated based on the amount of DNA and size of genome DNA). For example, a sample (e.g., plasma) is obtained from a patient with a disease accompanied by amyloid P aggregation or neuronal cell degeneration, and the gene copy number when the sample is used in the above method may be 10 or more. The 10 or more may be, for example, 10, 20, 30, 40, 50, 100, 1000, 10000, or 100000 or more, or may be within the range of any two of those values. For example, when 10 or more copies of the gene are detected per 1 mL of sample, it may be assessed that herpes virus is reactivated. The detection of the ApoE4 allele may be performed, for example, using a PCR method. The detecting step may include, for example, a step of analyzing the type of ApoE.The detecting step may include, for example, a step of analyzing the nucleotide sequence of ApoE. The amino acid sequence for ApoE4 may be the sequence set forth as SEQ ID NO: 2 (www.ncbi.nlm.nih.gov / protein / ARQ79462.1). The nucleic acid sequence encoding ApoE4 may be the sequence set forth as SEQ ID NO: 3 (www.ncbi.nlm.nih.gov / nuccore / KY924487.1).

[0035] In some embodiments, the treatment method further comprises diagnosing whether the subject has a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, or specifying the subject having the disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau as a candidate for treatment, wherein the specified subject is tested for reactivation of herpes virus. The subject with reactivated herpes virus does not include, for example, a subject diagnosed as being in a latent infection state of herpes virus. The subject with reactivated herpes virus may include, for example, a subject diagnosed as being in a lytic infection state of herpes virus. The treatment method may further include, for example, a step of administering an antiviral agent to the subject. In some embodiments, the identifying step includes selecting the subject.

[0036] In some embodiments, the method may not comprise testing for brain edema after administering BCV to the subject. Recanemab, a drug for the treatment of Alzheimer's disease, is known to cause brain edema as a side effect in humans. Therefore, it was necessary to test for brain edema after administering recanemab to humans. On the other hand, brain edema is unlikely to occur after BCV is administered to humans. Therefore, the treatment method using BCV is superior in terms of safety. In some embodiments, the subject may be a subject who is not suitable for receiving or continuing treatment with administration of an antibody against amyloid p. BCV acts through a different mechanism than antibodies against amyloid P and is superior in terms of safety, so it is suitable for administration to such subjects. In some embodiments, amyloid P in a subject may be tested after BCV is administered to the subject.

[0037] Brincidofovir (BCV) is a compound having the structure represented by the following formula. BCV may also be denoted by the IUPAC name of [(2S)-l-(4-amino-2-oxopyrimidin-l- yl)-3-hydroxypropan-2-yl]oxymethyl-(3-hexadecoxypropoxy)phosphinic acid. Known salt forms of BCV include sodium BCV and ammonium BCV. The term BCV may include the form of a salt of BCV or a solvate thereof having the formula:

[0038] In some embodiments, the aggregation, degeneration, or phosphorylation occurs due to pathogens including herpes virus. Herpes virus refers to Herpesviridae, a large family of DNA viruses. In the case of human subjects, the Herpes virus is a Herpes virus known to primarily infect humans. Examples of Herpes virus known to infect humans include herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), human herpesvirus 6 (HHV-6), varicella zoster virus (VZV), Epstein- Barr virus, and human cytomegalovirus. In some embodiments, aggregation, degeneration, or phosphorylation is the result of infection by HSV-1, HSV-2, or VZV.

[0039] The term “infected with herpes virus” refers to cells or a subject in which herpes virus virus particles have been detected. Methods of detecting herpes virus infection are described in greater detail herein in regard to diagnostic methods. Reducing herpes virus viral load refers to decreasing the number of herpes virus virus particles in cells or in a subject. Reducing the viral load can include reducing the viral load by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or in some cases even by 100%.

[0040] In some embodiments, an additional anti-herpes virus agent (i.e., drug for inhibiting herpes virus replication) is administered to the subject. A number of drugs are known to those skilled in the art for treating infection by HSV-1, HSV-2, or VZV. See Sadowski et al., Viruses, 13(7), 1228 (2021), the disclosure of which is incorporated herein by reference.

[0041] The above prevention method may include, for example, a step of identifying a subject with a risk of a neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau associated with herpes virus infection (e.g., Alzheimer’s disease) as a target for prevention, a step of administering to a subject a therapeutically effective amount of BCV, a step of inhibiting or suppressing herpes virus viral load in the subject, or a step of preventing the neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau associated with herpes virus infection in the subject. Examples of a subject who may be identified include a patient with a risk of neurological diseases

[0042] An embodiment of the present invention provides a method for prevention of neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau that are associated with herpes virus infection, comprising the step of administering to a subject a therapeutically effective amount of BCV. With this prevention method, neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau that are associated with herpes virus infection can be prevented by a novel therapeutic approach.

[0043] The disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau comprises, for example, a neurodegenerative disease. The disease treated by suppression of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau comprises, for example, a neurodegenerative disease. The neurodegenerative disease comprises, for example, Alzheimer's disease or dementia. The dementia comprises, for example, mild cognitive impairment or mild dementia. BCV is particularly suitable for treatment of mild cognitive impairment or mild dementia because it is effective in suppressing disease progression. The dementia comprises, for example, Alzheimer's disease or dementia with Lewy bodies.Diagnostic Methods

[0044] The method of treatment may include a diagnostic step.

[0045] An embodiment of the present invention provides a method of predicting susceptibility to BCV in a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, comprising testing for reactivation of herpes virus in a sample from a subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. This method can be used to predict or identify a subject with high or low susceptibility to BCV by utilizing reactivation of herpes virus as an indicator. Therefore, this method can be used to implement more appropriate BCV treatment. For example, if a subject's herpes virus is reactivated and it is predicted that the subject will be highly susceptible, it may be chosen to administer BCV. For example, if a subject's herpes virus is not reactivated and it is predicted that the subject will be less susceptible, it may be chosen not to administer BCV.

