Wild-derived mouse model of Alzheimer's disease

Abstract

The present disclosure provides wild-derived mouse models comprising a nucleic acid encoding human amyloid precursor protein (APP), a nucleic acid encoding mutant human presenilin 1 protein (PSEN1), and in some embodiments, human apolipoprotein E (APOE) or human amyloid beta and human tau. These mouse models are useful, for example, for Alzheimer's disease research. In some embodiments, the wild-derived humanized mouse models of AD express human amyloid precursor protein (APP), express mutant human presenilin 1 protein (PSEN1, also abbreviated as PSEN1), and express (a) human apolipoprotein (APOE) (e.g., human apolipoprotein E4, human apolipoprotein E3, or human apolipoprotein E2), or (b) human amyloid beta and / or human tau.

Description

[Technical field]

[0001] (Related Applications) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 318,313, filed March 9, 2022, which is incorporated by reference herein in its entirety.

[0002] (Reference to Electronic Sequence Listing) The contents of the electronic sequence listing (J022770116WO00-SEQ-EAS.xml; size: 4,984 bytes; and creation date: March 9, 2023) are incorporated by reference in their entirety herein. [Background technology]

[0003] (background) Transgenic mouse models expressing human amyloid precursor protein (APP) with or without expression of human presenilin 1 (PSEN1) have been used extensively to study Alzheimer's disease (AD) in vivo, leading to a better understanding of the pathogenesis of the disease in human patients. Nevertheless, such models often only poorly recapitulate the widespread neurodegeneration and focal brain atrophy that occurs in AD (Drummond et al., Acta Neuropathol., 2017 Feb;133(2):155-175). Moreover, such models have been generated based on limited background strains (e.g., C57BL6 / J). These transgenic mouse strains have shown significant parenchymal amyloid deposit burdens that may exceed those observed in human patients. In general, neuroinflammation, another key feature, has been limited to responses specific to parenchymal plaques. Due to all of the above reasons, existing transgenic mouse models expressing APP are limited in their ability to recapitulate human-associated AD pathology. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Drummond et al., Acta Neuropathol., 2017 Feb;133(2):155-175 Summary of the Invention [Means for solving the problem]

[0005] (overview) The present disclosure, in some aspects, provides an improved wild-derived "humanized" mouse model of Alzheimer's disease (AD). As known in the art, mice expressing human genes / proteins are often referred to as "humanized" mice. It should be understood that human genes include human nucleic acid sequences that code for human proteins or human protein domains. In some embodiments, the wild-derived humanized mouse model of AD expresses human amyloid precursor protein (APP), expresses mutant human presenilin 1 protein (PSEN1, also abbreviated as PSEN1), and expresses (a) human apolipoprotein (APOE) (e.g., human apolipoprotein E4, human apolipoprotein E3, or human apolipoprotein E2) or (b) human amyloid beta and / or human tau. Modeling AD in a wild-derived background allows a platform for studying interactions with amyloid in a more human-relevant genetically diverse background. This background also allows exploration of a range of neuroinflammatory responses.

[0006] The wild-derived humanized mouse model provided herein is based at least in part on the theory that studying the pathogenesis of AD by modulating known genetic risk factors in genetically diverse mouse backgrounds will result in a more clinically relevant model.This theory was tested by introducing human risk allele APOE4, as well as human amyloid beta and / or human tau, into a wild-derived humanized mouse model expressing human APP and mutant human PSEN1.First, a WSB.APOE4 mouse model was generated.The WSB.APP / PSEN1 model was then crossed with this WSB.APOE4 mouse model to generate a new wild-derived humanized mouse model expressing human APP, mutant human PSEN1 and human APOE4 ("WSB.APP / PSEN1 / APOE4" model).It was surprisingly shown that the wild-derived humanized mouse model of AD containing transgenic amyloid mutations leads to neurodegeneration in both the cortex and hippocampus of female mice as early as 8 months of age.

[0007] Accordingly, some aspects of the present disclosure provide a wild-derived humanized mouse comprising in its genome a nucleic acid encoding human amyloid precursor protein (APP), a nucleic acid encoding a mutant human presenilin 1 protein (PSEN1), and a nucleic acid encoding human apolipoprotein E.

[0008] In some embodiments, the human apolipoprotein E is human apolipoprotein E4 (APOE4).

[0009] Another aspect of the present disclosure provides a wild-derived humanized mouse comprising in its genome a nucleic acid encoding human amyloid precursor protein (APP), a nucleic acid encoding a mutant human presenilin 1 protein (PSEN1), a nucleic acid encoding human amyloid beta, and a nucleic acid encoding human tau.

[0010] In some embodiments, the nucleic acid encoding the human APP is a chimeric nucleic acid comprising a mouse coding sequence and a human coding sequence.

[0011] In some embodiments, the chimeric nucleic acid comprises a human coding sequence in the A-beta domain of the mouse APP coding sequence.

[0012] In some embodiments, the chimeric nucleic acid encodes the human mutation K595N and the human mutation M596L, as compared to a human APP comprising the amino acid sequence of SEQ ID NO:1.

[0013] In some embodiments, the nucleic acid encoding human APP is an APPswe transgene.

[0014] In some embodiments, the nucleic acid encoding the mutant PSEN1 comprises a human PSEN1 coding sequence that includes a deletion in exon 9.

[0015] In some embodiments, the nucleic acid encoding the mutant PSEN1 is a PSEN1de9 transgene.

[0016] In some embodiments, the mouse comprises the Tg(APPswe,PSEN1de9)85Dbo transgene insertion in its genome.

[0017] In some embodiments, the mouse expresses human APP, human PSEN1, and human apolipoprotein E (optionally human APOE4).

[0018] In some embodiments, the mouse expresses human amyloid beta and human tau.

[0019] In some embodiments, the mouse has a genetic background selected from WSB / EiJ, CAST / EiJ, and PWK / PhJ.

[0020] In some embodiments, the mouse has at least one characteristic of early onset Alzheimer's disease, e.g., the at least one characteristic of early onset Alzheimer's disease selected from neurodegeneration compared to a control, cognitive deficits compared to a control, and increased neuroinflammation in the brain compared to a control.

[0021] In some embodiments, the mice do not develop tumors or have a measurable tumor burden.

[0022] In some embodiments, the mouse is at least 1 year old.

[0023] Some aspects of the disclosure provide a wild-derived humanized WSB mouse comprising in its genome an APPswe transgene, a PSENde9 transgene, and a gene encoding human apolipoprotein E (optionally human apolipoprotein E4 (APOE4)).

