Genetic modification of mammalian cells to confer resistance to CSF1R antagonists

Abstract

Microglia / monocytes reside in a "niche" that limits the total number of microglia / monocytes / macrophages present in the mammalian central nervous system (CNS). Thus, there is a need for methods that can help therapeutically modify microglia, monocytes and macrophages or the cells that give rise to them that compete with endogenous microglia and partially or completely occupy the CNS niche. The present disclosure features therapeutic microglia, therapeutic monocytes or therapeutic macrophages that have a selective advantage in response to CSF1 R inhibitors compared to endogenous brain-resident microglia. Specifically, the therapeutic cells developed in the present disclosure are not killed by a given dose of CSF1 R inhibitor that is sufficient to kill endogenous microglia. The therapeutic cells described herein can be used to treat neurological diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 169,578, filed April 1, 2021, and U.S. Provisional Application No. 63 / 236,951, filed August 25, 2021, the contents of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing Reference Applicant asserts that the information submitted pursuant to Rule 13ter.1(a) and recorded in the form of an Annex C / ST.25 text file entitled "UCI 21.03 PCT Sequencing Listing_ST25" is identical to that which forms part of the international application as filed. The contents of the sequence listing are incorporated herein by reference in their entirety.

[0003] The present disclosure features methods and compositions for treating neurological disorders. In particular, the methods and compositions feature cells that have partial or complete resistance to CSF1R antagonists. [Background technology]

[0004] Nearly all human neurological diseases and injuries involve impairment or alteration of the function, gene expression and activation state of microglia, the brain's resident immune cells. In some cases, genetic mutations in microglia are the primary cause of neurological disease, whereas in others, polymorphisms in microglial genes increase the risk of developing the disease. Unfortunately, there are few, if any, effective treatments for many of these diseases and injuries.

[0005] Microglia, monocytes and macrophages reside in a "niche" that may limit the total number of microglia, monocytes and macrophages present in the mammalian central nervous system (CNS). Thus, there is a need for a method that can help therapeutically modify microglia, monocytes and macrophages or the cells that give rise to them that compete with endogenous microglia and partially or completely occupy the CNS niche. The objective of the present disclosure is to develop microglia-based therapeutics, including techniques for transplanting genetically modified human microglia or related monocyte lineage or progenitor cells into patients. Summary of the Invention

[0006] It is an object of the present disclosure to provide compositions and methods that allow for differential resistance (eg, partial, complete, increased, decreased) to CSF1R antagonists for the treatment of neurological disorders.

[0007] Microglia depend on CSF1R signaling through two ligands (CSF1 and IL-34) for survival, proliferation and self-renewal. Treatment of mammalian models with various CSF1R antagonists reduces the number of microglia within the central nervous system. Furthermore, when these compounds are removed, surviving microglia proliferate and rapidly refill the niche. In addition, human stem cell-derived microglia transplanted into mouse brains are also found to be sensitive to and die upon CSF1R inhibition (Figure 11F, Figure 11G, Figure 11H, Figure 11I, Figure 11J).

[0008] To compete with endogenous microglial nits, therapeutic microglia, therapeutic monocytes, therapeutic macrophages, or their progenitors or precursors must have a selective advantage in their response to CSF1R inhibitors compared to endogenous brain-resident microglia. Specifically, it is necessary to develop therapeutic cells that do not die at a given dose of CSF1R inhibitor that is sufficient to kill endogenous microglia. In some cases, this selective advantage should be partial so that even higher doses of CSF1R inhibitors can also kill therapeutic microglia, therapeutic macrophages, therapeutic monocytes, or their progenitors or precursors if necessary for safety purposes.

[0009] In some embodiments, the disclosure features a cell (e.g., a modified human cell) that includes a nucleic acid encoding a modified CSF1R protein (e.g., a human CSF1R protein) that exhibits differential resistance to a CSF1R antagonist. In other embodiments, the disclosure features a modified human cell that exhibits differential resistance to a CSF1R antagonist. In further embodiments, the disclosure features a modified human cell that includes a nucleic acid encoding a modified CSF1R protein that exhibits differential resistance to a CSF1R antagonist.

[0010] In some embodiments, the present disclosure may also feature a method of treating a subject. In some embodiments, the method may include administering a CSF1R antagonist to the subject in an amount sufficient to inhibit CSF1R signaling in the subject's cells, and contacting the subject with a modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte) that has differential resistance to the CSF1R antagonist. In other embodiments, the method may include contacting the subject with a modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte) that has differential resistance to the CSF1R antagonist, and administering a CSF1R antagonist to the subject in an amount sufficient to inhibit CSF1R signaling in the subject's cells.

[0011] In a further embodiment, the disclosure also features a method of treating a subject, the method including contacting the subject with a modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte) and differentially altering the proliferation or survival of the unmodified CSF1R-expressing cell (e.g., the modified CSF1R-expressing phagocyte) compared to the modified CSF1R-expressing cell (e.g., the modified CSF1R-expressing phagocyte).

[0012] In some embodiments, the disclosure may further feature nucleic acid compositions and vectors encoding modified CSF1R proteins that exhibit differential resistance to the CSF1R antagonists described herein.

[0013] The technical features of the present disclosure include the use of differential resistance (e.g., partial, complete, increased, or decreased resistance) to CSF1R antagonists to generate therapeutic microglia, therapeutic macrophages, therapeutic monocytes, or their progenitor cells or precursors for the treatment of neurological diseases. Without wishing to limit the present disclosure to any theory or mechanism, it is believed that the technical features of the present disclosure advantageously provide for the generation of microglia, monocytes, HSPCs (hematopoietic stem or progenitor cells) or primitive macrophages from pluripotent stem cells with differential resistance (e.g., CSF1R antagonist resistance) to CSF1R antagonists. As described herein, without the use of some form of pretreatment or selective advantage, transplantation of the aforementioned cells is likely to show only partial or limited engraftment into the central nervous system (CNS). The present disclosure exploits the techniques described herein that significantly promote therapeutic engraftment of microglia, macrophages or monocytes or their progenitor cells (HSPCs, PMPs) in the mammalian brain. None of the currently known prior references or works possess the unique inventive technical features of the present invention.