[0046] An embodiment of the present invention provides a method of determining a subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau to be administered BCV, comprising testing for reactivation of herpes virus in a sample from the subject. Utilizing this method provides more appropriate BCV treatment. From anotherperspective, an embodiment of the present invention provides a method of diagnosing whether a subject having a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau is a subject highly susceptible to BCV, comprising testing for reactivation of herpes virus in a sample from the subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. From another perspective, an embodiment of the present invention provides a method of predicting the efficacy of BCV in a subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, comprising testing for reactivation of herpes virus in a sample from the subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. From another perspective, an embodiment of the present invention provides a method of selecting a subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau who is expected to show a therapeutic effect of BCV, comprising testing for reactivation of herpes virus in a sample from the subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. From another perspective, an embodiment of the present invention provides a method of obtaining an indicator for determining whether to implement a treatment method using BCV, comprising testing for reactivation of herpes virus in a sample from the subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau. For example, the method may include (a) obtaining a sample from a subject, and / or (b) detecting reactivation of herpes virus by contacting a sample with a reagent for detecting reactivation of herpes virus. For example, the method may include an auxiliary method. The auxiliary may include assisting in the consideration or determination of a treatment target, a treatment method, or a diagnostic method. For example, the method may be carried out in vitro, ex vivo, or in vivo, depending on the purpose.

[0047] An embodiment of the present invention provides a method of detecting reactivation of herpes virus from a sample of a subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau susceptible to BCV, comprising (a) obtaining a sample from the subject and / or (b) detecting an antibody against herpes virus antigen or a herpes genome DNA in the sample, thereby detecting reactivation of herpes virus in the sample. This method can be used to detect reactivation of herpes virus in the sample of a subject with a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau susceptible to BCV. For example, by detecting reactivation of herpes virus, it is possible to treat the subject while anticipating that BCV will have a positive therapeutic effect.

[0048] The method can include, for example, the following steps. For example, it can include a step of identifying a subject with a neurological disease associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau associated with herpes virus infection as a target for treatment. Other diagnostic steps can include identifying a subject having neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, a subject not having neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, or a subject having a neurological diseases caused by infection with herpes virus. In some embodiments, the presence of herpes virus in the subject is detected before the administration of BCV. The method may include identifying a subject in which herpes virus is reactivated as a target for BCV treatment, or administering BCV to a subject in which herpes virus is reactivated.

[0049] The treatment methods described herein may include a diagnostic step. For example the methods can include the step of (i) identifying a subject with herpes virus infection as a target for anti-herpes virus treatment, (ii) identifying a subject with herpes virus infection with neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau as a target for anti-herpes virus treatment, (iii) identifying a subject with infection of herpes virus without neurological diseases associated with amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau as a target for anti-herpes virus treatment, (iv) detecting herpes virus in the subject, (v) detecting whether or not the subject is infected with herpes virus, (vi) detecting herpes virus in the samples collected, or (vii) detecting an ApoE4 allele in the subject. If one or more of these diagnostic steps are included in the method, it can be useful for identifying a particular population in order to provide increased therapeutic efficacy. When two or more of the above steps are employed, any order is acceptable. The order can be determined according to the desired method. These steps may be performed before or after the step of administering to a subject a therapeutically effective amount of BCV. The detection may be carried out using the gene level, the mRNA level, the protein level, or gene translocation as an indicator.

[0050] The methods can include the step of obtaining a biological sample from the subject. Biological samples include but are not limited to bodily fluids such as saliva, urine and blood- related samples (e.g., whole blood, serum, plasma, peripheral blood lymphocytes (PBMC) and other blood-derived samples), urine, cerebral spinal fluid, bronchoalveolar lavage, and the like. Another example of a biological sample is a cell sample or a tissue sample. The levels of an indicator in a biological sample taken from the subject can be assessed either quantitatively orqualitatively, and can be determined either in vivo or in vitro. In some embodiments, the biological sample comprises blood, plasma, or serum.

[0051] The biological sample can be provided by immediately obtaining a biological sample from the subject, or a biological sample can be provided that was previously obtained. A biological sample may be fresh or stored (e.g., blood or blood fraction stored in a blood bank). Samples can be stored for varying amounts of time, such as being stored for an hour, a day, a week, a month, more than a month, years and decades of storage. The biological sample may be a bodily fluid expressly obtained for use in the methods of this invention or can be a bodily fluid obtained for another purpose which can be subsampled for the assays of this invention.

[0052] In some embodiments, the indicator is a polynucleotide, while in other embodiments, the indicator is a polypeptide. The level of indicator in a subject can be measured using an analytic device. The analytic device used to measure the levels of indicator can be either a portable or a stationary device. In addition to including equipment used for detecting indicators, the analytic device can also include additional equipment to provide purification (i.e., physical separation) of analytes prior to analysis.

[0053] Polypeptide indicators may be determined by any of a variety of standard or novel recently developed protein analytic methods known in the art. These methods include absorbance, gel electrophoresis (e.g., SDS-PAGE gel purification), a protein immunoblot (e.g., western blot), chromatography (e.g., size exclusion chromatography, ion exchange chromatography, and affinity chromatography), precipitation, ultracentrifugation, an immunoassay, such as an enzyme-linked immunosorbent assays (ELISA), mass spectrometry, and other common techniques known to one of ordinary skill in the art. Novel recently developed assays include bead based immunofluorescence assays that can quantitatively measure different proteins in the same well with high sensitivity.

[0054] Polynucleotide indicators may be determined by any of a variety of standard analytic methods known in the art. In one embodiment of the invention, the expression level of an indicator gene (which is a polynucleotide) is determined by nucleic acid amplification of said gene using gene specific primer pairs, and quantifying the amplification results. In some embodiments, the nucleic acid amplification method used is real-time polymerase chain reaction (PCR) analysis.