[0024] Another aspect of the disclosure provides a wild-derived humanized mouse that comprises in its genome a nucleic acid encoding human apolipoprotein E, optionally human apolipoprotein E4 (APOE4).

[0025] Yet another aspect of the present disclosure provides a wild-derived humanized mouse that comprises humanized amyloid beta and humanized tau in its genome.

[0026] Yet another aspect of the disclosure provides a cell derived from the mouse according to any one of the above paragraphs.

[0027] A further aspect of the disclosure provides a mouse comprising a cell having the same genotype as a cell derived from a mouse according to any one of the above paragraphs.

[0028] Some aspects of the disclosure provide a mouse that is a progeny of a mouse described in any one of the above paragraphs.

[0029] Also provided herein, in some aspects, is a progeny mouse of a mouse described in any one of the above paragraphs. [Brief description of the drawings]

[0030] [Figure 1] Figure 1A and Figure 1B show immunohistochemistry and corresponding quantification of brain tissue from 8-month-old female mice of WSB, WSB.APOE4, WSB.APP / PSEN1 and WSB.APP / PSEN1 / APOE4 genotypes. Figure 1A shows brain tissue stained with DAPI and antibody NEUN in multiple coronal brain sections covering the anterior, central and posterior regions of the brain. Figure 1B shows the amount of NEUN measured in traced cortical regions (top) and a comparison of cortical surface area (bottom) averaged between each genotype.

[0031] [Diagram 2] FIG. 2 is a graph showing a comparison of cortical surface area of ​​brain tissue from 4-month-old female mice of the WSB genotype, WSB.APOE4 genotype, WSB.APP / PSEN1 genotype, and WSB.APP / PSEN1 / APOE4 genotype. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] (Detailed Description) Alzheimer's disease (AD) is the most common cause of dementia. AD currently affects 35 million people, and its global prevalence is expected to reach 115 million by 2050 due to the aging of the population. AD progresses through three stages: pre-symptomatic, mild cognitive impairment (MCI), and dementia. While people with MCI have cognitive deficits but no functional impairment, people with dementia show decline in two or more cognitive domains that have progressed gradually to the point of impairing function at work or in daily activities. Pathologically, AD diagnosis in humans is based on protein aggregates in the brain, including amyloid plaques composed of amyloid-β (Aβ) peptides and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau. In humans, the early spatial distribution of plaque pathology, including plaque pathology that first occurs in the hippocampus, strongly correlates with the diagnosis of dementia.

[0033] Mouse models of AD are limited in that none of the existing methods show the full range of clinical and pathological features of AD, including cognitive and behavioral deficits, amyloid plaques, neurofibrillary tangles, gliosis, synapse loss, axonopathy, neuronal loss, and neurodegeneration.Importantly, different mouse models provide different degrees of AD phenotype.For example, phenotypes such as cognitive deficits and amyloid plaques are observed in almost all mouse models of AD, but the human pathology of AD has not yet been reproduced.In the B6.APP / PSEN1 mouse model, for example, plaque deposition in the hippocampus and solid cortex is observed at early time points, which is in contrast to the human pathology in which plaques are mainly restricted to the hippocampus.Unlike the B6.APP / PSEN1 mouse model, the mouse model of the present disclosure (which models AD in a wild-derived background) shows severe neurodegeneration, which more closely resembles human AD pathology.

[0034] In some embodiments, the present disclosure provides a wild-derived humanized mouse model (e.g., a WSB / EiJ mouse model) that includes human amyloid precursor protein (APP), mutant human presenilin 1 protein (PSEN1), and human apolipoprotein E (e.g., human apolipoprotein E4, human apolipoprotein E3, or human apolipoprotein E2). In some embodiments, the present disclosure provides a wild-derived humanized mouse model (e.g., a WSB / EiJ mouse model) that includes human APP, mutant human PSEN1, and human amyloid beta and / or human tau.

[0035] (Parent lines and alleles) In some embodiments, the mouse models provided herein are generated in a wild-type derived genetic background (e.g., WSB background). In some embodiments, the mouse models provided herein are generated using a WSB / EiJ strain (e.g., WSB.Cg-Tg(APPswe,PSEN1dE9)85Dbo / How strain (WSB.APP / PS1)). WSB.APP / PS1 is a double transgenic mouse expressing chimeric mouse / human amyloid precursor protein (Mo / HuAPP695swe) and mutant human presenilin 1 (PS1-dE9), both to central nervous system (CNS) neurons. Both mutations are associated with early-onset Alzheimer's disease. The "humanized" Mo / HuAPP695swe transgene allows the mouse to secrete human Aβ peptide. Both the transgenic peptide and the holoprotein can be detected by an antibody specific for human sequences in this region (monoclonal 6E10 antibody from Signet Laboratories). The contained Swedish mutation (K595N / M596L) increases the amount of Aβ produced from the transgene by favoring processing via the β-secretase pathway. WSB.APP / PS1 female hemizygotes show increased loss of cortical regions and CA1 NEUN+DAPI+ (hippocampal) neurons, as well as short-term memory impairment, compared to controls. Cortical plaques are fewer in number compared to B6.APP / PS1 mice. WSB.APP / PS1 males, but not WSB.APP / PS1 females, show a reduced number of hippocampal plaques (compared to B6.APP / PS1 controls). WSB.APP1 / PS1 mice were generated by backcrossing B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo / Mmjax (JAX stock no. 34832-JAX) mice to WSB / EiJ (JAX stock no. 001145) mice for 12 generations.

[0036] In some embodiments, the mouse models provided herein are generated in a wild-type derived genetic background (e.g., PWK background). In some embodiments, the mouse models provided herein are generated using a PWK / PhJ strain (e.g., PWK.Cg-Tg(APPswe,PSEN1dE9)85Dbo / How strain (PWK.APP / PS1)). PWK.APP1 / PS1 mice were generated by backcrossing B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo / Mmjax (stock number 34832-JAX) mice to PWK / PhJ (stock number 003715) mice for 11 generations.

[0037] In some embodiments, the mouse models provided herein are generated in a wild-type derived genetic background (e.g., CAST background). In some embodiments, the mouse models provided herein are generated using the CAST / EiJ strain. This strain can be backcrossed for multiple generations to, for example, B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo / Mmjax (JAX stock number 34832-JAX).

