Genetically engineered immunodeficient mice expressing human APP or humanized APP and mutant human PSEN1

Abstract

The present disclosure provides an immunodeficient mouse model comprising a nucleic acid encoding human amyloid precursor protein (APP) or a nucleic acid encoding humanized amyloid precursor protein (APP), and in some models further comprising a nucleic acid encoding mutant human presenilin 1 protein (PSEN1). These mouse models are useful, for example, for Alzheimer's disease research. Thus, some aspects of the present disclosure provide an immunocompromised mouse comprising in its genome a loss-of-function mutation in mouse Prkdc gene, a loss-of-function mutation in mouse Il2rg gene, and a nucleic acid encoding human amyloid precursor protein (APP) or a nucleic acid encoding humanized amyloid precursor protein (APP).

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 / 126,457, filed December 16, 2020, which is incorporated by reference in its entirety herein. [Background technology]

[0002] (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 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 show dramatic differences in neuroinflammation over background. For example, the microglial response in one mouse model is blunted, with the mouse lacking disease-associated microglia, while another mouse model shows a robust microglial response. Yet other transgenic immunodeficient mouse models expressing APP are insufficient for AD research. This is because they are unable to develop large tumor burdens and age until beyond 8 months of age (Espuny-Camacho et al., Neuron, 2017;93(5):1066-81). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Drummond et al., Acta Neuropathol. (2017) 133(2):155~175 [Non-Patent Document 2] Espuny-Camacho et al. Neuron (2017) 93(5):1066~81 Summary of the Invention [Means for solving the problem]

[0004] (overview) The present disclosure provides, in some aspects, an improved immunodeficient mouse model of Alzheimer's disease (AD). In some embodiments, the immunodeficient mouse model of AD expresses human amyloid precursor protein (APP) or humanized amyloid precursor protein (APP). In other embodiments, the immunodeficient mouse model of AD also expresses mutant human presenilin 1 protein (PSEN1, also abbreviated as PSEN1). Unlike other AD mouse models, the models provided herein do not develop large tumor burdens (often associated with death at about 7-8 months), and thus, they can age to a time point that is more appropriate for studying specific mechanisms related to the development and progression of AD. Modeling AD on an immunodeficient background allows a platform for studying immune interactions with amyloid, providing insight into, for example, how immune compromise affects short-term memory and / or how immune compromise affects the development of hippocampal and cortical plaque deposits.

[0005] The mouse model provided herein is based, at least in part, on the theory that adaptive immunity plays a role in the pathogenesis of AD by regulating neuroinflammation in the brain in response to amyloid. This theory was tested by disrupting adaptive immunity using a two-step approach. Non-obese diabetic (NOD) mice expressing humanized APP and mutant PSEN1 were first generated ("NOD.APP / PSEN1" model). This NOD.APP / PSEN1 model was then transformed into NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ (NSG™) mouse model to generate a novel immunodeficient mouse model expressing humanized APP and mutant human PSEN1 (the "NSG.APP / PSEN1" model).

[0006] Accordingly, some aspects of the present disclosure provide an immunocompromised mouse comprising in its genome a loss-of-function mutation in the mouse Prkdc gene, a loss-of-function mutation in the mouse Il2rg gene, and a nucleic acid encoding human amyloid precursor protein (APP) or a nucleic acid encoding a humanized amyloid precursor protein (APP).

[0007] In some embodiments, the mouse has a non-obese diabetic (NOD) genetic background. In some embodiments, the loss-of-function mutation in the mouse Prkdc gene is a null mutation. For example, the null mutation is a Prkdc scid In some embodiments, the loss-of-function mutation in the mouse Il2rg gene is a null mutation. For example, the null mutation is tm1Wjl In some embodiments, the mouse is a NOD.Cg-Prkdc scid Il2rg tm1Wjl In another example, the null mutation is em26Cd22 In some embodiments, the mouse is a NOD-Prkdc em26Cd52 Il2rg em26Cd22 / NjuCrl genetic background.

[0008] In some embodiments, the mouse comprises a nucleic acid encoding a humanized APP. For example, the nucleic acid encoding the humanized APP can be a chimeric nucleic acid comprising a mouse coding sequence and a human coding sequence. In some embodiments, the chimeric nucleic acid comprises a human coding sequence in the A-beta domain of the mouse APP coding sequence. In some embodiments, the chimeric nucleic acid encodes the human mutation K595N and the human mutation M596L compared to the human APP comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mouse comprises an APPswe transgene in its genome.

[0009] In some embodiments, the mouse further comprises a nucleic acid encoding a mutant human presenilin 1 protein (PSEN1) in its genome. The nucleic acid encoding mutant PSEN1 can comprise, for example, a human PSEN1 coding sequence that includes a deletion in exon 9. In some embodiments, the mouse comprises a PSEN1de9 transgene in its genome. In some embodiments, the mouse comprises a Tg(APPswe,PSEN1de9)85Dbo transgene insertion in its genome.

[0010] In some embodiments, the mouse has characteristics of early onset Alzheimer's disease. For example, the characteristics of early onset Alzheimer's disease can be selected from cognitive deficits compared to controls, increased hippocampal plaque deposits compared to controls, and increased neuroinflammation in the brain compared to controls.

[0011] In some embodiments, the mice do not develop tumors (have no measurable tumor burden).

[0012] Some aspects of the present disclosure provide immunocompromised mice comprising a nucleic acid encoding human APP or a nucleic acid encoding a humanized APP, which have no measurable tumor burden.

[0013] Another aspect of the disclosure provides an immunocompromised mouse comprising a nucleic acid encoding a human APP or a nucleic acid encoding a humanized APP that is at least 1 year old (e.g., at least 12 months, 18 months, or 24 months old).

[0014] Yet another aspect of the present disclosure is a method for the preparation of Prkdc scid Mutations and Il2rg tm1Wjl A non-obese diabetic (NOD) mouse is provided that contains a mutation and an APPswe transgene and a PSEN1de9 transgene in its genome.

[0015] In some aspects, also provided herein is a cell derived from a mouse described in any one of the above paragraphs.

[0016] Further provided herein, in some aspects, is a mouse comprising a cell having the same genotype as a cell derived from a mouse described in any one of the above paragraphs.

[0017] Also provided herein, in some aspects, is a progeny mouse of a mouse described in any one of the above paragraphs.

[0018] Some aspects of the disclosure provide methods that include generating a mouse described in any one of the above paragraphs.

[0019] Another aspect of the present disclosure provides a method comprising introducing a null mutation in a mouse Prkdc gene, a null mutation in a mouse Il2rg gene, a nucleic acid encoding human amyloid precursor protein (APP) or a nucleic acid encoding a humanized amyloid precursor protein (APP), and a nucleic acid encoding a mutant human presenilin 1 protein (PSEN1) into a non-obese diabetic (NOD) mouse.

[0020] Yet another aspect of the present disclosure is a method for the preparation of a Prkdc scid Mutation, Il2rg tm1WjlA method is provided that includes introducing the mutation, an APPswe transgene, and a PSEN1de9 transgene into a non-obese diabetic (NOD) mouse.

[0021] A further aspect of the disclosure provides a method comprising: (a) mating a NOD mouse comprising (i) a loss-of-function mutation in the mouse Prkdc gene and (ii) a loss-of-function mutation in the mouse Il2rg gene to (b) a NOD mouse comprising (i) a nucleic acid encoding human APP or a nucleic acid encoding humanized APP and (ii) a nucleic acid encoding mutant human PSEN1 to generate immunocompromised progeny mice with characteristics of early-onset Alzheimer's disease.

[0022] Furthermore, in some aspects, the present disclosure relates to: (a) Prkdc scid Mutations and Il2rg tm1Wjl and (b) a non-obese diabetic (NOD) mouse comprising an APPswe transgene and a PSEN1de9 transgene to a NOD mouse comprising an APPswe transgene and a PSEN1de9 transgene. [Brief description of the drawings]

[0023] [Figure 1] 1 shows a graph depicting the results of cognitive assessment of both 7-month-old male and female NSG.APP / PSEN1 mice on a short-term memory Y-maze task, novel spatial recognition. Intact short-term memory is indicated when the animal spends a higher percentage of time in the novel arm.

[0024] [Diagram 2] Figure 2 shows immunofluorescence images depicting the results of amyloid deposition assessment using 1% Thioflavin S staining (diluted in a 1:1 water:ethanol ratio). These images showed that plaques were primarily confined to the hippocampus in NSG.APP / PSEN1 mice, with minimal cortical deposits.

[0025] [Diagram 3]FIG. 3 shows immunofluorescence images depicting the results of staining with markers of neuroinflammation (e.g., microglial activation and astrocyte reactivity), demonstrating that despite impaired adaptive immunity, NSG.APP / PSEN1 still exhibits robust neuroinflammation in the brain in response to amyloid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] (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.

[0027] 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 (Hall et al., 2012). Importantly, different mouse models provide different degrees of AD phenotype. 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 robust cortex is observed at early time points, which is in contrast to the human pathology where plaques are mainly restricted to the hippocampus. Unlike the B6.APP / PSEN1 mouse model, the mouse model of the present disclosure (a model of AD in an immunodeficient background) shows similar amyloid plaque deposition in the hippocampus and minimal cortical deposits, which more closely resembles human AD pathology. Additionally, the disclosed mouse model allows for a platform to study immune interactions with amyloid, providing insight into how immune compromise impacts short-term memory and / or how immune compromise impacts cognitive deficits.