[0014] The compositions and methods of the present disclosure represent a major departure from the current paradigm for using CSF1R antagonists in medicine. For example, the prior art teaches the use of CSF1R inhibitors to eliminate endogenous microglia and promote the infiltration of bone marrow-derived and / or blood-derived monocytes into the brain. However, bone marrow-derived and / or blood-derived monocytes that infiltrate the brain remain functionally and transcriptionally distinct from microglia even many months after infiltration. Furthermore, bone marrow-derived and / or blood-derived monocytes do not have the same sensitivity to CSF1R antagonists as microglia, demonstrating that these cells are not microglia. This teaches away from the present disclosure of promoting the expansion and proliferation of therapeutic microglia (e.g., human microglia) containing modified CSF1R proteins for the treatment of neurological diseases.

[0015] Moreover, most major pharmaceutical companies are developing CSF1R antagonist compounds to treat some forms of cancer. However, endogenous microglia are extremely sensitive to CSF1R antagonists and die in response to treatment with increasing doses of these compounds. This is also true for human stem cell-derived microglia transplanted into mouse brains (Figure 11F, Figure 11G, Figure 11H, Figure 11I, Figure 11J), and cultured human microglia (Figure 4A, Figure 4B, Figure 4C, Figure 4D, and Figure 4E). Paradoxically, the present disclosure utilizes compounds that are toxic to microglia (e.g., CSF1R antagonists) to improve long-term engraftment of transplanted microglia (e.g., transplanted therapeutic microglia; i.e., microglia containing modified CSF1R proteins). The methods of the present disclosure solve the problem of achieving widespread engraftment of transplanted microglia (e.g., transplanted therapeutic microglia; i.e., microglia containing modified CSF1R protein) in established microglial niches that are normally not amenable to such engraftment.

[0016] Moreover, the technical features of the present disclosure contribute to surprising results, illustrating the unpredictability of outcomes associated with these mutations. For example, two of the proposed point mutations result in complete (100%) protection against two CSF1R inhibitors (i.e., CSF1R antagonists) across multiple escalating doses when assessed 24 hours after inhibitor treatment (Figure 4A, Figure 4B, Figure 4C, Figure 4D, and Figure 4E). In contrast, the third mutation, G795V, prevented successful differentiation of iPSCs into microglia (Figure 1). These data demonstrate the importance of the specific DNA and resulting amino acid changes introduced into CSF1R, demonstrating that while some changes achieve the desired resistance to CSF1R inhibitors (i.e., CSF1R antagonists), other changes may instead impair the ability of microglia to differentiate or survive.

[0017] Thus, the present disclosure features methods and compositions that promote competitive engraftment of human microglia, human macrophages and human monocytes and / or their precursor cells (HSPCs, erythromyeloid progenitor cells (EMPs) or primitive macrophage progenitor cells (PMPs)). The cells can be obtained from a variety of sources, including, but not limited to, pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), bone marrow-derived hematopoietic stem cells, umbilical cord blood cells, blood-derived monocytes, fetal yolk sac macrophages or tissue macrophages, adult tissue-derived macrophages, or via direct reprogramming of another cell type into microglia, HSPCs, monocytes or macrophages. Additionally, the present disclosure may feature methods and compositions that promote competitive engraftment of CSF1R-expressing cells, including, but not limited to, cells from the heart, skin, liver, lung, kidney, eye, prostate and ovary.

[0018] Any feature or combination of features described herein is included within the scope of the present disclosure, unless the features included in any such combination are mutually inconsistent as is apparent from the context, the present specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present disclosure are apparent in the following detailed description and claims.

[0019] The features and advantages of the present disclosure will become apparent from consideration of the following detailed description, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0020] [Figure 1] In vitro proliferation rates of control and CSF1R mutant microglia are shown. Isogenic iPSC-derived hematopoietic progenitor cells were differentiated into microglia and cell densities were used for the first 14 days. The two wild-type isogenic control lines (Cntl-1 and Cntl-2) demonstrate some expected variability in iPSC-microglia proliferation rates. Both hCSF1R G795A and hCSF1R G785C mutants show proliferation rates comparable to the two control lines. In contrast, the hCSFR G794V mutant shows a rapid drop in cell density with no viable cells remaining beyond day 6 of differentiation. These data demonstrate that the specific choice of amino acid substitutions is critical for successful generation of viable microglia that can also exhibit CSF1R antagonist resistance.

[0021] [Figure 2A] We show that selected CSF1R mutations have little effect on the transcriptomic signature while simultaneously conferring resistance to PLX5622. [Figure 2B] We show that selected CSF1R mutations have little effect on the transcriptomic signature while simultaneously conferring resistance to PLX5622. [Figure 2C] We show that selected CSF1R mutations have little effect on the transcriptomic signature while simultaneously conferring resistance to PLX5622. [Figure 2D] We show that selected CSF1R mutations have little effect on the transcriptomic signature while simultaneously conferring resistance to PLX5622. [Figure 2E]We show that selected CSF1R mutations have little effect on the transcriptomic signature while simultaneously conferring resistance to PLX5622. [Figure 2F] We show that selected CSF1R mutations have little effect on the transcriptome signature while simultaneously conferring resistance to PLX5622. Figure 2A shows that principal component analysis using the top 2,000 genes reveals that the major source of variation in the dataset is the WT response to PLX5622. Figure 2B and Figure 2C show that linear regression analysis and calculation of Pearson correlation coefficients between DMSO-treated MUT1 (i.e., G795A; Figure 2B) or MUT2 (i.e., G795C; Figure 2C) and WT microglia confirmed a high degree of concordance when investigating the full transcriptome and a core microglial gene signature of 249 genes. Figures 2D, 2E, and 2F show that treatment of WT (Figure 2D), MUT1 (i.e., G795A; Figure 2E), and MUT2 (i.e., G795C; Figure 2F) microglia with PLX5622 for 24 h induced significant transcriptomic changes in WT cells (FDR ≤ 0.05; log2(FC) ≥ ± 1) when compared to DMSO-treated cells. However, treatment of both MUT1 and MUT2 lines failed to significantly alter gene expression, suggesting that these lines were resistant to the effects of PLX5622.