[0038] (Amyloid Precursor Protein) Amyloid precursor protein is a single-pass (type I) transmembrane precursor protein that is cleaved into amyloid beta (Aβ), the major component of amyloid plaques, and is associated with at least one feature of early-onset Alzheimer's disease. Knocking in chimeric mouse / human amyloid precursor protein can result in the secretion of human amyloid beta (Aβ) peptides. In some embodiments, the mouse model contains a chimeric nucleic acid that contains a human coding sequence in the A-β domain of the mouse APP coding sequence. In some embodiments, the chimeric nucleic acid encodes the human Swedish mutations K595N and M596L for human APP that contains the amino acid sequence of SEQ ID NO:1. The included Swedish mutations (K595N and M596L) increase the amount of Aβ produced from the transgene by favoring processing through the beta-secretase pathway (Shin et al., 2010). In some embodiments, the chimeric nucleic acid is an APPswe transgene, which encodes a chimeric amyloid beta (A4) precursor protein that contains the Swedish mutations K595N and M596L. See, e.g., Borchelt, David R., et al. Neuron 1996; 17(5): 1005-1013; JAX Stock No. 025970.

[0039] In some embodiments, the human amyloid precursor protein is SEQ ID NO:1: [ka] [ka] The present invention also includes sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of

[0040] (Presenilin 1) Presenilin 1 (PSEN1) is a subunit of the gamma (γ-) secretase complex involved in the cleavage of APP to generate amyloid β peptide. A mouse model expressing mutant human presenilin 1 and a human APP transgene is associated with at least one feature of early-onset Alzheimer's disease. In some embodiments, the nucleic acid encoding mutant PSEN1 comprises a human PSEN1 coding sequence that includes a deletion in exon 9 (ΔE9). In some embodiments, the nucleic acid is a PSEN1de9 transgene. In some embodiments, the PSEN1de9 transgene is a Tg(APPswe,PSEN1de9)85Dbo transgene insertion (see, e.g., Borchelt et al. 1996; JAX Stock No. 025970).

[0041] In some embodiments, the human presenilin 1 protein is SEQ ID NO:2: [ka] The present invention also includes sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of

[0042] (Apolipoprotein E (APOE)) The APOE gene provides the instructions for making apolipoprotein E. This protein combines with fats (lipids) in the body to form molecules called lipoproteins. Lipoproteins are responsible for packaging cholesterol and other fats and transporting them through the bloodstream. Maintaining normal levels of cholesterol is essential for the prevention of disorders that affect the heart and blood vessels (cardiovascular disease), including heart attacks and strokes.

[0043] There are at least three slightly different versions (alleles) of the APOE gene.The major alleles are APOE2, APOE3 and APOE4.The most common allele is APOE3, which is found in more than half of the general population.In some embodiments, the nucleic acid encodes human APOE2, human APOE3 or human APOE4.

[0044] Apolipoprotein E4 (APOE4) is a lipoprotein-binding protein involved in lipoprotein metabolism and is one of the greatest known genetic risk factors for late-onset sporadic AD. In some embodiments, the nucleic acid encodes human APOE4.

[0045] In some embodiments, the human APOE4 protein is SEQ ID NO:3: [ka] The present invention also includes sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of

[0046] (Amyloid β) Amyloid-β refers to a 36-43 amino acid peptide that is a major component of amyloid plaques found in the brains of people with AD. The peptide is derived from amyloid precursor protein (APP), which is cleaved by β-secretase and γ-secretase to generate Aβ in a cholesterol-dependent process and substrate presentation. In some embodiments, the nucleic acid encodes human amyloid-β.

[0047] (Tau) Tau refers to a soluble protein isoform generated by alternative splicing from the gene MAPT (microtubule-associated protein tau) and is associated with neurological pathologies and dementia (e.g., AD). In some embodiments, the nucleic acid encodes human tau.

[0048] (Assessment of neurodegeneration and other symptoms of Alzheimer's disease) Alzheimer's disease is a brain disorder that slowly destroys memory and thinking skills, eventually destroying the ability to perform the simplest tasks. In most people with Alzheimer's disease, symptoms first appear later in life. Estimates vary, but experts suggest that more than six million Americans, most of whom are age 65 or older, may have dementia caused by Alzheimer's disease. Alzheimer's disease currently ranks as the seventh leading cause of death in the United States and is the most common cause of dementia in older adults.

[0049] Dementia is the loss of cognitive function (thinking, remembering, and reasoning) and behavioral abilities to the extent that it interferes with a person's daily life and activities. Dementia ranges in severity from the mildest stage, where it just begins to affect a person's functions, to the most severe stage, where the person must be completely dependent on others for assistance with basic activities of daily living. The causes of dementia can vary, depending on the type of brain changes that may occur. Other dementias include Lewy body dementia, frontotemporal lobe disorder, and vascular dementia. It is common for people to have mixed dementia (a combination of two or more types of dementia). For example, some people have both Alzheimer's disease and vascular dementia.

[0050] As used herein, characteristics of early-onset Alzheimer's disease include, but are not limited to, neurodegeneration, vascular deficit, mitochondrial dysfunction, cognitive deficits, amyloid plaque deposition, cortical plaque deposition, and neuroinflammation in the brain.

[0051] In some embodiments, the wild-derived humanized mouse model of the present invention comprises in its genome a nucleic acid encoding human amyloid precursor protein (APP), a nucleic acid encoding mutant human presenilin 1 protein (PSEN1), and a nucleic acid encoding human apolipoprotein E (e.g., human apolipoprotein E4, human apolipoprotein E3, or human apolipoprotein E2) (e.g., WSB.APP / PSEN1 / APOE4 mouse and / or WSB.APOE4). Surprisingly, the mouse has at least 20%, at least 30%, or at least 40% greater, or about 20% to about 40% greater, neurodegeneration compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice) (see Figures 1A-1B and 2). As used herein, neurodegeneration refers to a decrease or deficiency of neurons. Neurodegeneration can be measured using immunofluorescent staining of NEUN (a substitute for neuron) in regions of the mouse brain. Immunofluorescence staining methods, which are well known in the art, are contemplated herein.Positive staining for NEUN (indicating neurons) can be present in coronal brain sections covering the anterior, central and posterior regions of the mouse brain of the present disclosure.The total immunofluorescence staining of neurodegeneration in one region of wild-derived mouse brain can be compared to the total immunofluorescence staining in different regions of the same wild-derived mouse brain.The total immunofluorescence staining of neurodegeneration in mouse brain can also be compared to the total immunofluorescence staining of neurodegeneration in control mouse brain.