[0055] Methods of the invention also include the step of comparing the amount of at least one indicator to a control level of the indicator. When more than one indicator is being evaluated, theindicator levels are compared with the control levels for the appropriate corresponding indicators; e.g., the level of herpes virus in the biological sample is compared with the control level of herpes virus. The control levels may be already available in the literature, they may be determined by evaluating biomarker levels in a pool of relatively healthy subjects, or they may be determined in an individual prior to the development of a disease or disorder.Formulation and Administration

[0056] The present invention provides methods comprising administration of a therapeutically effective amount of BCV in a pharmaceutical composition. Examples of pharmaceutical compositions include those for oral, intravenous, intramuscular, subcutaneous, transdermal, or intraperitoneal administration, or any other route known to those skilled in the art, and generally involves providing the BCV formulated together with a pharmaceutically acceptable carrier. The dosage form should be effective for therapeutic use and may be, for example, a solid preparation (e.g., a tablet(s)), a liquid preparation (e.g., suspension), or an injection (e.g., an intravenous injection). An embodiment of the present invention provides a pharmaceutical composition comprising BCV for use in any of the above methods. The methods include, for example, a method of suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau in a subject, the method comprising administering an effective amount of BCV to the subject. The methods include, for example, a method of treating a disease accompanied by amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau, comprising administrating an effective amount of BCV to the subject. An embodiment of the present invention provides a use of BCV for production of the pharmaceutical composition described above.

[0057] The dose, dosing interval, and administration method of a therapeutically effective amount of BCV may be selected. The amount of BCV that is administered and the dosage regimen for treating a disease condition with the compounds and / or compositions of this invention depends on a variety of factors, including the age, weight, sex, and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound employed, the location of the unwanted proliferating cells, as well as the pharmacokinetic properties of the individual treated, and thus may vary widely. The dose may be, for example, from 0.1 to 200 mg / kg body weight per administration. The dosing interval, for example, may be 1 to 28 days.

[0058] The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are may includeconventional additives, binders, disintegrators, or lubricants. The active ingredient may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier.

[0059] For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, the compound may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The formulations may be present in unit or multidose containers such as sealed ampoules or vials.

[0060] Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably made isotonic. Preparations for injections may also be formulated by suspending or emulsifying the compounds in non-aqueous solvent.

[0061] The BCV can also be provided as a pharmaceutically acceptable salt. The phrase “pharmaceutically acceptable salts” connotes salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds may be prepared from an inorganic acid or from an organic acid.

[0062] Some embodiments of the invention are within the scope of the following numbered paragraphs.1. A method of suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau in a subject in need thereof, the method comprising administering an effective amount of brincidofovir (BCV) to the subject.2. The method of paragraph 1, wherein the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau occurs in response to viral infection.3. The method of paragraph 2, wherein the virus is a herpes virus.4. The method of paragraph 3, wherein the herpes virus is Herpes simplex virus type I (HSV-1), Herpes simplex virus type 2 (HSV-2), or Varicella-zoster virus (VZV).5. The method of any one of paragraphs 1 to 4, which is a method of suppressing amyloid P aggregation.6. The method of any one of paragraphs 1 to 4, which is a method of suppressing neuronal cell degeneration.7. The method of any one of paragraphs 1 to 4, which is a method of suppressing phosphorylation of Tau.8. The method of any one of paragraphs 1 to 4, which is a method of suppressing amyloid P aggregation, neuronal cell degeneration, and phosphorylation of Tau.9. The method of any one of paragraphs 1 to 8, wherein the subject is in a state of reactivation of herpes virus.10. The method of any one of paragraphs 1 to 9, further comprising detecting reactivation of herpes virus in the subject.11. The method of paragraph 10, wherein the detecting comprises detecting an antibody against herpes virus antigen or detecting herpes virus genomic DNA.12. The method of any one of paragraphs 1 to 11, further comprising identifying a subject in a state of reactivation of a herpes virus as a treatment target for a neurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease.13. The method of any one of paragraphs 1 to 12, wherein the subject has an ApoE4 allele.14. The method of any one of paragraphs 1 to 13, further comprising detecting an ApoE4 allele in the subject.15. The method of any one of paragraphs 1 to 14, further comprising identifying a subject with an ApoE4 allele as a treatment target for a neurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease.16. The method of any one of paragraphs 1 to 15, further comprising diagnosing an onset of a neurodegenerative disease in the subject and identifying the subject with the onset of the neurodegenerative disease as a candidate for treatment, wherein the identified subject is tested for the presence or absence of reactivation of herpes virus.17. The method of any one of paragraphs 1 to 16, wherein the subject has a neurodegenerative disease.18. The method of any one of paragraphs 1 to 17, wherein the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau is accompanied by a neurodegenerative disease.19. The method of any one of paragraphs 1 to 18, wherein the suppression is carried out in a treatment of a neurodegenerative disease.20. The method of any one of paragraphs 1 to 19, wherein the suppression of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau results in a treatment of a neurodegenerative disease.21. The method of paragraph 20, wherein the neurodegenerative disease occurs as a result of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau.22. The method of paragraph 17, wherein the neurodegenerative disease is Alzheimer's disease or dementia.23. The method of paragraph 17, wherein the neurodegenerative disease is Alzheimer's disease.24. The method of any one of paragraphs 1 to 23, which does not comprise testing for edema after administering BCV to the subject.25. The method of any one of paragraphs 1 to 24, wherein the subject is not suitable for receiving or continuing treatment with administration of an antibody against amyloid p.26. A composition comprising BCV for use in the method of any one of paragraphs 1 to 25.27. A use of BCV for production of the composition of paragraph 26.Al. A pharmaceutical composition comprising brincidofovir (BCV) for use in a method of suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau in a subject.A2. The pharmaceutical composition of paragraph Al, wherein the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau occurs in response to viral infection.A3. The pharmaceutical composition of paragraph A2, wherein the virus is a herpes virus.A4. The pharmaceutical composition of paragraph A3, wherein the herpes virus is Herpes simplex virus type I (HSV-1), Herpes simplex virus type 2 (HSV-2), or Varicella-zoster virus (VZV).A5. The pharmaceutical composition of any one of paragraphs Al to A4, which is a pharmaceutical composition for use in a method of suppressing amyloid P aggregation.A6. The pharmaceutical composition of any one of paragraphs Al to A4, which is a pharmaceutical composition for use in a method of suppressing neuronal cell degeneration.A7. The pharmaceutical composition of any one of paragraphs Al to A4, which is a pharmaceutical composition for use in a method of suppressing phosphorylation of Tau.A8. The pharmaceutical composition of any one of paragraphs Al to A4, which is a pharmaceutical composition for use in a method of suppressing amyloid P aggregation, neuronal cell degeneration, and phosphorylation of Tau.A9. The pharmaceutical composition of any one of paragraphs Al to A8, wherein the subject is in a state of reactivation of herpes virus.A 10. The pharmaceutical composition of any one of paragraphs Al to A9, wherein the method further comprises detecting reactivation of herpes virus in the subject.Al l. The pharmaceutical composition of paragraph 10, wherein the detecting comprises detecting an antibody against herpes virus antigen or detecting herpes virus genomic DNA.A 12. The pharmaceutical composition of any one of paragraphs Al to Al l, wherein the method further comprises identifying a subject in a state of reactivation of a herpes virus as a treatment target for a neurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease.A13. The pharmaceutical composition of any one of paragraphs Al to A12, wherein the subject has an ApoE4 allele.A14. The pharmaceutical composition of any one of paragraphs Al to A13, wherein the method further comprises detecting an ApoE4 allele in the subject.A15. The pharmaceutical composition of any one of paragraphs Al to A14, wherein the method further comprises identifying a subject with an ApoE4 allele as a treatment target for aneurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease.A16. The pharmaceutical composition of any one of paragraphs Al to A15, wherein the method further comprises diagnosing an onset of a neurodegenerative disease in the subject and identifying the subject with the onset of the neurodegenerative disease as a candidate for treatment, wherein the identified subject is tested for the presence or absence of reactivation of herpes virus.A17. The pharmaceutical composition of any one of paragraphs Al to A16, wherein the subject has a neurodegenerative disease.A18. The pharmaceutical composition of any one of paragraphs Al to A17, wherein the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau is accompanied by a neurodegenerative disease.A19. The pharmaceutical composition of any one of paragraphs Al to A18, wherein the suppression is carried out in a treatment of a neurodegenerative disease.A20. The pharmaceutical composition of any one of paragraphs Al to A 19, wherein the suppression of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau results in a treatment of a neurodegenerative disease.A21. The pharmaceutical composition of paragraph A20, wherein the neurodegenerative disease occurs as a result of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau.A22. The pharmaceutical composition of paragraph A17, wherein the neurodegenerative disease is Alzheimer's disease or dementia.A23. The pharmaceutical composition of paragraph A17, wherein the neurodegenerative disease is Alzheimer's disease.A24. The pharmaceutical composition of any one of paragraphs Al to A23, wherein the method does not comprise testing for edema after administering BCV to the subject.A25. The pharmaceutical composition of any one of paragraphs Al to A24, wherein the subject is not suitable for receiving or continuing treatment with administration of an antibody against amyloid p.A26. A use of BCV for production of the pharmaceutical composition of any one of paragraphs Al to A25.