[0028] In some embodiments, the present disclosure provides a method for the treatment of amyloid precursor protein (APP) or humanized amyloid precursor protein (APP), in some embodiments, a mutant human presenilin 1 protein (PSEN1), in an immunodeficient mouse model (e.g., non-obese diabetic (NOD) (e.g., NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG™) mouse model).

[0029] (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 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 containing the Swedish mutations K595N and M596L (JAX Stock No. 025970). [ka]

[0030] (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. Mouse models expressing mutant human presenilin 1 and a human or humanized APP transgene are associated with 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) (JAX Stock No. 025970). In some embodiments, the nucleic acid is a PSEN1de9 transgene. In some embodiments, the PSEN1de9 transgene is a Tg(APPswe,PSEN1de9)85Dbo transgene insertion (JAX Stock No. 025970).

[0031] (Assessment of neuroinflammation and other AD symptoms) The APP / PSEN1 mouse model of the present disclosure has impaired adaptive immunity.Surprisingly, in some embodiments, the APP / PSEN1 mouse model of the present disclosure has intact innate immune signaling, and therefore can be used to evaluate immune interactions with amyloid through the introduction of substances from different lineage backgrounds or with human origin.In some embodiments, the substances from different lineage backgrounds or with human origin can include glial cells that are isolated from a first subject for transplantation in a second subject.As used herein, glial cells can refer to oligodendrocytes, astrocytes, ependymal cells and / or microglia.

[0032] In some embodiments, materials from different lineage backgrounds may include glial cells isolated from mouse models of other backgrounds with intact adaptive immunity. For example, materials may be derived from models of other backgrounds, such as WSB.APP / PSEN1 mice (JAX line number 025970) or PWK.APP / PSEN1 mice (JAX line number 025971), which are non-immunocompromised mouse models. In some embodiments, the APP / PSEN1 mouse model of the present disclosure is used to support transplantation of glial cells isolated from the WSB.APP / PSEN1 mouse model. In some embodiments, the APP / PSEN1 mouse model of the present disclosure is used to support transplantation of glial cells isolated from the PWK.APP / PSEN1 mouse model. In other embodiments, materials may be derived from C57BL / 6J, 129 / S1, A / J, CAST / EiJ, or collaborative lines, or diversity outbred mice.

[0033] In some embodiments, materials derived from human sources may include glial cells isolated from human microglia. In some embodiments, the APP / PSEN1 mouse model of the present disclosure is used to support transplantation of glial cells isolated from human microglia.

[0034] In some embodiments, the APP / PSEN1 mouse model has short-term memory impairment compared to age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice) (see, e.g., FIG. 1). As used herein, short-term memory impairment refers to changes to the function and structure of neurons in various brain regions. Short-term memory impairment in mice can be measured according to any of the following behavioral assays, including, but not limited to: delayed alternation, novel spatial recognition, matching to sample and matching to place, or contextual fear conditioning.

[0035] Delayed alternation refers to a task that utilizes the natural tendency of mice to explore and select alternative maze arms after being re-exposed to the task.The most common delayed alternation task is the Y-maze or T-path, in which animals begin the task at the stem of a "Y" or "T" and must choose between two arms, one of which has a food reward.Mice with short-term memory deficits show reduced spontaneous alternation on this task.A subtype of delayed alternation, novel spatial recognition, refers to a task that utilizes the natural tendency of mice to explore novel environments.In some embodiments, the APP / PSEN1 mice of the present disclosure can show reduced spontaneous alternation and / or reduced novel spatial recognition on this task compared to control mice.

[0036] Match-to-sample and match-to-place tasks require mice to remember the identity or location of a stimulus for longer than 2-3 seconds. For mice, this task concept is adapted to maze environments (e.g., delayed non-match-to-place in T-maze or water maze). In these tasks, mice are cued to make a choice response based on past recall to obtain the location of an escape platform or a food reward. In some embodiments, the APP / PSEN1 mice of the present disclosure can have delayed match-to-sample or match-to-place compared to control mice.

[0037] Contextual fear conditioning refers to a task where mice are conditioned with an aversive event and then tested for recall.Mice are usually given food shock (unconditioned aversive stimulus) in a specific environment (conditioned neutral stimulus) so that they will freeze when placed back in that environment after training.To test short-term memory, mice are placed in the shock environment until 1 hour after training.In some embodiments, the APP / PSEN1 mice of the present disclosure can show a reduced incidence of freezing compared to control mice.

[0038] Other assays known to measure short-term memory impairment in rodents are also contemplated herein.

[0039] In some embodiments, the APP / PSEN1 mouse model has greater cognitive deficits compared to age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice). As used herein, cognitive deficits are used to describe impairments in various cognitive domains and are used interchangeably with the term cognitive impairment. The 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: novel object recognition (NOR), passive inhibitory avoidance or Morris water maze test.

[0040] Novel object recognition (NOR) task is used to evaluate cognition, especially recognition memory, in mouse models of CNS disorders.This test is based on the spontaneous tendency of mice to explore novel objects longer than familiar objects.In some embodiments, APP / PSEN1 mice of the present disclosure can explore novel objects equal or shorter than familiar objects when compared with control mice.

[0041] Passive avoidance task is a fear-aggravated test that is used to evaluate learning and memory in mouse models of CNS disorders.In this test, mice learn to avoid the environment that aversive stimuli (e.g., food shock) have been previously applied to them.In some embodiments, the APP / PSEN1 mice of the present disclosure may not avoid the environment that aversive stimuli have been previously applied to them, compared with control mice that avoid the environment that aversive environment has been previously applied to them.

[0042] Morris water maze is one of the most widely used tasks in behavioral neuroscience to study the psychological processes and neural mechanisms of spatial learning and memory. Mice are placed in a large circular pool of water and are required to escape from the water to a hidden platform, whose location can usually be identified only by using spatial memory. In some embodiments, the APP / PSEN1 mice of the present disclosure may not find the hidden platform or may spend a long time finding the hidden platform compared to control mice.

[0043] In some embodiments, the APP / PSEN1 mouse model 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.

[0044] In some embodiments, in APP / PSEN1 mice, 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.

[0045] In some embodiments, the APP / PSEN1 mice of the present disclosure have less cortical plaque deposition compared to that of age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice). As used herein, cortical plaque deposition refers to plaque deposition in the cortical region of the brain. Cortical plaque deposition can be measured using immunofluorescence staining of amyloid precursor protein in the cortical region of the mouse brain. Immunofluorescence staining methods, which are well known in the art, are contemplated herein. Positive staining for amyloid precursor protein, indicative of cortical plaque deposition, can be present in the cortical region of the mouse brain of the present disclosure. The total immunofluorescence staining of cortical plaque deposition in the mouse brain is compared to the total immunofluorescence staining of cortical plaque deposition in the control mouse brain.

[0046] In some embodiments, in APP / PSEN1 mice of the present disclosure, cortical plaque deposition may be reduced 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 age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice).

[0047] In some embodiments, the plaque region specificity of the APP / PSEN1 mice of the present disclosure is different compared to age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice) (see, e.g., FIG. 2). As used herein, plaque region specificity refers to the mouse brain region (e.g., cortical or hippocampal regions, or both cortical or 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 hippocampus). The B6.APP / PSEN1 mouse model shows plaque region specificity in both the cortical and hippocampal regions of the mouse brain. In some embodiments, the plaque region specificity of the APP / PSEN1 mice of the present disclosure occurs in a manner similar to the plaque region specificity reported in humans (e.g., plaque pathology occurs first in the hippocampus). Furthermore, the NSG® mouse model does not show plaque pathology.

[0048] In some embodiments, neuroinflammation in APP / PSEN1 mice is different compared to age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice) (see, e.g., FIG. 3). 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.

[0049] In some embodiments, neuroinflammation (e.g., as indicated by immunofluorescent staining for astrocyte reactivity) is higher in APP / PSEN1 mice compared to age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice). In some embodiments, neuroinflammation (e.g., as indicated by immunofluorescent staining for astrocyte reactivity) in APP / PSEN1 mice may be increased by 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 age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice).

[0050] In some embodiments, neuroinflammation (e.g., as indicated by immunofluorescence staining for microglial activation) is higher in APP / PSEN1 mice compared to age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice). In some embodiments, neuroinflammation (e.g., as indicated by immunofluorescence staining for microglial activation) in APP / PSEN1 mice may be increased by 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 age-matched control mice (e.g., NSG® mice and / or B6.APP / PSEN1 mice).

[0051] (Method of use) The mouse model provided herein (e.g., APP / PSEN1 mouse model) can be used for many applications. In some embodiments, the mouse model of the present disclosure shows robust neuroinflammation in the brain in response to amyloid despite having impaired adaptive immunity, indicating that the mouse model has intact innate immune signaling. Therefore, the mouse model of the present disclosure can be used as a platform for the evaluation of immune interactions with amyloid through the introduction of substances from different lineage backgrounds or human origins as described above.

[0052] In some embodiments, the mouse model of the present disclosure can be used to evaluate immune interactions with amyloid in the context of Alzheimer's disease (AD). In some embodiments, the AD can be early-onset AD. In some embodiments, the mouse model of the present disclosure can be used to evaluate amyloid plaque deposition, cortical plaque deposition, plaque area specificity, and / or neuroinflammation as described above in the context of early-onset AD.