[0052] In some embodiments, in the wild-derived humanized mouse model of the present disclosure, neurodegeneration in a brain region may be increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% compared to neurodegeneration in a brain region of an age-matched control mouse. In some embodiments, neurodegeneration may be assessed using a spot counting immunohistochemistry assay. In some embodiments, in the wild-derived humanized mouse model, neurodegeneration in a brain region may be increased by at least 20% compared to neurodegeneration in a brain region of an age-matched control mouse. In some embodiments, neurodegeneration in a brain region may be increased by about 20% compared to neurodegeneration in a brain region of an age-matched control mouse. In other embodiments, neurodegeneration may be assessed based on surface area. In some embodiments, in the wild-derived humanized mouse model, neurodegeneration in a brain region may be increased by at least 35% or at least 40% compared to neurodegeneration in a brain region of an age-matched control mouse. In some embodiments, neurodegeneration in a brain region in a wild-derived humanized mouse model may be increased by about 35% or about 40% compared to neurodegeneration in a brain region in an age-matched control mouse.

[0053] In some embodiments, the wild-derived humanized mouse model of the present disclosure has greater vascular impairment compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice). As used herein, vascular impairment refers to any reduction in vascular function known to be associated with AD. Vascular impairment can be measured using immunofluorescence staining of known vascular markers. Immunofluorescence staining methods, which are well known in the art, are contemplated herein. Total immunofluorescence staining of vascular impairment in a region of a wild-derived humanized mouse brain can be compared to total immunofluorescence staining in a different region of the same wild-derived mouse brain. Total immunofluorescence staining of vascular impairment in mouse brain can also be compared to total immunofluorescence staining of vascular impairment in a control mouse brain.

[0054] In some embodiments, in a wild-derived humanized mouse model of the present disclosure, vascular damage in a brain region may be increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to vascular damage in a brain region of an age-matched control mouse.

[0055] In some embodiments, the wild-derived humanized mouse model of the present disclosure has greater mitochondrial dysfunction compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice). As used herein, mitochondrial dysfunction refers to any reduction in mitochondrial function known to be associated with AD. Mitochondrial dysfunction can be measured using immunofluorescence staining of known mitochondrial markers. Immunofluorescence staining methods, which are well known in the art, are contemplated herein. Total immunofluorescence staining of mitochondrial dysfunction in one region of a wild-derived mouse brain can be compared to total immunofluorescence staining in a different region of the same wild-derived mouse brain. Total immunofluorescence staining of mitochondrial dysfunction in mouse brain can also be compared to total immunofluorescence staining of mitochondrial dysfunction in control mouse brain.

[0056] In some embodiments, mitochondrial dysfunction in brain regions in a wild-derived humanized mouse model may be increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to mitochondrial dysfunction in brain regions of age-matched control mice.

[0057] In some embodiments, the wild-derived humanized mouse model of the present disclosure has a greater cognitive deficit compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice). As used herein, cognitive deficit is used to describe impairments in various cognitive domains and is used interchangeably with the term cognitive impairment. Cognitive deficits in the mice of the present disclosure can be measured according to any of the following behavioral assays, including, but not limited to: Y-maze measures for working and short-term memory, hole board, open field, and touch screen.

[0058] The Y-maze is used to assess short-term memory in mice. Spontaneous alternation (a measure of spatial working memory) can be assessed by allowing mice to explore all three arms of the maze, and spontaneous alternation is driven by rodents' innate curiosity to explore previously unvisited areas. Mice with intact working memory, and therefore intact prefrontal cortex function, show a tendency to recall previously visited arms and enter less recently visited arms. Spatial reference memory (which is emphasized by the hippocampus) can also be tested by placing test mice in a Y-maze with one arm closed during training. This test is based on the spontaneous tendency of mice to spend more time exploring novel arms than familiar arms. In some embodiments, the wild-derived humanized mouse model of the present disclosure can spend equal or less time exploring novel arms compared to familiar objects when compared to control mice.

[0059] The hole-board task is used to measure exploratory behavior, locomotor activity and cognitive function in mice. The test is based on the natural curiosity and attraction of mice to novelty. The hole-board consists of a small square arena with a removable platform as the floor, which has a set of circular holes equally spaced on its surface. Mice approach the holes spontaneously and explore them by briefly inserting their noses inside (a behavior defined as nose-poking (or head-dipping)). After a period of time, when mice are re-exposed to the hole-board, the novelty of the holes is reduced. Animals with intact long-term memory show a reduced frequency of nose-poking into holes. In some embodiments, the wild-derived humanized mouse model of the present disclosure can have increased nose-poking events compared to control mice, which avoid nose-poking into holes previously explored by themselves.

[0060] The open field task is a brief sensorimotor test used to determine general activity level, gross locomotor activity, and exploration behavior in rodent models of central nervous system (CNS) disorders. Evaluation is carried out in a rectangular, white Plexiglas box. Animals are placed in the arena and allowed to move freely for 10 minutes, during which time they are recorded by an overhead camera. The footage is then analyzed by an automated tracking system for the following parameters: distance traveled, speed, and time spent in predefined areas. In some embodiments, the wild-derived humanized mouse model of the present disclosure can show different levels of activity level, gross locomotor activity, and exploration behavior compared to control mice.

[0061] Touch screen task is a method for performing simple and complex cognitive neuroscience measurements in mice.Touch screen task uses an automated touch screen platform, and can test a wide variety of cognitive functions in mice.In some embodiments, the wild-derived humanized mouse model of the present disclosure can show lower cognitive function compared to control mice.

[0062] In some embodiments, the wild-derived humanized mouse model of the present disclosure has increased amyloid plaque deposition in the hippocampus region of the brain compared to the cortical region of the brain. As used herein, amyloid plaque deposition refers to Aβ protein deposition, which gradually accumulates to form plaque-like lesions throughout the entire length of the mouse. Amyloid plaque deposition can be measured using immunofluorescence staining of amyloid precursor protein in the cortical and / or hippocampus regions of the mouse brain. Immunofluorescence staining methods, which are well known in the art, are contemplated herein. Positive staining for amyloid precursor protein, which indicates amyloid plaque deposition, can be present in the cortical or hippocampus regions of the mouse brain of the present disclosure, or in both the cortical and hippocampal regions. The total immunofluorescence staining of amyloid plaque deposition in the cortical region can be compared to the total immunofluorescence staining in the hippocampus region of the same mouse. The total immunofluorescence staining of amyloid plaque deposition in the mouse brain can also be compared to the total immunofluorescence staining of amyloid plaque deposition in the control mouse brain.

[0063] In some embodiments, amyloid plaque deposition in the hippocampal region may be increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to amyloid plaque deposition in the cortical region in a wild-derived humanized mouse model of the present disclosure.