[0063] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.EXAMPLESExample 1: Effects of BCV on HSV-1 infection and resulting AD-like phenotypes in the 3D human brain-like tissue model

[0064] The purpose of these tasks is to study the antiviral effects of BCV in our established 3D human brain-like tissue model of HSV-1 -induced AD. The initial experiments were conducted in 2D monolayer cultures to determine relevant drug concentrations of BCV that demonstrate antiviral activity. Human induced neural stem cells (hiNSCs) were cultured in 2D or 3D conditions then exposed to either mock or HSV-1 infection. BCV (or vehicle) was added concurrently with infection at day 0, then cultured for either 7 (for monolayer cultures) or 10 days (for 3D cultures) then assayed for markers of HSV-1 infection as well as AD.MethodsExpansion and differentiation of hiNSCs

[0065] hiNSCs were generated as previously described. hiNSCs were expanded on mouse embryonic fibroblast (MEF) feeder layers that had been previously inactivated by mitomycin C, using hiNSC media: Knockout (KO) DMEM supplemented with 20% KO xeno-free serum replacement, 20 ng / mL recombinant bFGF, 1% Glutamax, 1% antibiotic-antimycotic, and O.lmM B-mercaptoethanol. hiNSC colonies were trypsinized off MEF feeder layers using TrypLE (Invitrogen), then dissociated by pipetting. Cell suspensions were passaged through a 40 pM cell strainer to remove larger aggregates. Dissociated hiNSCs were cultured on laminin-coated plates in Neurobasal media supplemented with 2% B27 (Invitrogen), 1% Glutamax, and 1% antibiotic- antimycotic.3D human brain-like tissue model

[0066] A 3D brain tissue model was generated as previously described. Diez-Domingo, J., et al., Dermatology and therapy, 2021. 11(4): p. 1119-1126. Briefly, silk protein sponges (pore size 500- 600 um) were prepared from 6% (wt / vol) Bombyx on-derived silk solution. Sponges were biopsy punched into 6 mm discs (2 mm in height), with 2 mm holes punched in the center to form donut shaped scaffolds. Scaffolds were autoclaved and coated with laminin (0.5 mg / ml) (Roche, Indianapolis, IN). Dissociated hiNSCs were seeded into the silk porous scaffolds at a density of 106 cells per scaffold and allowed to adhere overnight. The following day, collagen gels were prepared using type I rat tail collagen (Coming, Bedford, MA, USA) as previously described. Cairns, D.M., R.F. Itzhaki, and D.L. Kaplan, J Alzheimers Dis, 2022. 88(3): p. 1189-1200. 3D human brain tissue constructs were then cultured in neurobasal media (Invitrogen, Carlsbad, CA) supplemented with 2% B27 (Invitrogen, Carlsbad, CA), 0.5 mM Glutamax, and 1% antibiotic- antimycotic (Invitrogen, Carlsbad, CA) for 4 or 8 weeks to allow for mature network formation, with medium changes every 3 days.HSV-1 infection and treatment with BCV

[0067] HSV-1 McIntyre strain VR-539 was purchased from ATCC (Manassas, VA). We used purified HSV-1 to directly infect hiNSCs at an MOI of 0.0001 based on our previous studies (Cairns, D.M., R.F. Itzhaki, and D.L. Kaplan, J Alzheimers Dis, 2022. 88(3): p. 1189-1200), which was calculated according to initial seeding density. For HSV-1, the purified vims preparation titration was 2xl07PFU / mL, which was adjusted to the desired MOI through serial dilution, 1 pl per 2,000 ml cell culture media. Purified vims was highly diluted with cell culture medium (1:2,000,000); thus, any potential effects from non-viral contaminants in the virus preparations would have been negligible. For mock infections, an equal volume of control culture medium from uninfected viral production cells was used (ATCC). BCV was prepared according to instmctions provided by Symbio Pharmaceuticals Limited. Briefly, 10 mM stock concentrations were prepared by weighing appropriate amounts of lyophilized BCV powder and reconstituting in PBS (pH = 11.6). Stock concentrations were diluted in cell culture media to prepare experimental concentrations. Valacyclovir HC1 (Sigma) was used as an antiviral control as previously described. Cairns, D.M., et al., Science Advances, 2020. 6(19): p. eaay8828.Lactate Dehydrogenase (LDH) Assay