[0053] In some embodiments, the mouse model of the present disclosure may be used to test how a particular agent (e.g., a therapeutic agent) or medical procedure (e.g., cell or tissue transplant) affects neuroinflammation (e.g., microglial activation and astrocyte reactivity) in response to amyloid. In some embodiments, a particular agent (e.g., a therapeutic agent) may be delivered to the mouse model of the present disclosure, and changes in neuroinflammation as a result of the agent may be measured as described above, compared to a mouse model of the present disclosure that did not receive the agent. Changes in neuroinflammation as a result of treatment with the agent may be indicated by an increase or decrease in microglial activation and astrocyte reactivity as described above.

[0054] Non-limiting examples of agents include therapeutic agents (eg, anti-cancer agents and anti-inflammatory agents) and prophylactic agents (eg, immunogenic compositions (eg, vaccines)).

[0055] In some embodiments, the mouse model of the present disclosure may receive a medical procedure (e.g., cell or tissue transplantation), and the change in neuroinflammation as a result of the medical procedure may be measured as described above, compared to a mouse model of the present disclosure that did not receive the medical procedure. The change in neuroinflammation as a result of the medical procedure (e.g., cell or tissue transplantation) may be indicated by an increase or decrease in microglial activation and astrocyte reactivity as described above.

[0056] Non-limiting examples of medical procedures include transplantation of cells (e.g., microglia) from other mouse background strains or from human origin as described above. In some embodiments, the mouse models of the present disclosure may be used to evaluate the effect of transplantation of cells from different genetic backgrounds (e.g., microglia cells isolated from WSB.APP / PSEN1 and / or PWK.APP / PSEN1) as described above. In some embodiments, the mouse models of the present disclosure may be used to evaluate the effect of transplantation of cells from human microglia as described above. In some embodiments, transplantation from other mouse background strains includes, but is not limited to, mouse background strains C57BL / 6J, 129 / S1, A / J, CAST / EiJ, and diversity outbred (DO) mice. Transplantation from other mouse background strains and other cells of human origin are also contemplated.

[0057] In some embodiments, the mouse model of the present disclosure (e.g., APP / PSEN1 mouse model) can be used to evaluate the effect of a drug or medical procedure on neuroinflammation in response to amyloid.Thus, a method is provided herein that includes administering a drug or medical procedure to a mouse model and evaluating the drug or medical procedure on neuroinflammation in response to amyloid in the mouse.

[0058] Evaluating the effect of an agent or medical procedure on neuroinflammation in response to amyloid in a mouse model of the present disclosure (e.g., an APP / PSEN1 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 non-immunodeficient mouse expressing human APP and mutant human PSEN1 (e.g., B6.APP / PSEN1), or a wild-type mouse (e.g., a mouse that does not express human APP and mutant human PSEN1)).

[0059] In some embodiments, the mouse model of the present disclosure (e.g., APP / PSEN1 mouse model) can be used to evaluate the effect of a drug or medical procedure on amyloid plaque deposition.Therefore, 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 amyloid plaque deposition in the mouse.The change in amyloid plaque deposition as a result of the drug or medical procedure can be indicated by an increase or decrease in amyloid staining in the cortical and / or hippocampal regions of the mouse brain as described above.In some embodiments, the decrease in amyloid plaque deposition as a result of the drug or medical procedure can indicate a decrease in the progression of AD phenotype in the mouse.

[0060] Evaluating the effect of an agent or medical procedure on amyloid plaque deposition in a mouse model of the present disclosure (e.g., an APP / PSEN1 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 non-immunodeficient mouse expressing human APP and mutant human PSEN1 (e.g., B6.APP / PSEN1), or a wild-type mouse (e.g., a mouse that does not express human APP and mutant human PSEN1)).

[0061] In some embodiments, the mouse model of the present disclosure (e.g., APP / PSEN1 mouse model) can be used to evaluate the effect of a drug or medical procedure on cortical plaque deposition. The change in cortical plaque deposition as a result of the drug or medical procedure can be indicated by an increase or decrease in amyloid staining in the cortical region of the mouse brain as described above. In some embodiments, the decrease in amyloid plaque deposition as a result of the drug or medical procedure can indicate a decrease in the progression of the AD phenotype in the mouse.

[0062] Evaluating the effect of an agent or medical procedure on cortical plaque deposition in a mouse model of the present disclosure (e.g., an APP / PSEN1 mouse model) can include, 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 non-immunodeficient mouse expressing human APP and mutant human PSEN1 (e.g., B6.APP / PSEN1), or a wild-type mouse (e.g., a mouse that does not express human APP and mutant human PSEN1)).

[0063] In some embodiments, the mouse model of the present disclosure (e.g., APP / PSEN1 mouse model) can be used to evaluate the effect of a drug or medical procedure on plaque region specificity. The change in plaque region specificity as a result of the drug or medical procedure can be indicated by an increase or decrease in amyloid staining in the cortical and / or hippocampal regions of the mouse brain as described above. In some embodiments, the decrease in amyloid plaque deposition in the cortical and / or hippocampal regions of the mouse brain as a result of the drug or medical procedure can indicate a decrease in the progression of the AD phenotype in the mouse.

[0064] Evaluating the effect of an agent or medical procedure on plaque region specificity in a mouse model of the present disclosure (e.g., an APP / PSEN1 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 non-immunodeficient mouse expressing human APP and mutant human PSEN1 (e.g., B6.APP / PSEN1), or a wild-type mouse (e.g., a mouse that does not express human APP and mutant human PSEN1)).

[0065] In some embodiments, the mouse model of the present disclosure (e.g., APP / PSEN1 mouse model) can be used to evaluate the effect of a drug or medical procedure on short-term memory. The change in short-term memory as a result of the drug or medical procedure can be indicated by improved performance in any one of the behavioral assays used to measure short-term memory as described above. In some embodiments, improved performance in any one of the behavioral assays used to measure short-term memory can indicate a decrease in the progression of AD phenotype in the mouse.

[0066] Evaluating the effect of a drug or medical procedure on short-term memory in a mouse model of the present disclosure (e.g., an APP / PSEN1 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 non-immunodeficient mouse expressing human APP and mutant human PSEN1 (e.g., B6.APP / PSEN1), or a wild-type mouse (e.g., a mouse that does not express human APP and mutant human PSEN1)).

[0067] In some embodiments, the mouse model of the present disclosure (e.g., APP / PSEN1 mouse model) can be used to evaluate the effect of a drug or medical procedure on cognitive deficit. The change in cognitive deficit as a result of the drug or medical procedure can be indicated by improved performance in any one of the behavioral assays used to measure cognitive deficit as described above. In some embodiments, the improved performance in any one of the behavioral assays used to measure cognitive deficit can indicate a decrease in the progression of AD phenotype in the mouse.

[0068] Evaluating the effect of a drug or medical procedure on cognitive deficits in a mouse model of the present disclosure (e.g., an APP / PSEN1 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 non-immunodeficient mouse expressing human APP and mutant human PSEN1 (e.g., B6.APP / PSEN1), or a wild-type mouse (e.g., a mouse that does not express human APP and mutant human PSEN1)).

[0069] (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.

[0070] 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.

[0071] The mouse model provided herein is a transgenic mouse model that expresses human amyloid precursor protein (APP) or humanized amyloid precursor protein (APP).In some embodiments, the transgenic mouse model also expresses human presenilin 1 protein (PSEN1).Transgenic mouse is a mouse that has exogenous nucleic acid (e.g., transgene) in its genome.The method for producing transgenic mouse is well known.

[0072] 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.

[0073] 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).

[0074] 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.

[0075] 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.

[0076] In some embodiments, a NOD mouse comprising a loss-of-function mutation in mouse Prkdc gene and a loss-of-function mutation in mouse Il2rg gene is bred with a NOD mouse comprising a nucleic acid encoding human amyloid precursor protein or a nucleic acid encoding humanized amyloid precursor protein and a nucleic acid encoding mutant human presenilin 1 protein to generate immunocompromised progeny mice with characteristics of early-onset Alzheimer's disease.A method comprising breeding the progeny mice is also contemplated.

[0077] In some embodiments, Prkdc scid Mutations and Il2rg tm1WjlA method is also contemplated that includes crossing a non-obese diabetic (NOD) mouse containing the APPswe transgene and the PSENde9 transgene to a NOD mouse containing the APPswe transgene and the PSENde9 transgene, and breeding the offspring mice.

[0078] In some embodiments, male mice (JAX stock number 25967) comprising a NOD.Cg-Tg(APPswe,PSEN1dE9)85Dbo / How background are cloned into a NOD.Cg-Prkdc scid Il2rg tm1Wjl A method is also contemplated that includes mating the mice to female mice comprising an APP / PSEN1 / SzJ background (JAX stock number 005557) and then breeding the resulting male offspring, which are then genotyped for the presence of the APP / PSEN1 transgene and the gamma mutation, to female NSG® mice.

[0079] F1 hybrid mice are produced by crossing two different inbred strains of mice. These F1 hybrid mice are similar to inbred strains in that they are heterozygous at all loci where the parents have different alleles, but they are genetically and phenotypically uniform. F1 hybrids can be produced as long as the parent strains are present. However, unlike the parent strains, F1 hybrids are not bred true. All F2 progeny produced by mating F1 mice have a unique random mixture of alleles derived from both parent strains.

[0080] 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.