[0064] In some embodiments, the wild-derived humanized mouse model of the present disclosure has increased cortical plaque deposition compared to that of an age-matched control mouse (e.g., a WSB mouse (e.g., a WSB.APP / PSEN1 mouse), or a C57BL6 / J mouse). As used herein, cortical plaque deposition refers to plaque deposition in the cortical region of the brain. Cortical plaque deposition may be measured using immunofluorescent staining of amyloid precursor protein in the cortical region of the mouse brain. Immunofluorescent staining methods, which are well known in the art, are contemplated herein. Positive staining for amyloid precursor protein, indicative of cortical plaque deposition, may be present in the cortical region of the mouse brain of the present disclosure. Total immunofluorescent staining of cortical plaque deposition in the mouse brain is compared to total immunofluorescent staining of cortical plaque deposition in the control mouse brain.

[0065] In some embodiments, the wild-derived humanized mouse models of the present disclosure have a greater cerebral amyloid angiopathy compared to that of age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice). As used herein, cerebral amyloid angiopathy refers to amyloid β-peptide deposits in small to medium sized blood vessels of the brain and pia mater.

[0066] In some embodiments, in a wild-derived humanized mouse model of the present disclosure, cortical plaque deposition and / or cerebral amyloid angiopathy may be increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to cortical plaque deposition in an age-matched control mouse (e.g., a WSB mouse (e.g., a WSB.APP / PSEN1 mouse), or a C57BL6 / J mouse).

[0067] In some embodiments, the plaque region specificity of the wild-derived humanized mouse model of the present disclosure is different compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice). As used herein, plaque region specificity refers to the mouse brain region (e.g., cortical or hippocampal regions, or both cortical and hippocampal regions) in which amyloid plaque deposition can occur. In humans, plaque pathology occurs first in the hippocampus (i.e., plaque region specificity in humans occurs first in the hippocampal region). The wild-derived humanized mouse model exhibits plaque region specificity in both the cortical and hippocampal regions of the mouse brain.

[0068] In some embodiments, neuroinflammation in the wild-derived humanized mouse model of the present disclosure is altered compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice). As used herein, neuroinflammation is indicated by positive immunofluorescence staining of microglial activation and astrocyte reactivity in the brain. Microglial activation can be measured by staining brain tissue with a marker for microglia. Astrocyte reactivity can be measured by staining brain tissue with a marker for astrocytes. Total immunofluorescence staining of microglial activation and / or astrocyte reactivity in mouse brain can be compared to total immunofluorescence staining of microglial activation and / or astrocyte reactivity in control mouse brain.

[0069] In some embodiments, neuroinflammation (e.g., as indicated by immunofluorescent staining for astrocyte reactivity or microglial activation) in the wild-derived humanized mouse models of the present disclosure is higher compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice). In some embodiments, neuroinflammation (e.g., as indicated by immunofluorescent staining for astrocyte reactivity) in the wild-derived humanized mouse models of the present disclosure can be at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher compared to age-matched control mice (e.g., WSB mice (e.g., WSB.APP / PSEN1 mice), or C57BL6 / J mice).

[0070] In some embodiments, the wild-derived humanized mouse model of the present disclosure does not develop tumors. In some embodiments, the wild-derived humanized mouse model of the present disclosure does not have a measurable tumor burden. As used herein, "measurable tumor burden" refers to any tumor volume value that is not zero. Any mechanism for calculating tumor volume known in the art is contemplated herein.

[0071] (Method of use) The mouse model provided herein (e.g., wild-derived humanized mouse model) can be used for many applications.In some embodiments, the wild-derived humanized mouse model of the present disclosure shows neurodegeneration in the brain in response to human APOE (e.g., APOE4, APOE3, or APOE2), or human amyloid beta and / or human tau.Therefore, the wild-derived humanized mouse model of the present disclosure can be used to further evaluate the effect of human APOE (e.g., human APOE4), or human amyloid beta and / or human tau in more genetically diverse mouse models.

[0072] In some embodiments, the wild-derived humanized mouse model of the present disclosure can be used to test how a particular drug (e.g., therapeutic drug) or medical procedure (e.g., cell transplantation or tissue transplantation) affects the action of human APOE (e.g., APOE4, APOE3, or APOE2), or human amyloid beta and / or human tau as a risk factor for AD. In some embodiments, a particular drug (e.g., therapeutic drug) can be delivered to the wild-derived humanized mouse model of the present disclosure, and the neurodegenerative changes resulting from the drug can be measured as described above, compared to the wild-derived humanized mouse model of the present disclosure that did not receive the drug. The neurodegenerative changes resulting from treatment with the drug can be indicated by an increase or decrease in NEUN staining in brain regions as described above.

[0073] Non-limiting examples of agents include therapeutic agents (eg, anti-cancer and anti-inflammatory agents) and prophylactic agents (eg, vaccines).

[0074] In some embodiments, the wild-derived humanized mouse model of the present disclosure can be subjected to a medical procedure (e.g., cell transplantation or tissue transplantation), and the neurodegenerative changes resulting from the medical procedure can be measured as described above, compared to the wild-derived humanized mouse model of the present disclosure that has not received the medical procedure. The neurodegenerative changes resulting from the medical procedure (e.g., cell transplantation or tissue transplantation) can be shown by an increase or decrease in NEUN staining in brain regions as described above.

[0075] Non-limiting examples of medical procedures include transplantation of cells (eg, microglia) from other mouse background strains or from human sources.

[0076] In some embodiments, the wild-derived humanized mouse model of the present disclosure (e.g., WSB.APP / PSEN1 / APOE4 mouse model) can be used to evaluate the effect of a drug or medical procedure on neurodegeneration in response to human APOE (e.g., APOE4, APOE3, or APOE2), or human amyloid beta and / or human tau.Thus, a method is provided herein that includes administering a drug or medical procedure to a mouse model, and evaluating the effect of the drug or medical procedure on neurodegeneration in response to human APOE (e.g., APOE4, APOE3, or APOE2), or human amyloid beta and / or human tau in the mouse.

[0077] Evaluating the effect of an agent or medical procedure on neurodegeneration in response to human APOE (e.g., APOE4, APOE3, or APOE2), or human amyloid beta and / or human tau in a wild-derived humanized mouse model of the present disclosure (e.g., a WSB.APP / PSEN1 / APOE4 mouse model) includes, for example, comparing the results of the evaluation to an appropriate control (e.g., but not limited to, the effect of the compound on a control mouse (e.g., a WSB mouse (e.g., a WSB.APP / PSEN1 mouse), or a C57BL6 / J mouse)).

[0078] (Mouse model) For brevity, reference will be made herein to "mouse" and "mouse model" (e.g., a surrogate for the human condition). It should be understood that these terms may be used interchangeably throughout the specification to include "rodent" and "rodent model," including mice, rats and other rodent species, unless otherwise indicated.