[0068] Media samples were collected after 30 minutes, 1 day, 2 days, and 4 days during the first round of 3D experiments for assays of LDH levels. Samples were stored at -80 °C upon collection until assay completion. A Sigma- Aldrich Lactate Dehydrogenase Activity Assay Kit (MAK066) was used to perform the LDH assay following the company-provided protocol. Briefly, 50 pL of samples were added to a well along with 50 pL of a 25x dilution of LDH substrate mix. Samples were run alongside a provided standard curve. After incubation for 1 hour samples were read for absorbance at 450 nm using a plate reader. These values were used to calculate levels of LDH expression in each injury type. qRT-PCR

[0069] Total RNA was isolated using the RNeasy Mini kit (Qiagen). cDNA was generated using iScript (BioRad) according to the manufacturers’ protocols. Quantitative RT-PCR (qPCR) was performed using SYBR green and the CFX96 Real-Time PCR Detection System (BioRad) and normalized against the housekeeping gene GAPDH.Immunofluorescence

[0070] Cells grown in monolayer tissue culture plates or in 3D scaffolds were fixed in 4% paraformaldehyde and washed with IX phosphate-buffered saline (PBS). Samples were incubated with blocking buffer (PBS, 10% goat serum, and 0.1% Triton X-100). Primary antibodies were added to blocking buffer and incubated with samples overnight at 4 °C. The next day, samples were washed several times with PBS, and incubated with a corresponding fluorescently conjugated secondary antibody in blocking buffer for 1 h at room temperature. Nuclei were counterstained with DAPI (Invitrogen). Expression values were calculated by measuring pixel intensity using ImageJ and normalizing to cell number.Microscopy

[0071] Brightfield and fluorescent images were obtained using a Keyence BZ-X700 microscope and associated software.RESULTSBCV prevents HSV-1 infection in monolayer hiNSC cultures

[0072] We cultured hiNSCs in monolayer then exposed to mock or HSV-1 infection and treated concurrently with varying concentrations of BCV ranging from 0.001 - 100 pM, as well as positive antiviral control VCV 200 pM (Fig 2). Immunostaining results for HSV infection revealed complete protection against HSV-1 using BCV 0.1 pM (Fig. 2A, B). Importantly, some drug cytotoxicity was observed at BCV 10 pM and higher, however, these levels are not physiologically relevant. Naderer, O., et al., Open Forum Infect Dis, 2018. 5(Suppl 1): p. S438-9. Based on a dosing study in healthy patients, the highest IV dose of BCV (20 mg once per week) resulted in Cmax levels of 1720 ng / ml, which corresponds to roughly 3 pM.BCV prevents herpes-induced expression of AD markers in monolayer hiNSC cultures

[0073] Similar to the results of BCV on HSV-1 infection, the expression of herpes-induced beta amyloid (AB) was also inhibited by BCV, demonstrating total prevention at BCV 0.01 pM (middle pert, 3rd row) (Fig. 3). Treatment with BCV also inhibited alteration (i.e. cell atrophy and axon loss, etc.) of cell morphology by HSV-1 infection, indicating protective effects on resultant neuronal degeneration.Generation of 3D human brain-like tissues

[0074] We generated 3D human brain-like tissues using hiNSCs as previously described. Silk scaffolds were prepared by purifying and casting silk fibroin solution into cylindrical geometries (Fig. 4A). Sterilized scaffolds were coated with laminin then seeded with dissociated hiNSCs. Type I collagen was used to fill the scaffolds, and these brain-like tissue constructs could be grown in long-term culture.Pilot infection and BCV treatment studies using 3D human brain-like tissues

[0075] We conducted initial pilot experiments to test HSV-1 infection and BCV treatment in 3D human brain-like tissues (Fig. 4B). As an initial assay of cytotoxicity, we collected culture media from 3D constructs and ran a lactate dehydrogenase (LDH) assay (Fig. 4C). As expected, HSV-1- infected tissues showed higher LDH release suggesting cell damage. Interestingly, BCV treatment at various concentrations, restored LDH to mock levels.BCV attenuates HSV-1 infection and downstream Abeta expression in 3D human brain-like tissues

[0076] Similar to results seen in monolayer cultures, BCV also prevented HSV-1 infection and herpes-induced amyloid beta expression in 3D human brain-like tissues using concentrations as low as 0.01 pM (Fig. 5A, B). As described in monolayer cultures, we also observed cytotoxicity at BCV >= 10 pM in 3D tissue constructs (Fig. 5C).BCV suppresses herpes-induced expression of AD markers, gliosis and inflammation in 3D human brain-like tissues

[0077] We also used qRT-PCR to analyze mRNA expression in 3D tissues infected with HSV-1 and treated with BCV (Fig. 6). Our previous study to characterize the response of hiNSCs to HSV- 1 infection revealed AD-like responses including downregulation of AD mediators, amyloid precursor protein (APP) and beta secretase 1 (a.k.a. beta- site APP cleaving enzyme 1, BACE1) as well as upregulation of presenilin 2 (PSEN2) (Fig. 6A-D). Cairns, D.M., et al., Science Advances, 2020. 6(19): p. eaay8828. BCV treatment partially restores expression levels to those in mock- infected samples, with the exception of APP. The decrease in presenilin 2, a causative gene for Alzheimer's disease, due to BCV treatment was a particularly surprising result. Similarly, gliosis, as measured by glial acidic fibrillary protein (GFAP) expression, is also attenuated by BCV treatment (Fig. 6E). Various markers of inflammation that are typically upregulated in response to HSV-1 infection, including interleukin 1 beta (ILl-beta), interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) were similarly upregulated due to HSV-1 infection (Fig. 6F-H). Importantly, BCV treatment (at concentrations 0.01 - 1 pM) substantially reduced expression of these inflammatory markers in 3D human brain-like tissues.Example 2: Compare outcomes above with Valacyclovir or other therapy / controls including combinatorial treatment

[0078] The purpose of these tasks is to determine whether other therapeutic modalities including resveratrol, green tea catechins or fucoidan (Silveira, I. A., et al., Free Radic Biol Med, 2022) could outperform BCV in terms of antiviral activity using our hiNSC models. It was discovered that BCV exhibited very potent antiviral efficacy even with concentrations as low as 0.001 pM.