[0081] (immunodeficient mouse model) In some embodiments, immunodeficient mouse models are provided herein.As known in the art, immunodeficient mice have impaired or destroyed immune system (e.g., specific deficiency in MHC class I, specific deficiency in MHC class II or both, B cell deficiency or T cell deficiency, or both) and immunodeficiency due to knockdown of genes related to cytokines, cytokine receptors, TLR receptors, and various transducers and transcription factors of signal transduction pathways.Immune-deficient mouse models include single gene mutation models, such as nude mouse (nu) strains and severe combined immunodeficiency (scid) strains, non-obese diabetic (NOD) strains, RAG (recombination activating gene) strains with targeted gene deletion, and various hybrids made by crossing double mutant mouse strains and triple mutant mouse strains with additional deficiencies in innate immunity and adaptive immunity.

[0082] Non-limiting examples of naturally and transgenic immunodeficient mouse models include the following mouse strains: · nude (nu) [Flanagan SP, Genet Res, 1966;8:295-309; and Nehls M et al., Nature, 1994;372:103-7]; · Scid (scid) [Bosma GC et al., Nature, 1983;301:527-30; Mosier DE et al., Nature, 1988;335:256-9; and Greiner DL et al., Stem Cells, 1998;16:166-77]; · NOD [Kikutani H et al., Adv Immunol, 1992;51:285-322; and Anderson MS et al., Ann Rev Immunol, 2005;23:447-85]; · RAG1 and RAG2 (rag) [Mombaerts P et al., Cell, 1992;68:869-77; Shinkai U et al., Cell, 1992;68:855-67]; · NOD-scid [Greiner DL et al., 1998; Shultz LD et al., J Immunol, 1995;154:180-91; Melkus MW et al., Nature Med, 2006;12:1316-22; and Denton PW et al., PLoS Med, 2008;4(12):e357]; ·IL2rgnull[DiSanto JP et al., Proc Natl Acad Sci USA, 1995;92:377~81]; · B2mnull [Christianson SW et al., J Immunol, 1997;158:3578~86]; · NOD-scid IL2rγnull [Shultz LD et al., Nat Rev Immunol, 2007;7:118-30; Ito M et al., Blood, 2002;100:3175-82; Ishikawa I et al., Blood, 2005;106:1565-73; and Macchiarini F et al., J Exp Med, 2005;202:1307-11]; NOD-scid B2mnull [Shultz et al., 2007; Shultz LD et al., Transplantation, 2003;76:1036-42; Islas-Ohlmayer MA et al., J Virol, 2004;78:13891-900; and Macchiarini et al., 2005]; and HLA transgenic mice [Grusby MJ et al., Proc Natl Acad Sci USA, 1993;90(9):3913-7; and Roy CJ et al., Infect Immun, 2005;73(4):2452-60]. See, e.g., Belizario JE, The Open Immunology Journal, 2009;2:79-85.

[0083] In some embodiments, an immunodeficient mouse model having a non-obese diabetic (NOD) mouse genotype is provided herein. NOD mice (e.g., Jackson Labs Stock No. 001976, NOD-Shi LtJNOD mice are a polygenic mouse model of autoimmune (e.g., type 1) diabetes characterized by hyperglycemia and insulitis (leukocyte infiltration of islet cells). The NOD mouse is hypoinsulinemic and hyperglucagonemic, indicating selective destruction of islet β cells. A major component of diabetes susceptibility in NOD mice is the unique MHC haplotype. NOD mice also exhibit multiple abnormal immune phenotypes, including defective antigen-presenting cell immunoregulatory function, defective regulation of the T lymphocyte repertoire, defective NK cell function, defective cytokine production from macrophages (Fan et al., 2004), and impaired wound healing. NOD mice also lack the hemolytic complement C5. NOD mice are also severely deaf. Various mutations causing immune deficiencies, targeted mutations in cytokine genes, as well as transgenes affecting immune function have been backcrossed onto the NOD inbred background.

[0084] In some aspects of the present disclosure, the immunodeficient mice provided herein based on a NOD background are NOD-Cg.-Prkdc scid IL2rg tm1wJl / SzJ (NSG®), NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl / SzJ(NRG), and NOD.Cg-Prkdc scid Il2rg tm1Sug / ShiJic(NOG). Other immunodeficient mouse strains are contemplated herein.

[0085] In some embodiments, the immunodeficient mouse model has the NSG™ genotype. NSG® mice (e.g., Jackson Labs stock number: #005557) are immunodeficient mice that lack mouse T cells, mouse B cells, and mouse NK cells, are defective in multiple cytokine signaling pathways, and have numerous defects in innate immunity (see, e.g., Shultz, Ishikawa, and Greiner, 2007; Shultz et al., 2005; and Shultz et al., 1995, each of which is incorporated herein by reference). NSG® mice, derived from the NOD mouse strain NOD / ShiLtJ (see, e.g., Makino et al., 1980, which is incorporated herein by reference), ... the Prkdc gene. scid mutation (also called the "severe combined immunodeficiency" mutation or "scid" mutation) and Il2rg tm1Wjl This Prkdc gene is a novel gene that is involved in targeted mutations. scid The mutation is a loss-of-function (null) mutation in the mouse homolog of the human PRKDC gene - this mutation essentially eliminates adaptive immunity (see, e.g., (Blunt et al., 1995; Greiner, Hesselton, and Shultz, 1998), each of which is incorporated herein by reference). tm1Wjl The mutation is a null mutation in the gene encoding the interleukin 2 receptor gamma chain (IL2Rγ, homologous to IL2RG in humans), which blocks NK cell differentiation, thereby removing the obstacle that prevents efficient engraftment of primary human cells (Cao et al., 1995; Greiner et al., 1998; and Shultz et al., 2005, each of which is incorporated herein by reference).

[0086] In some embodiments, the immunodeficient mouse model has the NRG genotype. NRG mice (e.g., Jackson Labs stock number 007799) are severely immunodeficient. These mice are bred with a targeted knockout mutation in recombination activating gene 1 (Rag1) and a complete null allele of the IL2 receptor common gamma chain (IL2rg null), which contains two mutations in the NOD / ShiLtJ genetic background. null The mutation causes defects in mouse B cells and mouse T cells, and this IL2rg null The mutations disrupt cytokine signaling through multiple receptors, resulting in a lack of functional NK cells. The severe immune deficiency of NRG is due to the deletion of human CD34 + It allows for the efficient humanization of the mice by transplantation of hematopoietic stem cells (HSCs) and patient-derived xenografts (PDXs). Immunodeficient NRG mice are more resistant to irradiation and genotoxic drugs than mice with a scid mutation in the DNA repair enzyme Prkdc.

[0087] In some embodiments, the immunodeficient mouse model is NOG mouse. NOG mouse (Ito M et al., Blood, 2002) is a severe combined immunodeficient (scid) mouse established by combining NOD / scid mouse with IL-2 receptor-gamma chain knockout (IL2rγKO) mouse (Ohbo K. et al., Blood, 1996). NOG mouse lacks T cells and B cells, lacks natural killer (NK) cells, shows reduced dendritic cell function and reduced macrophage function, and lacks complement activity.

[0088] In some embodiments, the immunodeficient mouse model has NCG genotype. NCG mice (e.g., Charles River stock number 572) are generated by sequential CRISPR / Cas9 editing of Prkdc locus and Il2rg locus in NOD / Nju mice to generate coisogenic mice for the NOD / Nju. The NOD / Nju carries a mutation in Sirpa (SIRPα) gene that allows transplantation of foreign hematopoietic stem cells. The Prkdc knockout causes a SCID-like phenotype that lacks proper T cell and B cell formation. The knockout of Il2rg gene further aggravates this SCID-like phenotype, and also causes a concomitant decrease in NK cell production.

[0089] In some embodiments, immunodeficient mice that are deficient in MHC class I, immunodeficient mice that are deficient in MHC class II, or immunodeficient mice that are deficient in MHC class I and MHC class II are provided herein. MHC class I and / or MHC class II deficient mice do not express the same level of MHC class I protein (e.g., α-microglobulin and β2-microglobulin (B2M)) and / or MHC class II protein (e.g., α chain and β chain) as non-immunodeficient (e.g., MHC class I / II wild type) mice, and do not have the same level of MHC class I protein activity and / or MHC class II protein activity as non-immunodeficient (e.g., MHC class I / II wild type) mice. In some embodiments, the expression or activity of MHC class I protein and / or MHC class II protein is reduced (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more) compared to non-immunodeficient mice.

[0090] Immunodeficient mice deficient in MHC class I, immunodeficient mice deficient in MHC class II, and immunodeficient mice deficient in MHC class I and MHC class II are described in International Publication No. WO2018 / 209344, the contents of which are incorporated herein by reference.

[0091] (humanized mouse model) In some embodiments, a humanized immune-deficient mouse model and a method for making the model are provided herein. An immune-deficient mouse transplanted with functional human cells and / or functional human tissues is referred to as a "humanized mouse". As used herein, the terms "humanized mouse", "humanized immune-deficient mouse", "humanized immune-deficient mouse", and plural versions thereof are used interchangeably to refer to an immune-deficient mouse that has been humanized by transplantation of functional human cells and / or functional human tissues. For example, the mouse model can be transplanted with human hematopoietic stem cells (HSCs) and / or human peripheral blood mononuclear cells (PMBCs). In some embodiments, the mouse model is transplanted with human tissues (e.g., islets, liver, skin, and / or solid or blood cancers). In other embodiments, the mouse model can be genetically modified to convert endogenous mouse genes to human homologs (see, e.g., Pearson et al., Curr Protoc Immunol., 2008, Chapter:Unit-15.21).