[0022] [Diagram 3] Distance matrices for CSF1R mutant and control xMG (microglia) are shown. RNA-seq expression counts were normalized and transformed using a variance-stabilizing transformation. Pairwise Euclidean distances were calculated between all samples and samples were hierarchically clustered by distance. These data demonstrate that the transcriptome of G795A mutant microglia is most comparable to that of WT CSF1R microglia.

[0023] [Figure 4A]We show that human stem cell-derived microglia are also sensitive to and killed by CSF1R inhibition in vitro. [Figure 4B] We show that human stem cell-derived microglia are also sensitive to and killed by CSF1R inhibition in vitro. [Figure 4C] We show that human stem cell-derived microglia are also sensitive to and killed by CSF1R inhibition in vitro. [Figure 4D] We show that human stem cell-derived microglia are also sensitive to and killed by CSF1R inhibition in vitro. [Figure 4E]We show that human stem cell-derived microglia are also sensitive to CSF1R inhibition in vitro and die upon CSF1R inhibition. Figure 4A shows caspase 3 / 7 levels imaged over 24 hours in culture with complete medium containing 0.1% DMSO, 250 nm PLX3397, 500 nm PLX3397 and 1 μm PLX3397. Images captured using an Incucyte S3 live cell imager. Figure 4B shows caspase 3 / 7 levels imaged over 24 hours in culture with complete medium containing 0.1% DMSO, 250 nm PLX5622, 500 nm PLX5622 and 1 μm PLX5622. Images captured using an Incucyte S3 live cell imager. Figure 4C shows caspase 3 / 7 levels imaged over 24 hours upon culture with complete medium containing 0.1% DMSO, 250 nm edicotinib (JNJ-40346527), 500 nm edicotinib and 1 μm edicotinib. Images captured using an Incucyte S3 live cell imager. Figure 4D shows caspase 3 / 7 levels imaged over 24 hours upon culture with complete medium containing 0.1% DMSO, 250 nm BLZ945, 500 nm BLZ945 and 1 μm BLZ945. Images captured using an Incucyte S3 live cell imager. Figure 4E shows representative images of caspase 3 / 7 fluorescent activity for wild type, G795A and G795C microglia after 24 hours upon culture with PLX3397. For all panels, n=4 images in 6 independent wells were quantified. Data expressed as mean ± SEM.

[0024] [Figure 5A] 13 shows that G795A iPSC-derived microglia are resistant to CSF1R antagonist-induced cell death. [Figure 5B]We show that G795A iPSC-derived microglia are resistant to CSF1R antagonist-induced cell death. Because cells can die from processes other than caspase-mediated apoptosis, it is important to determine whether the G795A mutation provides a broader range of resistance to any form of cell death induced by CSF1R antagonists. Therefore, G795A iPSC-microglia and wild-type (WT) iPSC-microglia were treated for 24 hours with 500 nM (Figure 5A) or 1 uM (Figure 5B) DMSO vehicle control or one of four CSF1R antagonists, PLX3397, PLX5622, edicotinib, and BLZ945. The percentage of live versus dead cells was then quantified using a two-color viability / cytotoxicity approach (Thermo L3224; LIVE / DEAD™ Viability / Cytotoxicity Kit). After 24 hours of treatment with DMSO, very few dead microglia are observed. In contrast, treatment with CSF1R antagonists induces substantial cell death in wild-type microglia, but little or no cell death in G795A microglia. Live cells are indicated by circles.

[0025] [Figure 6]Figure 1 shows that treatment of mutant CSF1R expressing microglia with 25ng / ml Human Macrophage Colony Stimulating Factor (Peprotech #300-25) after 48 hours stimulates tyrosine phosphorylation of CSF-1R / M-CSF-R protein as detected by PathScan® Phospho-CSF-1R / M-CSF-R (panTyr) Sandwich ELISA Kit n=7. Compared to WT microglia, microglia expressing CSF1R G795A phosphorylate similarly, whereas microglia expressing CSF1R G795C show elevated levels of phosphorylation. These data suggest that both G795A and G795C confer resistance to CSF1R antagonists, but the G795C mutation may result in enhanced CSF1R signaling. Data presented as mean ± SEM.

[0026] [Figure 7A] 1 shows the crystal structure of the CSF1R receptor bound to a CSF1R antagonist. [Figure 7B] 1 shows the crystal structure of the CSF1R receptor bound to a CSF1R antagonist. [Figure 7C] 1 shows the crystal structure of the CSF1R receptor bound to a CSF1R antagonist. [Figure 7D]Crystal structures of CSF1R receptor bound to CSF1R antagonists are shown. Figure 7A shows human CSF1R receptor bound to PLX5622 CSF1R antagonist, and Figure 7B shows CSF1R receptor bound to PLX3397 CSF1R antagonist. The crystal structures were examined to predict amino acid substitutions that may sterically interfere with the binding of CSF1R antagonists without compromising the normal binding of ATP to the same binding pocket. The crystal structures of CSF1R antagonists PLX5622 (Figure 7A) or PLX3397 (Figure 7B) bound to human CSF1R were previously published. This publicly available data was used to investigate the ATP binding pocket. Through molecular modeling, single amino acid changes were predicted that would likely impair the binding of PLX5622 or PLX3397 or other CSF1R antagonists without disrupting normal ATP binding to CSF1R (Figure 7C). Specifically, the modeling suggests that substitution of amino acid G795 with either alanine (A), valine (V) or cysteine ​​(C) increases steric hindrance of PLX5622 and / or PLX3397 binding without disrupting the normal binding ability of ATP. Other mutations that prevent binding could also be explored (Figure 7D).

[0027] [Figure 8] 1 shows DNA chromatograms demonstrating successful production of isogenic human iPSC cell lines encoding a single amino acid point mutation at position 795 within the CSF1R coding sequence. In this particular case, each line was generated by CRISPR-mediated gene editing. However, similar changes can be introduced by a variety of other gene editing methods, including but not limited to TALEN, conventional homologous recombination, viral gene delivery or other CRISPR variants.