[0079] For comparison, for control antiviral Valacyclovir HC1, the concentration used is 200 pM. In our previous study to test potential neuroprotective compounds, we found that resveratrolalso exhibited protection against AD-like phenotypes, however, this compound too required a much higher concentration (100 pM). Silveira, I.A., et al., Free Radic Biol Med, 2022.Example 3: Effects of BCV at various concentrations / timing of treatment of HSV-1 infection

[0080] The purpose of these tasks is to study the kinetics of antiviral activity of BCV in our hiNSC model. We aimed to understand whether BCV treatment pre-infection and / or post-infection could exhibit the same response as concurrent treatment with HSV-1 infection.MethodsHSV-1 infection and alternating timing of BCV treatment

[0081] HSV-1 McIntyre strain VR-539 was purchased from ATCC (Manassas, VA). We used purified HSV-1 to directly infect hiNSCs at an MOI of 0.0001 based on our previous studies, which was calculated according to initial seeding density. For HSV-1, the purified virus preparation titration was 2xl07PFU / mL, which was adjusted to the desired MOI through serial dilution, 1 pl per 2,000 ml cell culture media. Purified virus was highly diluted with cell culture medium (1:2,000,000); thus, any potential effects from non-viral contaminants in the virus preparations would have been negligible. For mock infections, an equal volume of control culture medium from uninfected viral production cells was used (ATCC). BCV was prepared according to instructions provided by Symbio. Briefly, 10 mM stock concentrations were prepared by weighing appropriate amounts of lyophilized BCV powder and reconstituting in PBS (pH = 11.6). Stock concentrations were diluted in cell culture media to prepare final BCV concentration 1 pM. BCV was added one day prior to HSV-1 infection (Day -1), concurrently with infection (Day 0), or at days 1-3 post-infection (Fig. 7). In a separate experiment, BCV was added concurrently with infection (Day 0), or at days 1-3 post-infection to assess effects on phosphorylation of Tau (Fig. 9A). Media was not changed during BCV addition - drug was spiked into existing cultures at appropriate concentrations so that virus was still present during the course of the study.ResultsEarly BCV treatment post-infection prevents herpes-induced AD-like phenotypes in 3D human brain-like tissues

[0082] We treated 3D human brain-like tissue constructs with 1 pM BCV either one day prior to, concurrently with, or 1-3 days after infecting with HSV-1 to determine whether BCV sustained its antiviral capacity at different stages of infection. Importantly, pre- or concurrent BCV treatment completely prevented herpes-induced Abeta aggregation. Treating with BCV one or two days post-infection offered substantial protection, however, treatment 3 days post-infection resulted in higher Abeta aggregation and neuronal cell loss (Fig. 8A-C). In addition, there was still some level of protective effect relative to those HSV-1 -infected cultures with no drug treatment. Furthermore, while phosphorylation of Tau at Ser202 / Thr205 (pTau) was emerged by HSV-1 infection, treating with BCV concurrently with or 1 day after infecting with HSV-1 completely suppressed the pTau generation (Fig. 9A-B).Example 4: Investigation on ApoE4 per risk for AD outcomes or infections

[0083] The purpose of this task is to determine whether there are any differential effects of BCV in treating HSV-1 infected hiNSCs without the known AD-risk variant, APOE4. For this purpose, we utilized our current hiNSC line, which carries one APOE3 allele and one APOE4 allele. Apolipoprotein E protein is around 300aa. The amino acid sequence for APOE4 is mkvlwaallvtflagcqakveqavetepepelrqqtewqsgqrwelalgrfwdylrwvqtlseqvqeellssqvtqelralmdetmke Ikaykseleeqltpvaeetrarlskelqaaqarlgadmedvrgrlvqyrgevqamlgqsteelrvrlashlrklrkrllrdaddlqkrlavy qagaregaerglsairerlgplveqgrvraatvgslagqplqeraqawgerlrarmeemgsrtrdrldevkeqvaevrakleeqaqqirl qaeafqarlkswfeplvedmqrqwaglvekvqaavgtsaapvpsdnh (SEQ ID NO: 2). The target sequence of the shRNA construct to knockdown APOE expression is: GCAGACACTGTCTGAGCAGGT (SEQ ID NO: 1). This lentiviral knockdown construct likely prevents translation of both APOE3 and E4. This sequence binds to nucleotides that correspond to aa stretches from 73- 115 (depending on which specific sequence you query). The APOE4 variant (C130R) is downstream of this shRNA sequence. The established APOE2 variant is also downstream of this sequence (R176C). Future studies can be conducted using CRISPR-based approaches to specifically knockout the APOE4 allele only.MethodsLenti viral infection of hiNSCs

[0084] Lentiviral preparations were purchased from Sigma. These constructs included shAPOE (cat# SHCLNV), a control GFP construct (cat# SHC003V), and a non-mammalian control shRNA (cat# SHC002V) (Fig. 10). All lentivirus constructs used contained a puromycin resistance cassette, which allows for stable selection of clones with the gene of interest inserted.

[0085] For hiNSC cultures, we routinely use MEF-based feeder cells to expand hiNSCs. However, as the ultimate goal of this study was to generate stable lines using puromycin-based methods of selection, we opted to use a feeder-free system as the presence of puromycin could be cytotoxic to MEF feeder cells. Future studies can be performed to generate feeder plates using MEFs derived from transgenic mice with puromycin resistance.