[0092] Humanized mice are generated by starting with an immunodeficient mouse and, if necessary, depleting and / or suppressing any remaining mouse immune cells (e.g., chemically or by irradiation). That is, successful survival of the human immune system in an immunodeficient mouse may require suppression of the mouse's immune system to prevent GVHD (graft-versus-host disease) rejection. After the immune system of the immunodeficient mouse has been successfully suppressed, the mouse is transplanted with human cells (e.g., HSCs and / or PBMCs). As used herein, "transplanting" refers to the process of human cells migrating and incorporating in vivo into an existing tissue of interest. For humanized immunodeficient mice, the transplanted human cells provide functional mature human cells (e.g., immune cells). The model has a specific time frame of about 4-5 weeks after transplantation before GVHD begins. To increase the lifespan of the model, double knockout mice lacking functional MHC I and MHC II as described above can be used.

[0093] In some embodiments, the human cells (e.g., HSC or PMBC) transplanted for humanization are human leukocyte antigen (HLA) compatible with the human cancer cells of the mouse model. HLA compatibility refers to cells that express the same major histocompatibility complex (MHC) genes. Transplanting HLA-compatible human xenografts and human immune cells into mice reduces or prevents the immunogenicity of the human immune cells, for example. In some embodiments, the humanized mice provided in the present disclosure are transplanted with human PMBC or human HSC that are HLA compatible with PDX or human cancer cell lines.

[0094] (irradiation) As described above, in some embodiments, immunodeficient mice are irradiated prior to transplantation of human cells (e.g., human HSCs and / or PMBCs). Irradiation of immunodeficient mice destroys mouse immune cells in the peripheral blood, spleen, and bone marrow, which is thought to facilitate transplantation of human cells (e.g., human HSCs and / or PMBCs), as well as proliferation of other immune cells (e.g., by increasing human cell survival factors). Irradiation also shortens the time it takes to accumulate the number of human immune cells required to "humanize" the mouse model.

[0095] For immunodeficient mice (e.g., NSG® mice), this preparation is typically accomplished through whole-body gamma irradiation. The irradiator can vary in size depending on its intended use. Animals are typically irradiated for a short period of time (less than 15 minutes). The amount of time spent in the irradiator varies depending on the radioisotope decay chart, the dose required, and the ionizing energy source (i.e., X-rays vs. gamma rays, for which a cesium or cobalt source is required).

[0096] Myeloablative radiation doses are typically between 700 cGy and 1300 cGy, although in some embodiments lower doses may be used, such as between 1 cGy and 100 cGy (eg, about 2 cGy, 5 cGy, or 10 cGy), or between 300 cGy and 700 cGy.

[0097] By way of example, a mouse may be irradiated with 100 cGy of X-rays (or 75 cGy to 125 cGy of X-rays). In some embodiments, the dose is about 1 cGy, 2 cGy, 3 cGy, 4 cGy, 5 cGy, 10 cGy, 20 cGy, 100 cGy, 200 cGy, 300 cGy, 400 cGy, 500 cGy, 600 cGy, 700 cGy, 800 cGy, 900 cGy, 1000 cGy, 1100 cGy, 1200 cGy, or 1300 cGy, or between any two doses described herein (e.g., 100 cGy to 300 cGy, 200 cGy to 500 cGy, 600 cGy to 1000 cGy, or 700 cGy to 1300 cGy). In some embodiments, the immunodeficient mice are irradiated about 15 minutes, 30 minutes, 45 minutes, 1 hour, or more prior to transplantation of the human HSCs and / or human PMBCs. In some embodiments, the immunodeficient mice are transplanted with the human HSCs and / or human PMBCs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, or 18 days after irradiation.

[0098] (transplant) As described above, in some embodiments, irradiated immunodeficient mice are transplanted with HSCs and / or PBMCs and the mice are humanized. Transplantation refers to the process of human cells migrating and incorporating in vivo into existing tissues of interest. The PBMCs can be transplanted after irradiation and prior to human cancer cell transplantation, after irradiation and simultaneously with human cancer cell transplantation, or after irradiation and subsequent to human cancer cell transplantation.

[0099] Peripheral blood mononuclear cells (PBMCs) are peripheral blood cells with round nuclei. These mononuclear blood cells recirculate between tissues and blood and are important components in the immune system that fights infection and adapts to invaders. There are two major types of mononuclear cells: lymphocytes and monocytes. The lymphocyte population of PBMCs typically includes T cells, B cells, and NK cells.

[0100] PBMCs can be isolated, for example, from a whole blood sample (e.g., a Ficoll gradient). In some embodiments, PBMCs from a subject (e.g., a human subject) with a current or previous diagnosis of a pathogen or pathogenic disease can be used.

[0101] Hematopoietic stem cells (HSCs) are stem cells that give rise to other blood cells during a process called hematopoiesis. Hematopoietic stem cells give rise to various types of blood cells of lineages called myeloid and lymphoid. Both the myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes-platelets. Lymphoid cells include T cells, B cells, natural killer cells, and innate lymphocytes.

[0102] Methods for transplanting HSCs and / or PBMCs into immunodeficient mice to generate humanized mouse models include, but are not limited to, intraperitoneal or intravenous injections (Shultz et al., J Immunol, 2005, 174:6477-6489; Pearson et al., Curr Protoc Immunol., 2008;15-21; Kim et al., AIDS Res Hum Retrovirus, 2016, 32(2):194-202; Yamaguchi et al., Cell & Mol Immunol, 2018, 15:953-962). In some embodiments, mice are injected with 1.0×10 6 pieces~3.0×10 7 HSCs and / or PBMCs are transplanted.