[0028] [Figure 9A] We show that selected CSF1R mutations have little effect on the transcriptomic signature in vivo. [Figure 9B]We show that selected CSF1R mutations have little effect on the transcriptomic signature in vivo. [Figure 9C] We show that selected CSF1R mutations have little effect on the transcriptomic signature in vivo. [Figure 9D] We show that selected CSF1R mutations have little effect on the transcriptomic signature in vivo. [Figure 9E] We show that selected CSF1R mutations have little effect on the transcriptome signature in vivo. Figure 9A shows that principal component analysis using the top 2,000 genes reveals that the majority of the variation between G795A, G795C and WT microglia is due to individual animals and not CSF1R mutations. In contrast, comparing G795V, there is some separation based on this mutation, further indicating that G795V may not allow for the generation of microglia without substantially altering gene expression. Figures 9B and 9C show that linear regression analysis and calculation of the Pearson correlation coefficient between G795A (Figure 9B) or G795C (Figure 9C) and WT microglia confirmed a high degree of concordance when investigating the full transcriptome and the 190-gene core microglial signature. Figure 9D and Figure 9E show that volcano plots of explanted human microglia 2 months after transplantation in MITRG mice show minimal significant transcriptomic changes (FDR < 0.05; log2(FC) > ± 1) in mutant cells when compared to WT cells.

[0029] [Figure 10A] We show that G795A iPSC-derived microglia exhibit a transcriptomic signature following xenotransplantation in vivo that is highly similar to that of isogenic wild-type iPSC-derived microglia. [Figure 10B]We show that G795A iPSC-derived microglia exhibit a transcriptomic signature following xenografting in vivo that is highly similar to that of isogenic wild-type iPSC-derived microglia. Xenograft-compatible mice were engrafted with wild-type (WT) microglia or G795A iPSC-microglia, and after 2 months, human cells were isolated and investigated by bulk RNA sequencing. Figure 10A shows the correlation across all detected genes for xMG isolated from individual WT- and G795A-engrafted mice. Although differences between individual mouse recipients introduce some variability, WT xMG and G795 xMG remain highly correlated, with R2 between 0.80 and 0.95. Figure 10B shows that a similar comparison was performed using an additionally selected microglia-specific gene list. This comparison similarly revealed a strong correlation between wild-type and G795A xMG, with R2 ranging from 0.78 to 0.97. Consistent with previous in vitro RNA sequencing analyses, these results demonstrate that the G795A CSF1R mutation has little to no effect on the underlying transcriptome of human microglia following in vivo engraftment, with most of the variance detected being due to differences between individual mouse graft recipients.

[0030] [Figure 11A] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11B] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11C] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11D]We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11E] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11F] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11G] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11H] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11I] We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. [Figure 11J]We show that G795A iPSC-derived microglia are resistant to CSF1R agonist treatment and enable robust engraftment of human microglia in the adult mammalian brain. Xenograft-compatible mice received bilateral stereotaxic intrahippocampal injections of 500,000 iPSC-derived human microglia (xMG) at 2 months of age. Mice received xMG differentiated from homozygous CSF1R-resistant G795A human iPSC lines or isogenic unmodified wild-type human iPSCs. When mice were 4 weeks of age, they were allowed ad libitum access to 600 mg / kg PLX3397 in rodent chow for 4 weeks. Mice were then sacrificed and half of the brains examined by fluorescent immunohistochemistry and confocal microscopy. Figures 11A-11E show that over the course of 1 month of PLX3397 treatment, G795A xMG proliferate and expand from the initial injection site within the hippocampus and cortex. We labeled microglia with IBA-1 (Figures 11A and 11F) and co-labeled the nuclei of human microglia using the human specific markers Ku80 (Figures 11B and 11G) and Ki67 (Figures 11C and 11H) to show a "wavefront" of proliferative human xMG migrating toward remaining unoccupied niches within the cortex and thalamus (arrows). Figure 11E shows a high magnification image of the boxed area in Figure 11D demonstrating that all IBA1+ microglia co-express Ku80. In contrast, in wild type human xMG, only a handful survive 1 month of PLX3397 treatment (Figures 11F-11I). For example, the arrows in Figure 11J reveal the only four viable xMGs within the boxed area shown in Figure 11I.

[0031] [Figure 12]Quantification of CSF1R-G795A human iPSC-microglia engraftment following transplantation into adult hCSF1 mice. Transplantation of CSF1R-G795A iPSC-microglia (without treatment with CSF1R antagonist) into fully occupied adult microglial niches results in extremely limited engraftment of human microglia (5-10% of total microglia) as measured by colocalization of the microglial marker IBA1 with the human specific nuclear marker Ku80 (Ku80+ / Iba1+). In contrast, treatment of mice with PLX3397-containing mouse chow (600 mg / kg PLX3397 in food) for 10 days after CSF1R-G795A iPSC-microglia transplantation increases the percentage of human microglia to nearly 25%. After 30 days of PLX3397 treatment (600 mg / kg PLX3397 in food), 90-95% of total microglia in the brain are human, as evidenced by co-expression of Ku80 and IBA1. By transplanting CSF1R-G795A iPSC-microglia and varying the concentration and duration of CSF1R antagonist treatment either before, during or after transplantation, the degree of human iPSC-microglia engraftment can be controlled. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Although embodiments of the present disclosure have been described in detail, including embodiments that do not provide all of the features and advantages described herein, certain variations and modifications will be apparent to those skilled in the art. It will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and / or uses and their obvious modifications and equivalents. Furthermore, while numerous variations have been shown and described in varying degrees of detail, other modifications that are within the scope of the present disclosure will be readily apparent to those skilled in the art based on the present disclosure. Also, various combinations or subcombinations of specific features and aspects of the embodiments may be made and are still considered to be within the scope of the present disclosure. Thus, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for each other to form various aspects of the present disclosure. It is therefore intended that the scope of the present disclosure disclosed herein should not be limited by the specific disclosed embodiments described herein.

[0033] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used anywhere in the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0034] term

[0035] As used herein, "CSF1R expressing cells" may refer to cells that express CSF1R, or cells that can be induced to express CSF1R, or cells that differentiate into cells that express CSF1R. Non-limiting examples of CSF1R expressing cells may include, but are not limited to, microglia, monocytes, HSPCs (hematopoietic stem or progenitor cells), macrophages, dendritic cells, Langerhans cells, Kupffer cells, Hofbauer cells, extravillous trophoblast cells, phagocytes, or primitive macrophages derived from pluripotent stem cells. Additionally, CSF1R expressing cells may refer to microglia that have been directly reprogrammed from iPSCs, monocytes, or fibroblasts via either genetic or small molecule approaches.