[0079] It should also be understood that the standard genetic nomenclature used herein provides a unique identification for various rodent strains, and the strain symbol conveys basic information about the type of strain or stock used and the genetic content of the strain. Rules for symbolizing strains and stocks have been promulgated by the International Committee on Standardized Genetic Nomenclature for Mice. These rules are available online at the Mouse Genome Database (MGD; informatics.jax.org) and have been published in print (Lyon et al., 1996). Strain symbols typically include a Laboratory Registration Code (Lab Code). The registry is maintained at the Institute for Laboratory Animal Research (ILAR) at the National Academy of Sciences, Washington, DC. The Lab Code is available electronically at the ILAR website: nationalacademies.org / ilar / institute-for-laboratory-animal-research. See also Davisson MT, Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic-Phenotypic Definition, National Research Council (US) International Committee of the Institute for Laboratory Animal Research, Washington (DC): National Academies Press (US); 1999.

[0080] The mouse model provided herein is a transgenic mouse model that expresses human amyloid precursor protein (APP) and human presenilin 1 protein (PSEN1). In some embodiments, the transgenic mouse model expresses human apolipoprotein E4 (APOE4). In some embodiments, the transgenic mouse model expresses human amyloid beta. In some embodiments, the transgenic mouse model expresses human tau. A transgenic mouse is a mouse that has exogenous nucleic acid (e.g., transgene) in its genome. Methods for producing transgenic mice are well known.

[0081] Three conventional methods used for the production of transgenic mice include DNA microinjection (Gordon and Ruddle, Science, 1981:214:1244-124, incorporated herein by reference), embryonic stem cell-mediated gene transfer (Gossler et al., Proc. Natl. Acad. Sci., 1986, 83:9065-9069, incorporated herein by reference), and retrovirus-mediated gene transfer (Jaenisch, Proc. Natl. Acad. Sci., 1976, 73:1260-1264, incorporated herein by reference), any of which may be used as provided herein. For example, genome editing methods using clustered regularly interspaced palindromic repeats (CRISPR / Cas) nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) are described elsewhere herein.

[0082] Following delivery of the nucleic acid into a fertilized embryo (e.g., a one-cell embryo (e.g., a zygote) or a multicellular embryo (e.g., a post-zygote developmental stage (e.g., a blastocyst)), the fertilized embryo is introduced into a pseudopregnant female, which then gives birth to offspring. The presence or absence of nucleic acid encoding human FcRn and / or a chimeric IgG antibody can be confirmed, for example, using a number of genotyping methods (e.g., sequencing and / or genomic PCR).

[0083] New mouse models can also be made by crossing parental lines, as described in the examples herein.Using various available mutants, knockouts, knock-ins, transgenics, Cre-lox, Tet induction system and other mouse lines, multiple mutations and transgenes can be combined to make new mouse models.Multiple mouse lines can be crossed together to make double, triple, or even quadruple or more multiple mutant / transgenic mice.

[0084] In some embodiments, parent mice are crossed to generate F1 mice.Parent mice can be, for example, homozygous, heterozygous, hemizygous, or homozygous null at a particular allele.Homozygous describes the genotype of two identical alleles at a given locus, heterozygous describes the genotype of two different alleles at a locus, hemizygous describes the genotype of only one copy of a particular gene in an otherwise diploid organism, and homozygous null refers to an otherwise diploid organism in which both copies of that gene are missing.

[0085] In some embodiments of the present disclosure, one or more cells can be isolated from the mouse described by the present disclosure. In some embodiments, one or more cells isolated from the mouse of the present disclosure comprise the same genotype as the cell derived from the mouse.

[0086] In some embodiments, the WSB.APOE4 mouse is generated by backcrossing APOE4 into the WSB background for multiple (e.g., 5) generations. Methods including breeding the progeny mice are also contemplated.

[0087] In some embodiments, WSB.APP / PSEN1 / APOE4 mice are generated by backcrossing APOE4 to WSB background for multiple (e.g., 5) generations. The offspring WSB.APOE4 mice are then crossed with congenic WSB.APP / PSEN1 mice. The offspring are crossed to allow for the generation of WSB.APP / PSEN1 / APOE4 genotypes. Methods including breeding the offspring mice are also contemplated.

[0088] (Wild-derived humanized mouse model) In some embodiments, wild-derived humanized mouse models are provided herein. As known in the art, wild-derived mice have a genetically heterogeneous background. The introduction of transgenic mutations in the genetically diverse background of wild-derived mice can be more human-like and reproduce human pathology in a more appropriate manner. Non-limiting examples of wild-derived humanized mouse models include the following mouse strains: WSB / EiJ, CAST / EiJ, and PWK / PhJ. Other wild-derived humanized mouse models are also contemplated herein.

[0089] In some embodiments, the wild-derived humanized mouse has a WSB / EiJ genotype (e.g., Jackson Labs stock number 001145). Watkins Star Line B (WSB) was derived from a wild mouse captured on the Eastern Shore of Maryland. Wild-derived mice are genetically distinct from common laboratory mice for many complex phenotypic characteristics and are useful tools for gene mapping, evolution and phylogenetic studies. Other wild-derived humanized mouse strains are contemplated herein.

[0090] In some embodiments, the wild-derived humanized mouse has a PWK / PhJ genotype (eg, Jackson Labs stock number 003715).

[0091] In some embodiments, the wild-derived humanized mouse has a CAST / EiJ genotype (eg, Jackson Labs stock number 000928).

[0092] Nucleic Acids: Manipulation and Delivery The mouse model described herein comprises a nucleic acid encoding human APP, and in some embodiments, comprises a nucleic acid encoding mutant human PSEN1. In some embodiments, the mouse model described herein also comprises a mouse App allele and / or a mouse Psen1 allele. In some embodiments, the mouse model comprises a human APP transgene and a mutant human PSEN1 transgene. In some embodiments, the transgene (e.g., human APP transgene and / or mutant human PSEN1 transgene) is integrated into the mouse genome. Human APP and mutant human PSEN1 transgenes have been described (JAX Stock No. 025970) and are incorporated herein by reference. In some embodiments, the mouse model comprises human apolipoprotein E (e.g., human APOE4, human APOE3, or human APOE2). In some embodiments, the mouse model comprises human amyloid beta and human tau.

[0093] The nucleic acids provided herein are engineered in some embodiments. Engineered nucleic acids are nucleic acids that do not occur in nature (e.g., at least two nucleotides covalently linked together, in some cases including phosphodiester bonds, referred to as phosphodiester backbones). Engineered nucleic acids include recombinant and synthetic nucleic acids. Recombinant nucleic acids are molecules that are constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that are amplified or synthesized by chemical or other means. Synthetic nucleic acids include those that have been chemically or otherwise modified but can base pair (bind) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules that result from any of the above replications.