[0103] For example, a mouse has a resolution of 1.0×10 6 pieces, 1.1×10 6 pieces, 1.2×106 pieces, 1.3×10 6 pieces, 1.4×10 6 pieces, 1.5×10 6 pieces, 1.6×10 6 pieces, 1.7×10 6 pieces, 1.8×10 6 pieces, 1.9×10 6 pieces, 2.0×10 6 pieces, 2.5×10 6 pieces, 3.0×10 6 10 or more HSCs and / or PBMCs. In some embodiments, mice may be transplanted with 1.0×10 6 pieces~1.1×10 6 pieces, 1.0×10 6 pieces~1.2×10 6 pieces, 1.0×10 6 pieces~1.3×10 6 pieces, 1.0×10 6 pieces~1.4×10 6 pieces, 1.0×10 6 pieces~1.5×10 6 pieces, 1.0×10 6 pieces~1.6×10 6 pieces, 1.0×10 6 pieces~1.7×10 6 pieces, 1.0×10 6 pieces~1.8×10 6 pieces, 1.0×10 6 pieces~1.9×10 6 pieces, 1.0×10 6 pieces~2.0×10 6 pieces, 1.0×10 6 pieces~2.25×10 6 pieces, 1.0×10 6 pieces~2.5×10 6 pieces, 1.0×10 6 pieces~2.75×10 6 pieces, 1.0×10 6 pieces~3.0×10 6 pieces, 1.1×10 6 pieces~1.2×10 6 pieces, 1.1×10 6 pieces~1.3×10 6 pieces, 1.1×10 6 pieces~1.4×10 6 pieces, 1.1×106 pieces to 1.5×10 6 pieces, 1.1×10 6 pieces to 1.6×10 6 pieces, 1.1×10 6 pieces to 1.7×10 6 pieces, 1.1×10 6 pieces to 1.8×10 6 pieces, 1.1×10 6 pieces to 1.9×10 6 pieces, 1.1×10 6 pieces to 2.0×10 6 pieces, 1.1×10 6 pieces to 2.25×10 6 pieces, 1.1×10 6 pieces to 2.5×10 6 pieces, 1.1×10 6 pieces to 2.75×10 6 pieces, 1.1×10 6 pieces to 3.0×10 6 pieces, 1.2×10 6 pieces to 1.3×10 6 pieces, 1.2×10 6 pieces to 1.4×10 6 pieces, 1.2×10 6 pieces to 1.5×10 6 pieces, 1.2×10 6 pieces to 1.6×10 6 pieces, 1.2×10 6 pieces to 1.7×10 6 pieces, 1.2×10 6 pieces to 1.8×10 6 pieces, 1.2×10 6 pieces to 1.9×10 6 pieces, 1.2×10 6 pieces to 2.0×10 6 pieces, 1.2×10 6 pieces to 2.25×10 6 pieces, 1.2×10 6 pieces to 2.5×10 6 pieces, 1.2×10 6 pieces to 2.75×10 6 pieces, 1.2×10 6 pieces to 3.0×10 6 pieces, 1.3×10 6 pieces to 1.4×10 6 pieces, 1.3×10 6 pieces to 1.5×10 6pieces, 1.3×10 6 pieces ~ 1.6×10 6 pieces, 1.3×10 6 pieces ~ 1.7×10 6 pieces, 1.3×10 6 pieces ~ 1.8×10 6 pieces, 1.3×10 6 pieces ~ 1.9×10 6 pieces, 1.3×10 6 pieces ~ 2.0×10 6 pieces, 1.3×10 6 pieces ~ 2.25×10 6 pieces, 1.3×10 6 pieces ~ 2.5×10 6 pieces, 1.3×10 6 pieces ~ 2.75×10 6 pieces, 1.3×10 6 pieces ~ 3.0×10 6 pieces, 1.4×10 6 pieces ~ 1.5×10 6 pieces, 1.4×10 6 pieces ~ 1.6×10 6 pieces, 1.4×10 6 pieces ~ 1.7×10 6 pieces, 1.4×10 6 pieces ~ 1.8×10 6 pieces, 1.4×10 6 pieces ~ 1.9×10 6 pieces, 1.4×10 6 pieces ~ 2.0×10 6 pieces, 1.4×10 6 pieces ~ 2.25×10 6 pieces, 1.4×10 6 pieces ~ 2.5×10 6 pieces, 1.4×10 6 pieces ~ 2.75×10 6 pieces, 1.4×10 6 pieces ~ 3.0×10 6 pieces, 1.5×10 6 pieces ~ 1.6×10 6 pieces, 1.5×10 6 pieces ~ 1.7×10 6 pieces, 1.5×10 6 pieces ~ 1.8×10 6 pieces, 1.5×10 6 pieces ~ 1.9×10 6 pieces, 1.5×10 6 pieces ~ 2.0×106 pieces, 1.5×10 6 pieces ~ 2.25×10 6 pieces, 1.5×10 6 pieces ~ 2.5×10 6 pieces, 1.5×10 6 pieces ~ 2.75×10 6 pieces, 1.5×10 6 pieces ~ 3.0×10 6 pieces, 1.6×10 6 pieces ~ 1.7×10 6 pieces, 1.6×10 6 pieces ~ 1.8×10 6 pieces, 1.6×10 6 pieces ~ 1.9×10 6 pieces, 1.6×10 6 pieces ~ 2.0×10 6 pieces, 1.6×10 6 pieces ~ 2.25×10 6 pieces, 1.6×10 6 pieces ~ 2.5×10 6 pieces, 1.6×10 6 pieces ~ 2.75×10 6 pieces, 1.6×10 6 pieces ~ 3.0×10 6 pieces, 1.7×10 6 pieces ~ 1.8×10 6 pieces, 1.7×10 6 pieces ~ 1.9×10 6 pieces, 1.7×10 6 pieces ~ 2.0×10 6 pieces, 1.7×10 6 pieces ~ 2.25×10 6 pieces, 1.7×10 6 pieces ~ 2.5×10 6 pieces, 1.7×10 6 pieces ~ 2.75×10 6 pieces, 1.7×10 6 pieces ~ 3.0×10 6 pieces, 1.8×10 6 pieces ~ 1.9×10 6 pieces, 1.8×10 6 pieces ~ 2.0×10 6 pieces, 1.8×10 6 pieces ~ 2.25×10 6 pieces, 1.8×10 6 pieces ~ 2.5×10 6 pieces, 1.8×106 pieces~2.75×10 6 pieces, 1.8×10 6 pieces~3.0×10 6 pieces, 1.9×10 6 pieces~2.0×10 6 pieces, 1.9×10 6 pieces~2.25×10 6 pieces, 1.9×10 6 pieces~2.5×10 6 pieces, 1.9×10 6 pieces~2.75×10 6 pieces, 1.9×10 6 pieces~3.0×10 6 pieces, 2.0×10 6 pieces~2.25×10 6 pieces, 2.0×10 6 pieces~2.5×10 6 pieces, 2.0×10 6 pieces~2.75×10 6 pieces, 2.0×10 6 pieces~3.0×10 6 pieces, 2.25×10 6 pieces~2.5×10 6 pieces, 2.25×10 6 pieces~2.75×10 6 pieces, 2.25×10 6 pieces~3.0×10 6 pieces, 2.5×10 6 pieces~2.75×10 6 pieces, 2.5×10 6 pieces~3.0×10 6 pieces or 2.75 x 10 6 pieces~3.0×10 6 HSCs and / or PBMCs are transplanted.

[0104] In some embodiments, the mouse is 7 pieces, 1.1×10 7 pieces, 1.2×10 7 pieces, 1.3×10 7 pieces, 1.4×10 7 pieces, 1.5×10 7 pieces, 1.6×10 7 pieces, 1.7×10 7 pieces, 1.8×10 7 pieces, 1.9×107 pieces, 2.0×10 7 pieces, 2.5×10 7 pieces, 3.0×10 7 10 or more HSCs and / or PBMCs. In some embodiments, mice may be transplanted with 1.0×10 7 pieces~1.1×10 7 pieces, 1.0×10 7 pieces~1.2×10 7 pieces, 1.0×10 7 pieces~1.3×10 7 pieces, 1.0×10 7 pieces~1.4×10 7 pieces, 1.0×10 7 pieces~1.5×10 7 pieces, 1.0×10 7 pieces~1.6×10 7 pieces, 1.0×10 7 pieces~1.7×10 7 pieces, 1.0×10 7 pieces~1.8×10 7 pieces, 1.0×10 7 pieces~1.9×10 7 pieces, 1.0×10 7 pieces~2.0×10 7 pieces, 1.0×10 7 pieces~2.25×10 7 pieces, 1.0×10 7 pieces~2.5×10 7 pieces, 1.0×10 7 pieces~2.75×10 7 pieces, 1.0×10 7 pieces~3.0×10 7 pieces, 1.1×10 7 pieces~×10 7 pieces~1.2×10 7 pieces, 1.1×10 7 pieces~1.3×10 7 pieces, 1.1×10 7 pieces~1.4×10 7 pieces, 1.1×10 7 pieces~1.5×10 7 pieces, 1.1×10 7 pieces~1.6×10 7 pieces, 1.1×10 7 pieces~1.7×10 7 pieces, 1.1×10 7pieces to 1.8×10 7 pieces, 1.1×10 7 pieces to 1.9×10 7 pieces, 1.1×10 7 pieces to 2.0×10 7 pieces, 1.1×10 7 pieces to 2.25×10 7 pieces, 1.1×10 7 pieces to 2.5×10 7 pieces, 1.1×10 7 pieces to 2.75×10 7 pieces, 1.1×10 7 pieces to 3.0×10 7 pieces, 1.2×10 7 pieces to 1.3×10 7 pieces, 1.2×10 7 pieces to 1.4×10 7 pieces, 1.2×10 7 pieces to 1.5×10 7 pieces, 1.2×10 7 pieces to 1.6×10 7 pieces, 1.2×10 7 pieces to 1.7×10 7 pieces, 1.2×10 7 pieces to 1.8×10 7 pieces, 1.2×10 7 pieces to 1.9×10 7 pieces, 1.2×10 7 pieces to 2.0×10 7 pieces, 1.2×10 7 pieces to 2.25×10 7 pieces, 1.2×10 7 pieces to 2.5×10 7 pieces, 1.2×10 7 pieces to 2.75×10 7 pieces, 1.2×10 7 pieces to 3.0×10 7 pieces, 1.3×10 7 pieces to 1.4×10 7 pieces, 1.3×10 7 pieces to 1.5×10 7 pieces, 1.3×10 7 pieces to 1.6×10 7 pieces, 1.3×10 7 pieces to 1.7×10 7 pieces, 1.3×10 7 pieces to 1.8×10 7 pieces, 1.3×107 pieces to 1.9×10 7 pieces, 1.3×10 7 pieces to 2.0×10 7 pieces, 1.3×10 7 pieces to 2.25×10 7 pieces, 1.3×10 7 pieces to 2.5×10 7 pieces, 1.3×10 7 pieces to 2.75×10 7 pieces, 1.3×10 7 pieces to 3.0×10 7 pieces, 1.4×10 7 pieces to 1.5×10 7 pieces, 1.4×10 7 pieces to 1.6×10 7 pieces, 1.4×10 7 pieces to 1.7×10 7 pieces, 1.4×10 7 pieces to 1.8×10 7 pieces, 1.4×10 7 pieces to 1.9×10 7 pieces, 1.4×10 7 pieces to 2.0×10 7 pieces, 1.4×10 7 pieces to 2.25×10 7 pieces, 1.4×10 7 pieces to 2.5×10 7 pieces, 1.4×10 7 pieces to 2.75×10 7 pieces, 1.4×10 7 pieces to 3.0×10 7 pieces, 1.5×10 7 pieces to 1.6×10 7 pieces, 1.5×10 7 pieces to 1.7×10 7 pieces, 1.5×10 7 pieces to 1.8×10 7 pieces, 1.5×10 7 pieces to 1.9×10 7 pieces, 1.5×10 7 pieces to 2.0×10 7 pieces, 1.5×10 7 pieces to 2.25×10 7 pieces, 1.5×10 7 pieces to 2.5×10 7 pieces, 1.5×10 7 pieces to 2.75×107 pieces, 1.5×10 7 pieces ~ 3.0×10 7 pieces, 1.6×10 7 pieces ~ 1.7×10 7 pieces, 1.6×10 7 pieces ~ 1.8×10 7 pieces, 1.6×10 7 pieces ~ 1.9×10 7 pieces, 1.6×10 7 pieces ~ 2.0×10 7 pieces, 1.6×10 7 pieces ~ 2.25×10 7 pieces, 1.6×10 7 pieces ~ 2.5×10 7 pieces, 1.6×10 7 pieces ~ 2.75×10 7 pieces, 1.6×10 7 pieces ~ 3.0×10 7 pieces, 1.7×10 7 pieces ~ 1.8×10 7 pieces, 1.7×10 7 pieces ~ 1.9×10 7 pieces, 1.7×10 7 pieces ~ 2.0×10 7 pieces, 1.7×10 7 pieces ~ 2.25×10 7 pieces, 1.7×10 7 pieces ~ 2.5×10 7 pieces, 1.7×10 7 pieces ~ 2.75×10 7 pieces, 1.7×10 7 pieces ~ 3.0×10 7 pieces, 1.8×10 7 pieces ~ 1.9×10 7 pieces, 1.8×10 7 pieces ~ 2.0×10 7 pieces, 1.8×10 7 pieces ~ 2.25×10 7 pieces, 1.8×10 7 pieces ~ 2.5×10 7 pieces, 1.8×10 7 pieces ~ 2.75×10 7 pieces, 1.8×10 7 pieces ~ 3.0×10 7 pieces, 1.9×10 7 pieces ~ 2.0×10 7 pieces, 1.9×107 pieces~2.25×10 7 pieces, 1.9×10 7 pieces~2.5×10 7 pieces, 1.9×10 7 pieces~2.75×10 7 pieces, 1.9×10 7 pieces~3.0×10 7 pieces, 2.0×10 7 pieces~2.25×10 7 pieces, 2.0×10 7 pieces~2.5×10 7 pieces, 2.0×10 7 pieces~2.75×10 7 pieces, 2.0×10 7 pieces~3.0×10 7 pieces, 2.25×10 7 pieces~2.5×10 7 pieces, 2.25×10 7 pieces~2.75×10 7 pieces, 2.25×10 7 pieces~3.0×10 7 pieces, 2.5×10 7 pieces~2.75×10 7 pieces, 2.5×10 7 pieces~3.0×10 7 , or 2.75 × 10 7 pieces~3.0×10 7 In some embodiments, mice may be transplanted with 2×10 HSCs and / or PBMCs. 7 According to some embodiments, mice are transplanted with 4.5×10 HSCs and / or PBMCs. 7 pieces~5.5×10 7 pieces (4.5×10 7 pieces~5.0×10 7 pieces, 5.0×10 7 pieces~5.5×10 7 1) HSCs and / or PBMCs are transplanted.