[0036] As used herein, "CSF1R-expressing phagocyte" may refer to a cell that expresses the CSF1R receptor and has the ability for phagocytosis, a process in which a cell uses its plasma membrane to engulf extracellular particles or bacteria, substrates, proteins, lipids or aggregates, and then internalizes the area to form a phagosome. Non-limiting examples of CSF1R-expressing phagocytes may include, but are not limited to, microglia, macrophages, monocytes, dendritic cells or other phagocytes. In some embodiments, the CSF1R-expressing phagocyte is a CSF1R-expressing cell.

[0037] As used herein, "differential resistance" may refer to a state that occurs in modified cells where inhibition of CSF1R signaling by a CSF1R antagonist exhibits a dose-response relationship or magnitude of response that differs from that observed in unmodified cells. In other embodiments, differential resistance may refer to altering the proliferation or survival of unmodified CSF1R-expressing cells compared to modified CSF1R-expressing cells. In some embodiments, differential resistance may include partial, complete, increased, or decreased resistance to a CSF1R antagonist.

[0038] As used herein, "CSF1R signal inhibition" may refer to a dose-dependent reduction caused by a CSF1R antagonist at a step in the CSF1R signal transduction cascade, such as phosphorylation or dephosphorylation events, gene expression, or the like.

[0039] As used herein, "partial resistance" refers to modified cells that are still destroyed by increased doses of a CSF1R antagonist. Partial resistance may be due to an increase in the binding constant (Kd) or ATP, a decrease in kinase activity, a decrease in binding affinity to the CSF1 ligand, or a decrease in signal transduction.

[0040] As used herein, "complete resistance" refers to modified cells in which CSF1R signal inhibition never occurs at any concentration of antagonist.

[0041] As used herein, "increased resistance" refers to modified cells in which CSF1R signal inhibition occurs at increased concentrations of antagonist compared to that observed in unmodified cells.

[0042] As used herein, "reduced resistance" refers to modified cells in which CSF1R signal inhibition occurs at reduced concentrations of receptor antagonist compared to that observed in unmodified cells.

[0043] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a non-primate (e.g., cow, pig, horse, cat, dog, rat, mouse, etc.) or a primate (e.g., monkey, ape, and human). In certain embodiments, a subject is a human. In one embodiment, a subject is a mammal (e.g., human) having a disease, disorder, or condition described herein. In another embodiment, a subject is a mammal (e.g., human) at risk of developing a disease, disorder, or condition described herein. In certain cases, the term patient refers to a human receiving medical care or an animal receiving veterinary care.

[0044] As used herein, the term "neurological disorder" refers to an injury, trauma, disorder or disease that affects the brain and nerves found throughout the body and spinal cord.

[0045] The term "treat" or "treatment" or "treating" as used herein refers to both therapeutic treatment and prophylactic measures, the purpose of which is to prevent or delay the onset of a disease (e.g., a neurological disease), e.g., to delay the onset of a disorder, or to reduce at least one adverse effect or symptom of a pathology, disease or disorder, e.g., any disorder characterized by insufficient or undesirable organ or tissue function. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, a treatment is "effective" if the progression of a disease is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or the reduction of markers of a disease, but also the cessation of symptoms or the delay or worsening of progression that would be expected in the absence of treatment. Beneficial or desirable clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (e.g., not worsening), delay in progression of the disease, improvement or alleviation of the disease state, and remission (whether partial or total). "Treatment" can also mean extending survival compared to the expected survival in the absence of treatment. "Treatment" also includes ameliorating the disease, reducing the severity of its complications, preventing it from appearing, preventing it from recurring, simply preventing it from worsening, alleviating the inflammatory response involved therein, or therapeutic efforts that affect any of the foregoing, even if such therapeutic efforts ultimately fail.

[0046] Referring now to Figures 1-12, the present disclosure features methods and compositions for making and using therapeutically engineered CSF1R-expressing cells (e.g., therapeutic microglia) that have differential resistance to CSF1R antagonists for the treatment of neurodegenerative diseases.

[0047] Cells within the central nervous system may reside within a "niche," which may limit the total number of cells that may reside within the mammalian CNS. The present disclosure aims to develop new treatments for neurological diseases and neurological injuries using CSF1R-expressing cells (e.g., CSF1R-expressing phagocytes, HSPCs, dendritic cells, erythromyeloid progenitor cells, microglia, macrophages, or monocytes). However, in some diseases, the engraftment and / or maturation of such cells within the CNS is likely limited by the presence of pre-existing endogenous microglia. For such indications, methods that can provide a selective survival or proliferation advantage to transplanted mammalian cells would improve engraftment and, therefore, therapeutic activity. Importantly, such approaches must also be safe and not induce uncontrolled proliferation.

[0048] The disclosure features modified CSF1R-expressing cells or their progenitor or stem cells that differentiate into cells expressing CSF1R, and the modified CSF1R-expressing cells have differential resistance to CSF1R antagonists. Also encompassed are methods of making modified cells according to the disclosure, which, in the case of modified cells derived from induced pluripotent stem cells (iPSCs) or modified cells derived from hematopoietic stem and progenitor cells (HSPCs), may include introducing the modification into an undifferentiated cell, optionally screening or selecting for the modification, and differentiating the modified cell into a cell or cell lineage, such as a monocyte or macrophage lineage.

[0049] The present disclosure features cells (e.g., human cells) that exhibit differential resistance to CSF1R antagonists. In some embodiments, the present disclosure features modified human cells that exhibit differential resistance to CSF1R antagonists. The present disclosure may also feature cells (e.g., human cells) that include a nucleic acid encoding a modified CSF1R protein that exhibits differential resistance to CSF1R antagonists. In some embodiments, the present disclosure features modified human cells that include a nucleic acid encoding a modified CSF1R protein that exhibits differential resistance to CSF1R antagonists. In some embodiments, the cells (e.g., human cells) of the present disclosure express CSF1R and may be CSF1R-expressing cells. In other embodiments, the cells (e.g., human cells) of the present disclosure may be induced or differentiated to express CSF1R.