[0086] iMatrix511(Reprocell), a laminin-based matrix used for culturing feeder- free iPSC lines, was used to coat tissue culture plates according to manufacturer’s instructions. hiNSCs were cultured for several passages before infecting with each construct at MOI = 1. Eentivirally transduced cultures were allowed to grow for several days before puromycin selection.Puromycin selection of lenti virally transduced hiNSC lines

[0087] Once hiNSCs were transduced with various lentiviruses and allowed to grow for several days, titration experiments were initially conducted to determine optimal concentration for puromycin concentration to allow for stable selection of transduced hiNSCs. Concentrations of 0.01, 0.1 and 1 pg / ml puromycin were added to both untransduced as well as lentivirally transduced hiNSC lines to determine which concentration effectively removed untransduced cells without compromising the growth of cells containing the gene of interest.HSV-1 infection of lentivirally transduced hiNSCs and treatment with BCV

[0088] HSV-1 McIntyre strain VR-539 was purchased from ATCC (Manassas, VA). We used purified HSV-1 to directly infect hiNSCs at an MOI of 0.0001 based on our previous studies, which was calculated according to initial seeding density. For HSV-1, the purified virus preparation titration was 2xl07PFU / mE, which was adjusted to the desired MOI through serial dilution, 1 pl per 2,000 ml cell culture media. Purified virus was highly diluted with cell culture medium (1:2,000,000); thus, any potential effects from non-viral contaminants in the viruspreparations would have been negligible. For mock infections, an equal volume of control culture medium from uninfected viral production cells was used (ATCC). BCV was prepared according to instructions provided by Symbio. Briefly, 10 mM stock concentrations were prepared by weighing appropriate amounts of lyophilized BCV powder and reconstituting in PBS (pH = 11.6). Stock concentrations were diluted in cell culture media to prepare 0.1 pM working solutions for monolayer experiments. All virus work was approved by Tufts Institutional Biosafety Committee.RESULTSInfection of hiNSCs with lentiviral constructs

[0089] HiNSCs were infected with shAPOE SHCLNV (Fig. 11A) at varying MOI and assessed for GFP expression. The higher MOI=1 lentiviral transduction resulted in higher GFP expression (Fig. 11B).Puromycin of lentivirally transduced hiNSC lines

[0090] Puromycin titration experiments were conducted to determine optimal concentration to allow for stable selection of transduced hiNSCs. Concentrations of 0.01, 0.1 and 1 pg / ml puromycin were added to both wildtype (WT) untransduced (Fig. 11C) as well as shAPOE hiNSC lines (Fig. 1 ID) to determine which concentration effectively removed untransduced cells without compromising the growth of cells containing the gene of interest. We found that 1 pg / ml puromycin treatment effectively killed most WT hiNSCs (Fig. 11C) suggesting that this concentration is useful for removing any cells lacking a puromycin resistance cassette. In parallel, we found that 1 pg / ml puromycin treatment in shAPOE hiNSCs (Fig. 1 ID) allowed for expansion of mostly GFP+ stably expressing cells, suggesting that most non-transduced cells were effectively removed with this treatment.

[0091] Lentivirally transduced hiNSC lines were expanded for several passages in the presence of 1 pg / ml puromycin to enhance the purity of the respective lines.APOE Knockdown hiNSCs do not develop AD-like morphology in response to HSV-1 infection

[0092] We used HSV-1 (or mock) to infect hiNSCs that were untransduced (Fig. 12A), expressing GFP (Fig. 12B), expressing non-mammalian control shRNA (Fig. 12C) or expression shAPOE (Fig. 12D) using similar protocols as described previously. All three control hiNSC lines tested exhibited the typical disrupted morphology (as evidenced by TUJ1 immunostaining) observed inresponse to HSV-1 infection including neuronal cell loss, blebbed nuclei, and syncytia formation as well as morphological changes (i.e. cell atrophy and the collapse of intracellular fibrous structure) (Fig. 12A-C). In all control cases, treatment with 0.1 pM BCV restored neuronal morphology to normal as previously observed (Fig. 12A-C. The fourth row at the top part has recovered compared to the third row at the top part.). Importantly, the shAPOE hiNSC line demonstrated no discernible response to infection (Fig. 12D. The third row in the top part does not show a typical destroyed form.), suggesting that knockdown of APOE somehow prevents the detrimental effects caused by HSV-1.APOE Knockdown hiNSCs do not express amyloid beta in response to HSV-1 infection

[0093] To further validate the finding that HSV-1 does not produce AD-like phenotypes in APOE knockdown lines, we analyzed additional samples for the expression of herpes-induced amyloid beta (Fig. 13). As shown previously, control hiNSC lines express robust Abeta in response to HSV- 1 expression (Fig. 13A. The staining of A|3 is shown in the second row at the top pert.). Lines expressing shAPOE did not demonstrate any detectable Abeta expression resulting from infection (Fig. 13A. The staining of A|3 is not shown in the fourth row at the top pert.).APOE4 is potentially unregulated in response to HSV-1 infection in untransduced hiNSCs

[0094] As stated previously, the genotype of the current hiNSC line is E3 / E4, meaning that the cells possess one APOE-e4 allele. We assessed expression of APOE-e4 to confirm knockdown in our shAPOE line and confirmed that indeed no APOE-e4 (E4) was detectable (Fig. 13B). Interestingly, we were also able to detect the presence of E4 in untransduced hiNSCs, which seemed to become more highly expressed in response to HSV-1 infection.Example 5: Investigation in animal model for HSV-1 infection-induced AD

[0095] The purpose of this task is to determine in vivo effects of BCV in a mouse model showing HSV-1 infection-induced AD-like pathology and impaired cognitive function. For this purpose, we utilize one of the standard mouse models for AD, 5xFAD transgenic mice, and accelerates development of amyloid pathology and decline of cognitive function by intracranial inoculation of HSV-1.MethodsAnima model and drug treatment

[0096] The 5xFAD transgenic mice are originally obtained from the Jackson laboratory and maintained on the C57BL / 6 background. Bilateral injections of HSV-1 (1 x 105PFUs, Mckrae strain, ATCC) are conducted in 3-month-old male 5xFAD mice at coordinates AP, - 2.0; ML, ± 1.5; DV, - 2.0. This injection was carried out using a 5 pL syringe equipped with a 30- gauge needle attached to a digital stereotaxic apparatus and an infusion pump, administrated at a rate of 0.2 pL / min. Following the completion of each injection, the needle was retained in place for 5 min before gradual withdrawal. Three weeks after the injections, the mice are subjected to the behavioral tests and immunofluorescence analysis of amyloid P deposition.