[0094] An engineered nucleic acid can comprise DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or a hybrid molecule (e.g., the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine).

[0095] In some embodiments, the nucleic acid is complementary DNA (cDNA). cDNA is synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0096] The engineered nucleic acids of the present disclosure can be made using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are made using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, DG et al., Nature Methods, 343-345, 2009; and Gibson, DG et al., Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzyme activities in a single tube reaction: a 5' exonuclease, a 3' extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity chews back the 5' terminal sequence, exposing the complementary sequence for annealing. The polymerase activity then fills the gaps in the annealed domains. DNA ligase then seals the nicks and covalently joins the DNA fragments together. The overlapping sequences of the adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. Other methods of generating engineered nucleic acids can be used in accordance with the present disclosure.

[0097] A gene is a distinct nucleotide sequence, the order of which determines the order of monomers in a polynucleotide or polypeptide. A gene typically encodes a protein. A gene may be endogenous (naturally occurring in a host organism) or exogenous (naturally transferred to a host organism or transferred through genetic engineering). An allele is one of two or more alternative forms of a gene that arise by mutation and are found at the same locus on a chromosome. In some embodiments, a gene includes a promoter sequence, a coding region (e.g., exons), a non-coding region (e.g., introns), and a regulatory region (also called a regulatory sequence).

[0098] A mouse containing a human gene is considered to contain a human transgene. A transgene is a gene that is exogenous to the host organism. That is, a transgene is a gene that is transferred to the host organism naturally or through genetic engineering. A transgene does not naturally occur in the host organism (the organism that contains the transgene, e.g., a mouse).

[0099] A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds and initiates transcription. A promoter is typically located immediately upstream (at its 5' end) from the transcription initiation site. In some embodiments, the promoter is an endogenous promoter. An endogenous promoter is a promoter that is naturally present in the host animal.

[0100] An open reading frame is a contiguous series of codons beginning with a start codon (e.g., ATG) and ending with a stop codon (e.g., TAA, TAG, or TGA) that encodes a polypeptide (e.g., a protein). An open reading frame is operably linked to a promoter if the promoter controls transcription of the open reading frame.

[0101] Exons are regions of genes that code for amino acids. Introns (and other non-coding DNA) are regions of genes that do not code for amino acids.

[0102] The nucleotide sequence encoding the product (e.g., a protein) has a length of 200 base pairs (bp) to 100 kilobases (kb) in some embodiments. The nucleotide sequence has a length of at least 10 kb in some embodiments. For example, the nucleotide sequence may have a length of at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, or at least 35 kb. In some embodiments, the nucleotide sequence has a length of 10 kb to 100 kb, 10 kb to 75 kb, 10 kb to 50 kb, 10 kb to 30 kb, 20 kb to 100 kb, 20 kb to 75 kb, 20 kb to 50 kb, 20 kb to 30 kb, 30 kb to 100 kb, 30 kb to 75 kb, or 30 kb to 50 kb.

[0103] Any one of the nucleic acids provided herein may have a length of 200bp to 500kb, 200bp to 250kb, or 200bp to 100kb. The nucleic acid, in some embodiments, has a length of at least 10kb. For example, the nucleic acid may have a length of at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 50kb, at least 100kb, at least 200kb, at least 300kb, at least 400kb, or at least 500kb. In some embodiments, the nucleic acid has a length of 10kb to 500kb, 20kb to 400kb, 10kb to 300kb, 10kb to 200kb, or 10kb to 100kb. In some embodiments, the nucleic acid has a length of 10 kb to 100 kb, 10 kb to 75 kb, 10 kb to 50 kb, 10 kb to 30 kb, 20 kb to 100 kb, 20 kb to 75 kb, 20 kb to 50 kb, 20 kb to 30 kb, 30 kb to 100 kb, 30 kb to 75 kb, or 30 kb to 50 kb. The nucleic acid may be circular or linear.

[0104] The nucleic acids described herein include modifications in some embodiments. Modification, in terms of nucleic acid, is any manipulation of the nucleic acid compared to the corresponding wild-type nucleic acid (e.g., naturally occurring nucleic acid). Thus, genomic modification is any manipulation of the nucleic acid in the genome (e.g., in the coding region, non-coding region, and / or regulatory region) compared to the corresponding wild-type nucleic acid in the genome (e.g., naturally occurring (unmodified) nucleic acid). Non-limiting examples of nucleic acid (e.g., genomic) modifications include deletions, insertions, "indels" (deletions and insertions), and substitutions (e.g., point mutations). In some embodiments, deletions, insertions, indels, or other modifications in a gene result in frameshift mutations, such that the gene no longer codes for a functional product (e.g., a protein). Modifications also include chemical modifications, such as chemical modifications of at least one nucleic acid base. Methods of nucleic acid modification (e.g., methods that result in gene inactivation) are known and include, but are not limited to, RNA interference, chemical modification, and gene editing (e.g., using recombinases or other programmable nuclease systems, such as CRISPR / Cas, TALEN, and / or ZFN).

[0105] Loss-of-function mutation, as known in the art, produces a gene product with little or no function. Null mutation, which is one type of loss-of-function mutation, produces a gene product with no function. In some embodiments, the inactivating allele is a null allele. Other examples of loss-of-function mutation include missense mutation and frameshift mutation.

[0106] A nucleic acid (e.g., an allele or alleles of a gene) can be altered such that the nucleic acid does not produce detectable levels of a functional gene product (e.g., a functional protein). Thus, an inactivated allele is an allele that does not produce detectable levels of a functional gene product (e.g., a functional protein). A detectable level of protein is any level of protein that is detected using a standard detection assay (e.g., flow cytometry and / or ELISA). In some embodiments, an inactivated allele is not transcribed. In some embodiments, an inactivated allele does not encode a functional protein.

[0107] Vectors used for delivery of nucleic acids include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. However, it should be understood that a vector may not be necessary. For example, a circularized or linear nucleic acid can be delivered to an embryo without its vector backbone. The vector backbone is small (approximately 4 kb), but the donor DNA that can be circularized can range from, for example, >100 bp to 50 kb.