[0050] The disclosure may also feature modified cells described herein that express CSF1R. In some embodiments, the disclosure features modified human cells described herein that express CSF1R. The cells may be induced and / or differentiated to express CSF1R and may be selected from the group consisting of pluripotent stem cells, hematopoietic stem cells, erythromyeloid progenitor cells, and hematopoietic progenitor cells.

[0051] In some embodiments, CSF1R-expressing cells (e.g., CSF1R-expressing human) cells may include, but are not limited to, microglia, macrophages, monocytes, or other phagocytes. In other embodiments, cells that can be induced or differentiated to express CSF1R may include, but are not limited to, pluripotent stem cells, hematopoietic stem cells, erythromyeloid progenitor cells, or hematopoietic progenitor cells. In some embodiments, modified cells (e.g., modified human cells) are precursor cells, including, but not limited to, microglia, monocytes, macrophages, hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs), erythromyeloid progenitor cells (EMPs), primitive macrophages and primitive macrophage progenitor cells (PMPs), or umbilical cord blood hematopoietic stem cells.

[0052] In some embodiments, the engineered cells are differentially resistant to CSF1R antagonists. In some embodiments, CSF1R antagonists include, but are not limited to, PLX5622, PLX3397 (pexidartinib), BLZ945, Ki20227, JNJ-40346527; JNJ-527 (edicotinib), cFMS receptor inhibitor II, AZ304, ARRY-382, YM-90709, GW2580, PLX108-01, PL These may include X7486, PLX647, ARRY-382, JNJ-40346527, Emactuzumab (RG7155), AMG820, IMC-CS4 (LY3022855), MCS110, BPR1R024, AZD7507, JTE-952, JNJ-28312141, c-FMS-IN-8, or CSF1R-IN-22.

[0053] In other embodiments, CSF1R antagonists may include any compound that inhibits CSF1R interaction with its cognate ligand (e.g., CSF1 or IL-34), substrate or downstream effector. Non-limiting examples of compounds that inhibit CSF1R interaction with its cognate ligand, substrate or downstream effector include, but are not limited to, antibodies or drugs that block the interaction between CSF1R and phospholipase C gamma 2 (PLCg2), spleen tyrosine kinase (Syk) and Grb2 / Gab2 / Shp2 complex. In some embodiments, differential CSF1R antagonist resistance is conferred by modifying genes downstream of CSF1R. Non-limiting examples may include, but are not limited to, INPP5D (SHIP1), TREM2, DAP12, PLCG2, PI3K, AKT, PKC, FIMP, JNK, A-SMase, spleen tyrosine kinase (Syk) and Grb2 / Gab2 / Shp2 complex. In other embodiments, partial CSF1R antagonist resistance is conferred by pre-treating microglia with an agonist or antagonist of CSF1R signaling. In further embodiments, CSF1R antagonists may include any compound that inhibits CSF1R signaling by blocking the ATP binding site (i.e., the ATP binding pocket).

[0054] Table 1 shows non-limiting examples of wild-type CSF1R sequences and modified CSF1R amino acid sequences (i.e., modified CSF1R proteins as described herein), as well as non-limiting examples of nucleic acid sequences encoding modified CSF1R proteins as described herein. Examples of such nucleic acids include those that have one or more silent or conservative nucleic acid substitutions compared to wild-type nucleic acid sequences. Bolded nucleic acids (NA) are examples of where one or more silent or conservative nucleic acid substitutions can be incorporated into the sequence. Potential nucleic acids are highlighted herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0055] In some embodiments, the cell (e.g., a modified human cell) comprises one or more genetic modifications in a gene for the CSF1R receptor (i.e., the CSF1R protein). In some embodiments, the cell (e.g., a modified human cell) comprises one or more genetic modifications in the CSF1R protein (i.e., the CSF1R receptor).

[0056] In some embodiments, one or more genetic modifications of the CSF1R receptor (i.e., CSF1R protein) do not induce constitutively active CSF1R signaling. The modified CSF1R receptor (i.e., CSF1R protein) can still be activated (e.g., phosphorylated) by a ligand of the CSF1R protein. In some embodiments, the ligand of the CSF1R protein can include, but is not limited to, CSF1, IL-34, an agonist or activating antibody of CSF1R, or a combination thereof. In some embodiments, the modified CSF1R protein is activated by a CSF1 ligand. In some embodiments, the CSF1 ligand induces phosphorylation of the modified CSF1R protein. In other embodiments, the modified CSF1R protein is activated by an IL-34 ligand. In some embodiments, the IL-34 ligand induces phosphorylation of the modified CSF1R protein.

[0057] In some embodiments, the one or more genetic modifications result in a modified ATP binding pocket. The modified ATP binding pocket of the CSF1R receptor (i.e., CSF1R protein) may have a reduced binding space (compared to wild-type CSF1R protein). In some embodiments, the modified ATP binding pocket cannot bind to a drug (e.g., a CSF1R antagonist or a CSF1R agonist). In other embodiments, the modified ATP binding pocket can bind to ATP. In some embodiments, the one or more genetic modifications of the CSF1R receptor do not interfere with the ATP binding activity of the CSF1R protein. In some embodiments, the one or more genetic modifications of the CSF1R receptor lack a pocket for a drug (e.g., a CSF1R antagonist or a CSF1R agonist) to bind, but do not interfere with the normal ATP binding activity of the CSF1R protein.

[0058] In some embodiments, the one or more genetic modifications are point mutations. In some embodiments, the one or more genetic modifications result in a single amino acid substitution. In other embodiments, the one or more genetic modifications result in a single amino acid insertion. In further embodiments, the one or more genetic modifications result in a single amino acid deletion.

[0059] In some embodiments, the one or more genetic modifications cause changes in the amino acid sequence of CSF1R protein. The changes in amino acid sequence may include substitutions of amino acid residues selected from G795, L785, M637, E633 and V647. In some embodiments, the one or more genetic modifications cause amino acid substitutions. In some embodiments, the one or more genetic modifications cause single amino acid substitutions. The single amino acid point mutation may be selected from the group consisting of G795A, G795V and G795C.