[0105] Nucleic Acids: Manipulation and Delivery The mouse model described herein comprises a nucleic acid encoding human APP or a nucleic acid encoding humanized 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 or a humanized APP transgene and a mutant human PSEN1 transgene. In some embodiments, the transgene (e.g., a human APP transgene and / or a mutant human PSEN1 transgene) is integrated into the mouse genome. Human APP or humanized APP, and mutant human PSEN1 transgenes have been described (JAX Stock No. 025970) and are incorporated herein by reference.

[0106] 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.

[0107] 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).

[0108] 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.

[0109] 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.

[0110] 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).

[0111] 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).

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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).

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] (Genome editing) The engineered nucleic acid (e.g., guide RNA, donor polynucleotide, and other nucleic acid coding sequences, etc.) can be introduced into the genome of an embryo or cell (e.g., stem cell) using any suitable method. The present application contemplates the use of various gene editing techniques to introduce nucleic acid into the genome of an embryo or cell, for example, to generate a transgenic rodent. Non-limiting examples include programmable nuclease-based systems, such as clustered regularly interspaced short palindromic repeats (CRISPR) systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., Carroll D, Genetics., 2011;188(4):773-782; Joung JK et al., Nat Rev Mol Cell Biol., 2013;14(1):49-55; and Gaj T et al., Trends Biotechnol., 2013 Jul;31(7):397-405, each of which is incorporated by reference herein.

[0123] In some embodiments, CRISPR system is used to edit genome of mouse embryo provided herein.See, for example, Harms DW et al., Curr Protoc Hum Genet., 2014;83:15.7.1-15.7.27; and Inui M et al., Sci Rep., 2014;4:5396 (each of which is incorporated herein by reference).For example, Cas9 mRNA or Cas9 protein, one or more guide RNAs (gRNAs), and / or donor nucleic acid can be directly delivered (e.g., injected or electroporated) into one-cell (zygote) stage or later stage mouse embryo, and homologous recombination repair (HDR) can be promoted, for example, engineered nucleic acid (e.g., donor nucleic acid) can be introduced into genome.

[0124] The CRISPR / Cas system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA targeting platform for gene editing. Engineered CRISPR systems contain two main components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (e.g., a Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence for nuclease binding and a user-defined nucleotide spacer (e.g., about 15-25 nucleotides, or about 20 nucleotides) that defines the genomic target (e.g., gene) to be modified. Thus, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA. In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes (NGG PAM) or Staphylococcus aureus (NNGRRT or NNGRR(N) PAM), although other Cas9 homologs, Cas9 orthologs, and / or Cas9 variants (e.g., evolved versions of Cas9) may be used as provided herein. Further non-limiting examples of RNA-guided nucleases that can be used as provided herein include Cpf1 (TTN PAM); SpCas9 D1135E variant (NGG (reduced NAG binding) PAM); SpCas9 VRER variant (NGCG PAM); SpCas9 EQR variant (NGAG PAM); SpCas9 VQR variant (NGAN or NGNG PAM); Cas9 from Neisseria meningitidis (NM) (NNNNGATT PAM); Cas9 from Streptococcus thermophilus (ST) (NNAGAAW PAM); and Cas9 from Treponema denticola (TD) (NAAAAC). In some embodiments, the CRISPR-associated endonuclease is selected from Cas9, Cpf1, C2c1, and C2c3. In some embodiments, the Cas nuclease is Cas9.

[0125] Guide RNA comprises at least a spacer sequence that hybridizes (binds) to target nucleic acid sequence and a CRISPR repeat sequence that binds to its endonuclease and guides its endonuclease to its target nucleic acid sequence.As understood by those skilled in the art, each gRNA is designed to comprise a spacer sequence that is complementary to its genome target sequence.See, for example, Jinek et al., Science, 2012;337:816-821 and Deltcheva et al., Nature, 2011;471:602-607 (each of which is incorporated herein by reference).

[0126] In some embodiments, the RNA-guided nuclease and the gRNA are complexed to form a ribonucleoprotein (RNP) prior to delivery to the embryo.

[0127] The concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease may vary. In some embodiments, the concentration is 100ng / μl to 1000ng / μl. For example, the concentration may be 100ng / μl, 150ng / μl, 200ng / μl, 250ng / μl, 300ng / μl, 350ng / μl, 400ng / μl, 450ng / μl, 500ng / μl, 550ng / μl, 600ng / μl, 650ng / μl, 700ng / μl, 750ng / μl, 800ng / μl, 850ng / μl, 900ng / μl, 950ng / μl, or 1000ng / μl. In some embodiments, the concentration is between 100 ng / μl and 500 ng / μl, or between 200 ng / μl and 500 ng / μl.

[0128] The concentration of gRNA can also be varied. In some embodiments, the concentration is 200ng / μl to 2000ng / μl. For example, the concentration can be 200ng / μl, 300ng / μl, 400ng / μl, 500ng / μl, 600ng / μl, 700ng / μl, 800ng / μl, 900ng / μl, 1000ng / μl, 1100ng / μl, 1200ng / μl, 1300ng / μl, 1400ng / μl, 1500ng / μl, 1600ng / μl, 1700ng / μl, 1700ng / μl, 1900ng / μl, or 2000ng / μl. In some embodiments, the concentration is 500ng / μl to 1000ng / μl. In some embodiments, the concentration is 100ng / μl to 1000ng / μl. For example, the concentration can be 100ng / μl, 150ng / μl, 200ng / μl, 250ng / μl, 300ng / μl, 350ng / μl, 400ng / μl, 450ng / μl, 500ng / μl, 550ng / μl, 600ng / μl, 650ng / μl, 700ng / μl, 750ng / μl, 800ng / μl, 850ng / μl, 900ng / μl, 950ng / μl, or 1000ng / μl.

[0129] In some embodiments, the ratio of the concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease to the concentration of the gRNA is 2: 1. In other embodiments, the ratio of the concentration of the RNA-guided nuclease or the nucleic acid encoding the RNA-guided nuclease to the concentration of the gRNA is 1: 1.

[0130] Donor nucleic acid typically comprises a sequence of interest flanked by homology arms. Homology arms are ssDNA regions that are homologous to genomic DNA regions located in genomic loci. One homology arm is located on the left (5') side of the genomic region of interest (where the sequence of interest is inserted) (left homology arm), and another homology arm is located on the right (3') side of the genomic region of interest (right homology arm). These homology arms allow homologous recombination between the ssDNA donor and genomic locus, resulting in the insertion of the sequence of interest into the genomic locus of interest (e.g., via CRISPR / Cas9-mediated homology-directed repair (HDR)).