[0097] Brincidofovir is administered to the treatment group of mice twice per week for up to 3 weeks via intraperitoneal injection at 5, 10 and 20 mg / kg. Saline is administered to control animals in a similar manner (n = 10 mice per group).Behavioral test (Morris water maze test)

[0098] A circular water tank (diameter: 120 cm) is filled with water to a depth of 27 cm, and the water is made opaque with non-toxic white paint. A 13-cm in diameter round platform is hidden 1 cm beneath the surface of the water at the center of a given quadrant of the water tank. Mice receive training for five consecutive days from one day after the last BCV or vehicle dose, with each session comprising four trails starting from different sites. For each trail, the mouse is gently released from the wall of the tank and allowed to explore, locate and stand on the platform for a duration of 20 s, within the 60-s trail period. Following the completion of training, a probe test is performed 24 h later. During the probe test, the platform is removed and task performance including swimming tracks, speed, time spent and entries into the platform are recorded for analysis. Animal’s behaviors are recorded in videos and analyzed by an application equivalent to Smart V3.0.03 software (Panlab)Immunofluorescence analysis of amyloid B deposition

[0099] Mice are anesthetized with 5% chloral hydrate and perfused with cold PBS, followed by 4% paraformaldehyde (Aladdin, PH 7.4). Subsequently, mouse brains are harvested and coronally sectioned into 40 pm-thick serial sections. The brain sections are then washed three times with PBS and blocked for 2 h with a blocking buffer (3% BSA containing 0.1% Triton X-100) at roomtemperature, followed by overnight incubation with primary antibodies against oligomeric AB42 (M0AB2, Abeam) with 1:1000 dilution at 4 °C. After washing, the sections are labeled with fluorescent secondary antibodies conjugated to Alexa Fluor 568 / 594 / 647 in blocking buffer containing DAPI (Invitrogen). The slides are then observed with a fluorescence microscopy (OLYMPUS).Statistical analysis

[0100] All statistical analyses are performed using Prism 8.0 (GraphPad Software). Datasets are analyzed for p-values using either unpaired Student’s t tests or ANOVA multiple comparison tests; all data are presented as means ± SEM. Statistical significance is represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: p > 0.05.RESULTS

[0101] During the training phase, each group shows improved latency to the platform, but compared with the control group, the HSV-1 infected group exhibits a significant delay to locate the platform. In the probe test, compared to the control group, the 5xFAD-HSV group exhibits a significantly longer latency to locate the platform, but fewer target crossings. There is no significant difference in swimming speeds between the two groups of mice, thus excluding impairments in motor function. At 3 months of age, 5xFAD mice do not exhibit significant deficits in spatial learning and memory, thus these results indicate that HSV-1 infection causes impairments in spatial learning and memory. In contrast, the animal group treated with BCV, such impairments in cognitive functions are not observed.

[0102] Representative images of A[3 staining and quantification of A|3 plaques in the hippocampus of 5xFAD mice infected with HSV-1 show significant increase in A[3 depositions compared to control animals. However, treatment with BCV significantly reduces the total A|3 deposition in 5xFAD mice with HSV-1 infection.

[0103] These results indicate that BCV can suppress the decline of cognitive impairment caused by HSV-1 infection in the brain through blocking A|3 deposition accelerated by HSV-1 infection.

Claims

CLAIMSWhat is claimed is:

1. A method of suppressing amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau in a subject in need thereof, the method comprising administering an effective amount of brincidofovir (BCV) to the subject.

2. The method of claim 1, wherein the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau occurs in response to viral infection.

3. The method of claim 2, wherein the virus is a herpes virus.

4. The method of claim 3, wherein the herpes virus is Herpes simplex virus type I (HSV-1), Herpes simplex virus type 2 (HSV-2), or Varicella-zoster virus (VZV).

5. The method of claim 1, which is a method of suppressing amyloid P aggregation.

6. The method of claim 1, which is a method of suppressing neuronal cell degeneration.

7. The method of claim 1, which is a method of suppressing phosphorylation of Tau.

8. The method of claim 1, which is a method of suppressing amyloid P aggregation, neuronal cell degeneration, and phosphorylation of Tau.

9. The method of claim 1, wherein the subject is in a state of reactivation of herpes virus.

10. The method of claim 1, further comprising detecting reactivation of herpes virus in the subject.

11. The method of claim 10, wherein the detecting comprises detecting an antibody against herpes virus antigen or detecting herpes virus genomic DNA.

12. The method of claim 1, further comprising identifying a subject in a state of reactivation of a herpes virus as a treatment target for a neurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease.

13. The method of claim 1, wherein the subject has an ApoE4 allele.

14. The method of claim 1, further comprising detecting an ApoE4 allele in the subject.

15. The method of claim 1, further comprising identifying a subject with an ApoE4 allele as a treatment target for a neurodegenerative disease, wherein the suppression is carried out in a treatment of a neurodegenerative disease.

16. The method of claim 1, further comprising diagnosing an onset of a neurodegenerative disease in the subject and identifying the subject with the onset of the neurodegenerative disease as a candidate for treatment, wherein the identified subject is tested for the presence or absence of reactivation of herpes virus.

17. The method of claim 1, wherein the subject has a neurodegenerative disease.

18. The method of claim 1, wherein the amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau is accompanied by a neurodegenerative disease.

19. The method of claim 1, wherein the suppression is carried out in a treatment of a neurodegenerative disease.

20. The method of claim 1, wherein the suppression of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau results in a treatment of a neurodegenerative disease.

21. The method of claim 20, wherein the neurodegenerative disease occurs as a result of amyloid P aggregation, neuronal cell degeneration, or phosphorylation of Tau.

22. The method of claim 17, wherein the neurodegenerative disease is Alzheimer's disease or dementia.

23. The method of claim 17, wherein the neurodegenerative disease is Alzheimer's disease.

24. The method of claim 1, which does not comprise testing for edema after administering BCV to the subject.

25. The method of claim 1, wherein the subject is not suitable for receiving or continuing treatment with administration of an antibody against amyloid p.

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