[0060] In some embodiments, the CSF1R gene is modified to confer partial or complete resistance to CSF1R antagonists in modified cells.In some embodiments, the CSF1R gene is modified by single point mutation.In other embodiments, the single point mutation of the CSF1R gene can include, but is not limited to, these single amino acid substitutions at positions G795, L785, M637, E633 and / or V647. As non-limiting examples, substitutions may include G795A, G795V, or G795C, or G795D, or G795E, or G795F, or G795G, or G795H, or G795I, or G795K, or G795L, or G795M, or G795N, or G795P, or G795Q, or G795R, or G795S, or G795T, or G795W, or G795Y.

[0061] Without wishing to be bound by any theory or mechanism, substitution of these residues, or any other amino acid residues that directly contact small molecule inhibitors bound to the ATP binding site, is predicted to alter their binding. Amino acid substitutions that contain slightly larger functional groups: serine; threonine; cysteine; valine; leucine; isoleucine; and methionine are predicted to introduce bulky substituents that clash with plexicon binding but still allow ATP binding for CSF1R function. Even larger amino acid substitutions: phenylalanine, tyrosine, lysine, arginine, and tryptophan are predicted to sterically clash with both inhibitors and ATP, thereby inhibiting normal CSF1R function. In contrast, substitution with amino acids that have less bulky side chains, such as glycine or alanine, may improve binding to antagonist species that are only weakly bound by the native sequence due to steric hindrance from slightly larger or incompatible polar side chains. Accordingly, one of skill in the art will understand that these and other residues which define the binding site of the allosteric antagonist may be subject to modification in accordance with the purposes of the present disclosure.

[0062] In other embodiments, the one or more genetic modifications result in multiple amino acid changes in the CSF1R protein. In some embodiments, the multiple amino acid changes are adjacent amino acids. In other embodiments, the multiple amino acid changes are non-adjacent amino acids.

[0063] In some embodiments, the one or more genetic modifications (e.g., point mutations) are within the ATP-binding pocket of the CSF1R protein (i.e., the CSF1R receptor). In other embodiments, the one or more genetic modifications (e.g., point mutations) are outside the ATP-binding pocket of the CSF1R protein (i.e., the CSF1R receptor). Without wishing to limit the present disclosure to any theory or mechanism, it is believed that mutations outside the ATP-binding pocket of the CSF1R protein may act via allosterism to inhibit antagonist binding.

[0064] In some embodiments, one or more genetic modifications are introduced ex vivo. In other embodiments, one or more genetic modifications are induced by transfecting or introducing a nucleic acid encoding a transgene into a cell (e.g., a human cell). In further embodiments, one or more genetic modifications are introduced by transduction or introduction of a targeted nuclease, nickase, or base-editing effector or ribonucleoprotein complex. In some embodiments, introducing a nucleic acid encoding a transgene into a cell comprises contacting the cell (e.g., a human cell) with a viral vector.

[0065] In some embodiments, one or more genetic modifications (e.g., point mutations) of the CSF1R gene are generated by site-directed mutagenesis or random mutagenesis methods known in the art, including, but not limited to, CRISPR-Cas genome editing system, TALEN genome editing system and ZFN genome editing system.Alternatively or additionally, modified CSF1R transgene can be introduced into cells (e.g., human cells) by, for example, DNA or RNA vectors, such as naked nucleic acid, liposomes or other encapsulated nucleic acid vectors, artificial chromosomes, and / or viral vectors, such as lentivirus vectors and adeno-associated virus vectors.

[0066] In some embodiments, cells (e.g., human cells) comprising the modified CSF1R proteins described herein retain the gene expression profile of cells (e.g., human cells) comprising wild-type CSF1R proteins. In some embodiments, in vitro cells (e.g., in vitro human cells) comprising modified CSF1R proteins have a similar gene expression profile to in vitro cells (e.g., in vitro human cells) comprising wild-type CSF1R proteins. In other embodiments, cells (e.g., human cells) comprising modified CSF1R proteins transplanted in vivo have a similar gene expression profile to transplanted cells (e.g., human cells) that do not compromise the modified CSF1R proteins. In further embodiments, modified CSF1R-expressing cells (e.g., CSF1R-expressing phagocytes) described herein retain the gene expression profile of wild-type CSF1R-expressing cells (e.g., CSF1R-expressing phagocytes).

[0067] The present disclosure may also feature a composition comprising a CSF1R-expressing cell as described herein. The present disclosure may also feature a composition comprising a CSF1R-expressing phagocyte as described herein. In some embodiments, the composition comprises a plurality of cells. In some embodiments, at least 0.1%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 99%, or at least 99.5% of the plurality of cells are CSF1R-expressing cells that have differential resistance to CSF1R antagonists. In other embodiments, at least 0.1%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 99%, or at least 99.5% of the plurality of cells are CSF1R-expressing phagocytes that have differential resistance to a CSF1R antagonist.

[0068] The present disclosure features a method of treating a subject. The method includes administering a CSF1R antagonist to the subject in an amount sufficient to inhibit CSF1R signaling in the subject's cells, and contacting the subject with a modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte) that has differential resistance to the CSF1R antagonist. In other embodiments, the method includes contacting the subject with a modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte) that has differential resistance to the CSF1R antagonist, and administering a CSF1R antagonist to the subject in an amount sufficient to inhibit CSF1R signaling in the subject's cells. In some embodiments, the differential resistance to the CSF1R antagonist is partial resistance or complete resistance to the CSF1R antagonist.

[0069] The modified CSF1R-expressing cells (e.g., modified human CSF1R-expressing cells) described herein may further comprise one or more modifications (e.g., one or more genetic modifications) in another gene (e.g., outside the CSF1R gene). In some embodiments, the modified CSF1R-expressing cells (e.g., modified CSF1R-expressing phagocytes) express a gene product that is useful for treating, curing, ameliorating, preventing, or alleviating a neurological disease. In some embodiments, the gene product is not normally expressed by the CSF1R-expressing cells (e.g., modified CSF1R-expressing phagocytes). In other embodiments, the modified CSF1R-expressing cells (e.g., modified CSF1R-expressing phagocytes) express other genetic modifications that are useful for treating, curing, ameliorating, preventing, or alleviating a neurological disease. In other embodiments, the other genetic modifications (outside the CSF1R gene) are used to correct the mutated gene to treat a disease caused by mutations in the other genes.