[0131] The homology arms can vary in length. For example, each homology arm (left and right homology arms) can have a length of 20 to 1000 nucleotide bases. In some embodiments, each homology arm has a length of 20 to 200 nucleotide bases, 20 to 300 nucleotide bases, 20 to 400 nucleotide bases, 20 to 500 nucleotide bases, 20 to 600 nucleotide bases, 20 to 700 nucleotide bases, 20 to 800 nucleotide bases, or 20 to 900 nucleotide bases. In some embodiments, each homology arm has a length of 20 nucleotide bases, 30 nucleotide bases, 40 nucleotide bases, 50 nucleotide bases, 60 nucleotide bases, 70 nucleotide bases, 80 nucleotide bases, 90 nucleotide bases, 100 nucleotide bases, 150 nucleotide bases, 200 nucleotide bases, 250 nucleotide bases, 300 nucleotide bases, 350 nucleotide bases, 400 nucleotide bases, 450 nucleotide bases, 500 nucleotide bases, 550 nucleotide bases, 600 nucleotide bases, 650 nucleotide bases, 700 nucleotide bases, 750 nucleotide bases, 800 nucleotide bases, 850 nucleotide bases, 900 nucleotide bases, 950 nucleotide bases, or 1000 nucleotide bases. In some embodiments, the length of one homology arm is different from the length of the other homology arm. For example, one homology arm can have a length of 20 nucleotide bases, and the other homology arm can have a length of 50 nucleotide bases. In some embodiments, the donor DNA is single-stranded. In some embodiments, the donor DNA is double stranded. In some embodiments, the donor DNA is modified, for example, via phosphorothiolation. Other modifications can be made. EXAMPLES

[0132] Example 1. Generation of NSG.APP / PSEN1 mouse model NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(APPswe,PSEN1dE9)85Dbo / How(JR# 29513)(NSG.APP / PSEN1) and male NOD.Cg-Tg(APPswe,PSEN1dE9)85Dbo / How(JR# 25967) were transgenic for female NOD.Cg-Prkdc. scid Il2rg tm1Wjl / SzJ (JR# 005557). Male offspring were genotyped for the presence of the APP / PSEN1 transgene and gamma mutations and subsequently crossed to female NSG mice. Cohorts of male NSG.APP / PSEN1 and female NSG.APP / PSEN1 were generated at N11 for evaluation. All mice were maintained on Sulfatrim antibiotic water. These mice represent a unique platform for the evaluation of immune interactions with amyloid through the introduction of substances from different strain backgrounds or human origin.

[0133] (behavioral phenotype) Cognition of the NSG.APP / PSEN1 mouse model was assessed at 7 months of age for the short-term memory Y-maze task novel spatial recognition as previously described (Sukoff Rizzo, 2018). Animals were placed in a Y-maze with visual cues located at the end of each arm. The animals explored the maze for 10 min with one of the arms (the novel arm) blocked off. After a 30 min delay, the animals were reintroduced to the maze for 5 min with all arms available. The results are shown in Figure 1. Intact short-term memory is indicated when the animal spends a higher percentage of time in the novel arm. Both male and female NSG animals showed intact short-term memory, while transgenic NSG.APP / PSEN1 littermates failed the task, spending an equal percentage of time exploring all arms. These findings are in contrast to the B6.APP / PSEN1 mouse model, which does not show cognitive deficits in this task at 7 months of age. Furthermore, the B6.APP / PSEN1 mice have not been reported to show deficits in other cognitive tasks, such as the Morris water maze, up to 10 months of age (AlzForum). These results highlight the utility of the NSG.APP / PSEN1 line for evaluation of immune-related interventions that may prevent cognitive deficits.

[0134] (Plaque deposits) Animals were sacrificed at 8 months of age via cardiac perfusion with 1x PBS, and brains were fixed overnight in 4% paraformaldehyde, placed in 15% sucrose followed by 30% sucrose for 24 hours, blocked in OCT, and cryosectioned every 25 microns for assessment of neuropathology. Amyloid deposition was assessed using 1% Thioflavin S staining (diluted in a 1:1 water:ethanol ratio), which showed that plaques were primarily confined to the hippocampus with minimal cortical deposits (see Figure 2). As this behavioral task requires the hippocampus, these results suggest a direct link to short-term memory deficits. This plaque regional specificity is in stark contrast to that observed in the B6.APP / PSEN1 mouse model, which shows hippocampal and robust cortical plaque deposition at this time point (Jackson et al., 2013; Onos et al., 2019). These results are also consistent with that seen in human patients, where plaque pathology occurs primarily in the hippocampus. As expected, non-transgenic NSG littermates did not exhibit plaque pathology.

[0135] (glial neuroinflammation) Immunofluorescence staining with a marker of astrocytes (anti-chicken GFAP, ACRIS / Origene, AP31806PU-N, 1:300) and microglia (anti-rabbit IBA1, Wako, 019-19741, 1:300) shows that despite impaired adaptive immunity, NSG.APP / PSEN1 mice still exhibit robust neuroinflammation in the brain in response to amyloid (see Figure 3). These results suggest that NSG.APP / PSEN1 mice retain intact innate immune signaling. Given these findings, the NSG.APP / PSEN1 mouse model may be useful to analyze the effects of transplanted glia from other lineage backgrounds or of human origin on cognitive deficits.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0141] When a range of values ​​is provided, each value between the upper and lower limits of the range is specifically contemplated and described herein.

[0142] (References) [ka] [ka] [ka] [ka] [ka] [ka] [ka]

Claims

1. An immunocompromised mouse comprising in its genome a loss-of-function mutation in the mouse Prkdc gene, a loss-of-function mutation in the mouse Il2rg gene, and a nucleic acid encoding a human amyloid precursor protein (APP) or a nucleic acid encoding a humanized amyloid precursor protein (APP).

2. 2. The immunocompromised mouse of claim 1 having a non-obese diabetic (NOD) genetic background.

3. 3. The immunocompromised mouse of claim 1 or 2, wherein the loss-of-function mutation in the mouse Prkdc gene is a null mutation in the mouse Prkdc gene, optionally wherein the null mutation in the mouse Prkdc gene is a Prkdc scid mutation.

4. 4. The immunocompromised mouse of claim 1, wherein the loss-of-function mutation in the mouse Il2rg gene is a null mutation in the mouse Il2rg gene, and optionally, the null mutation in the mouse Il2rg gene is an Il2rg tm1Wjl mutation or an Il2rg em26Cd22 mutation.

5. NOD. Cg-Prkdc ｓｃｉｄ Il2rg ｔｍ１Ｗｊｌ 5. The immunocompromised mouse of claim 4 having a NOD-Prkdc<em>Cd52 Il2rg<em>Cd22 / SzJ genetic background or a NOD-Prkdc<em>Cd52 Il2rg<em>Cd22 / NjuCrl genetic background.

6. The immunocompromised mouse of any one of claims 1 to 5, wherein the nucleic acid encoding the humanized APP is a chimeric nucleic acid comprising a mouse coding sequence and a human coding sequence, optionally wherein the chimeric nucleic acid comprises a human coding sequence in the A-β domain of the mouse APP coding sequence, and optionally 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 immunocompromised mouse of claim 6 , comprising an APPswe transgene in its genome.

8. 8. The immunocompromised mouse of any one of claims 1 to 7, further comprising in its genome a nucleic acid encoding a mutant human presenilin 1 protein (PSEN1), optionally wherein the nucleic acid encoding the mutant PSEN1 comprises a human PSEN coding sequence comprising a deletion in exon 9.

9. The immunocompromised mouse of claim 8 , comprising a PSENde9 transgene in its genome.

10. 10. The immunocompromised mouse of claim 9, comprising a Tg(APPswe, PSEN1de9)85Dbo transgene insertion in its genome.

11. An immunocompromised mouse described in any one of claims 1 to 10, optionally having at least one characteristic of early-onset Alzheimer's disease selected from cognitive deficits compared to controls, increased hippocampal plaque deposits compared to controls, and increased neuroinflammation in the brain compared to controls.

12. An immunocompromised mouse comprising a nucleic acid encoding human amyloid precursor protein (APP) or a nucleic acid encoding a humanized amyloid precursor protein (APP), which has no measurable tumor burden and / or is at least 1 year old.

13. Prkdc ｓｃｉｄ Mutations and Il2rg ｔｍ１Ｗｊｌ A non-obese diabetic (NOD) mouse carrying the mutation and the APPswe transgene and the PSENde9 transgene in its genome.

14. A descendant mouse of the mouse according to any one of claims 1 to 13.

15. A method comprising the step of producing a mouse according to any one of claims 1 to 14. (a) introducing into a non-obese diabetic (NOD) mouse a null mutation in a mouse Prkdc gene, a null mutation in a mouse Il2rg gene, a nucleic acid encoding a human amyloid precursor protein (APP) or a nucleic acid encoding a humanized amyloid precursor protein (APP), and a nucleic acid encoding a mutant human presenilin 1 protein (PSEN1); or (b) introducing the Prkdc scid mutation, the Il2rg tm1Wjl mutation, the APPswe transgene, and the PSENde9 transgene into non-obese diabetic (NOD) mice; A method comprising:

17. (a) mating a NOD mouse comprising (i) a loss-of-function mutation in the mouse Prkdc gene and (ii) a loss-of-function mutation in the mouse Il2rg gene to (b) a NOD mouse comprising (i) a nucleic acid encoding a human amyloid precursor protein or a nucleic acid encoding a humanized amyloid precursor protein and (ii) a nucleic acid encoding a mutant human presenilin 1 protein to generate immunocompromised progeny mice with characteristics of early onset Alzheimer's disease; or (a) crossing non-obese diabetic (NOD) mice containing the Prkdc scid and Il2rg tm1Wjl mutations with (b) NOD mice containing the APPswe transgene and the PSENde9 transgene. A method comprising:

18. A method comprising breeding the offspring mouse of claim 17.

19. 20. A method comprising breeding progeny mice from the progeny mice of claim 18.

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