[0108] Methods for delivering nucleic acids into mouse embryos for the generation of transgenic mice include electroporation (see, e.g., Wang W et al., J Genet Genomics, 2016;43(5):319-27; WO2016 / 054032; and WO2017 / 124086, each of which is incorporated by reference herein), DNA microinjection (see, e.g., Gordon and Ruddle, Science, 1981:214:1244-124, each of which is incorporated by reference herein), embryonic stem cell-mediated gene transfer (see, e.g., Gossler et al., Pr , 1986;83:9065-9069, herein incorporated by reference), and retroviral-mediated gene transfer (see, e.g., Jaenisch, Proc. Natl. Acad. Sci., 1976;73:1260-1264, herein incorporated by reference), any of which may be used as provided herein. EXAMPLES

[0109] Example 1. Generation of a WSB.APP / PSEN1 / APOE4 Mouse Model WSB.APP / PSEN1 / APOE4 was generated by first backcrossing APOE4 to the WSB background for five generations. The offspring WSB.APOE4 mice were then crossed with congenic WSB.APP / PSEN1 mice. The offspring were crossed to allow for wild-type (WT), APOE4 and APOE4.APP / PSEN1 genotypes.

[0110] (immunohistochemistry) WSB, WSB.APOE4, WSB.APP / PSEN1, and WSB.APP / PSEN1 / APOE4 female mice were allowed to reach 8 months of age, sacrificed, and brain tissue was collected for immunohistochemistry. Brain tissue was stained with DAPI and antibody NEUN in multiple coronal brain sections covering the anterior, central, and posterior regions of the brain. The stained sections were then scanned with a Versa slide scanner, and the images were converted for use within IMARIS software. Cortical and hippocampal regions were traced, and the amount of NEUN that coincided with DAPI was then assessed. Surface area was also measured in each of the traced regions. Representative images of the anterior brain regions for 8-month-old female WSB of all genotypes are shown in Figure 1A. The top graph shown in Figure 1B shows the amount of NEUN measured in the traced cortical regions averaged across each genotype. WSB.APOE4 has significantly less NEUN (which is a substitute for neurons) compared to wild-type (WT) WSB. WSB.APOE4.APP / PSEN1 also has significantly less NEUN compared to WSB.APP / PSEN1. The bottom graph in Figure 1B is a comparison of surface area. The cortical surface area is smaller in WSB.APOE4.APP / PSEN1 compared to WSB.APP / PSEN1 and WSB.APOE4. Taken together, this suggests that the addition of APOE4 to the WSB context contributes significantly to neurodegeneration even in the absence of amyloid drivers (e.g., APP / PSEN1).

[0111] Data from these 4-month-old mice show that WSB.APOE4 females have significantly more neurons than WSB, and WSB.APP / PSEN1 / APOE4 have significantly more neurons than WSB.APP / PSEN1 in central cortical regions (Figure 2). This mouse data correlates with human data suggesting that APOE4 carriers have more synapses early in life due to less pruning during development. Neither of these findings is present in the B6 background when APOE4 is present.

[0112] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which may include the entirety of that document. The indefinite articles "a" and "an," as used herein in the specification and claims, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0113] It is also to be understood that, unless expressly indicated to the contrary, in any method claimed in this document that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0114] In the claims and the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0115] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0116] When a range of values ​​is provided, each value between the upper and lower limits of the range is specifically contemplated and described herein.

Claims

1. A wild-derived humanized mouse comprising in its genome a nucleic acid encoding human amyloid precursor protein (APP), a nucleic acid encoding mutant human presenilin 1 protein (PSEN1), and a nucleic acid encoding human apolipoprotein E.

2. The nucleic acid encoding the human APP is a chimeric nucleic acid comprising a mouse coding sequence and a human coding sequence. the nucleic acid encoding the mutant PSEN1 comprises a human PSEN1 coding sequence comprising a deletion in exon 9, and / or The human apolipoprotein E is human apolipoprotein E4 (APOE4). The wild-derived humanized mouse of claim 1.

3. A wild-derived humanized mouse comprising in its genome a nucleic acid encoding human amyloid precursor protein (APP), a nucleic acid encoding mutant human presenilin 1 protein (PSEN1), a nucleic acid encoding human amyloid beta, and a nucleic acid encoding human tau.

4. the nucleic acid encoding human APP is a chimeric nucleic acid comprising a mouse coding sequence and a human coding sequence; and / or the nucleic acid encoding the mutant PSEN1 comprises a human PSEN1 coding sequence containing a deletion in exon 9; The wild-derived humanized mouse of claim 3 .

5. The wild-derived humanized mouse of claim 2 or 4, wherein the chimeric nucleic acid comprises a human coding sequence in the A-β domain of the mouse APP coding sequence.

6. The wild-derived humanized mouse of claim 5, wherein the chimeric nucleic acid encodes the human mutation K595N and the human mutation M596L compared to human APP comprising the amino acid sequence of SEQ ID NO:

1.

7. The wild-derived humanized mouse of claim 6 , wherein the nucleic acid encoding human APP is an APPswe transgene.

8. The wild-derived humanized mouse of claim 2 or 4, wherein the nucleic acid encoding the mutant PSEN1 is a PSEN1de9 transgene.

9. 9. The wild-derived humanized mouse of claim 8, comprising a Tg(APPswe, PSEN1de9)85Dbo transgene insertion in its genome.

10. The wild-derived humanized mouse of any one of claims 1 to 4, having a genetic background selected from WSB / EiJ, CAST / EiJ, and PWK / PhJ.

11. The wild-derived humanized mouse of any one of claims 1 to 4, which has at least one characteristic of early-onset Alzheimer's disease.

12. 12. The wild-derived humanized mouse of claim 11, wherein the at least one characteristic of early-onset Alzheimer's disease is selected from neurodegeneration compared to controls, cognitive deficits compared to controls, and increased neuroinflammation in the brain compared to controls.

13. The wild-derived humanized mouse of any one of claims 1 to 4, which does not develop tumors and has no measurable tumor burden.

14. The wild-derived humanized mouse of any one of claims 1 to 4, which is at least 1 year old.

15. A wild-derived humanized WSB mouse containing an APPswe transgene, a PSENde9 transgene, and a gene encoding human apolipoprotein E in its genome.

16. A wild-derived humanized WSB mouse as described in claim 15, wherein the gene encoding human apolipoprotein E is a gene encoding human apolipoprotein 4 (APOE4).

17. A wild-derived humanized mouse that contains a nucleic acid encoding human apolipoprotein E in its genome.

18. A wild-derived humanized mouse as described in Claim 17, wherein the nucleic acid encoding human apolipoprotein E is a nucleic acid encoding human apolipoprotein 4 (APOE4).

19. A wild-derived humanized mouse that contains humanized amyloid beta and humanized tau in its genome.

20. A cell derived from a mouse described in any one of claims 1 to 4.

21. A mouse comprising cells having the same genotype as cells derived from a mouse described in any one of claims 1 to 4.

22. A descendant mouse of a mouse described in any one of claims 1 to 4.

23. A method comprising a step of producing a mouse described in any one of claims 1 to 4.

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