[0070] In some embodiments, the CSF1R signal inhibited by the CSF1R antagonist is the proliferation or survival of CSF1R-expressing cells endogenous to the subject (e.g., CSF1R-expressing phagocytes). In other embodiments, the CSF1R antagonist is administered to the subject in an amount sufficient to reduce the proliferation or survival of the subject's endogenous CSF1R-expressing cells (e.g., CSF1R-expressing phagocytes) compared to the modified CSF1R-expressing cells (e.g., modified CSF1R-expressing phagocytes).

[0071] The present disclosure may also feature a method of treating a subject. The method may include contacting the subject with a modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte) and differentially altering the proliferation or survival of a non-modified CSF1R-expressing cell (e.g., a non-modified CSF1R-expressing phagocyte) compared to the modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte). In some embodiments, the modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte) is partially resistant to a CSF1R antagonist. In some embodiments, differentially altering the proliferation or survival of a non-modified CSF1R-expressing cell (e.g., a non-modified CSF1R-expressing phagocyte) includes administering a CSF1R antagonist to the subject in an amount sufficient to reduce the proliferation or survival of a non-modified CSF1R-expressing cell (e.g., a non-modified CSF1R-expressing phagocyte) compared to the modified CSF1R-expressing cell (e.g., a modified CSF1R-expressing phagocyte).

[0072] The methods described herein may kill about 0.1%, or about 1%, or about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 99%, or about 99.5% of the endogenous CSF1R-expressing phagocytes in the CNS of the subject. In other embodiments, the methods described herein may kill all endogenous CSF1R-expressing phagocytes in the CNS of the subject.

[0073] The methods described herein may further allow for engraftment of about 0.1%, or about 1%, or about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 99%, or about 99.5% of the modified CSF1R-expressing phagocytes within the CNS of the subject. In some embodiments, the methods described herein allow for complete engraftment of the modified CSF1R-expressing phagocytes within the CNS of the subject.

[0074] In some embodiments, the methods described herein relate to treating, preventing, alleviating, or ameliorating neurological disorders by contacting a subject with modified cells or vectors of the present disclosure. In some embodiments, the neurological disorder comprises a disease of the nervous system. Non-limiting examples of neurological disorders may include, but are not limited to, dementia, neurodegenerative disorders, genetic disorders, spinal cord injuries, traumatic brain injuries, diseases caused or exacerbated by exposure to chemicals or other agents, and the like.

[0075] The disclosure may also feature a composition for treating a neurological disease, the composition comprising a plurality of modified cells having differential (e.g., increased, decreased, partial, or complete) resistance to a CSF1R antagonist. In some embodiments, the modified cells are CSF1R-expressing cells.

[0076] The present disclosure may further feature nucleic acid compositions and vectors encoding modified CSF1R proteins that exhibit differential resistance to CSF1R antagonists as described herein. In some embodiments, the nucleic acid compositions and vectors may include a payload, including, but not limited to, a transgene, a marker or a reporter gene, and the like. In some embodiments, an expression vector includes the nucleic acid composition as described herein operably linked to an expression control sequence.

[0077] In some embodiments, the nucleic acid composition comprises a modified CSF1R protein that exhibits differential resistance to a CSF1R antagonist. In some embodiments, the differential resistance to a CSF1R antagonist is partial resistance, complete resistance, increased resistance, or decreased resistance to a CSF1R antagonist.

[0078] In other embodiments, the nucleic acid composition comprises a modified CSF1R protein comprising one or more genetic modifications. In some embodiments, the one or more genetic modifications result in an amino acid substitution selected from the group consisting of G795A, G795V, G795C, L785, M637, E633 and V647.

[0079] In some embodiments, the disclosure features an expression vector that includes a nucleic acid composition that includes a modified CSF1R protein that exhibits differential resistance to a CSF1R antagonist.

[0080] In some embodiments, the expression vectors described herein can be used to modify cultured cells or endogenous cells. In some embodiments, the expression vectors described herein are used to transfect cultured cells so that the cultured cells, or the progeny of the cells, express the polypeptide. In other embodiments, the expression vectors described herein are used to modify endogenous CSF1R-expressing cells by transducing nucleic acid (e.g., by infecting with a viral vector to generate CSF1R antagonist-resistant cells in situ).

[0081] The disclosed methods and compositions can be advantageously used in human or veterinary applications, where modified cells or vectors or genome editing systems (e.g., CRISPR-Cas, TALEN or ZFN systems) configured to introduce the disclosed mutations and thereby generate modified cells are used to prevent, alleviate, ameliorate or cure diseases or disorders. In general, these methods include contacting a subject with modified cells or vectors or genome editing systems configured to generate such cells, and contacting the subject with a CSF1R antagonist.

[0082] Turning first to subjects for which the methods of the present disclosure may be suitable, while any subject may be a candidate for treatment by these methods, it may be advantageous to treat subjects who suffer from, are predicted to suffer from, or are prone to suffer from, a disease, such as a neurological disease that may be prevented, alleviated, ameliorated, or cured by contacting the subject with cells of the present disclosure that are characterized by a differential response to a CSF1R antagonist compared to the subject's native or endogenous cells. Without limiting the above, neurological diseases suitable for treatment by the methods of the present disclosure may include dementia, neurodegenerative diseases, demyelinating diseases, mood or personality disorders, traumatic brain injury, genetic diseases, malignant or benign tumors, metastatic tumors or metastatic growths, and the like.

[0083] A subject may be contacted with the modified cells or vectors of the present disclosure at any suitable time by any suitable route of administration, including, but not limited to, intravenous, intramuscular, intraperitoneal, intraparenchymal, intrathecal, intercranial, interosseous or other injection or infusion, percutaneous administration (in the case of vectors), and / or by surgical implantation or transplantation. Any suitable number of cells, or any suitable titer of vector, for example, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 10, 20, 30, 40, 50, 60, 70, 80 14, 10 13 , 10 12 , 10 11 , 10 10 , 10 9 One or fewer virions or viral genomes may be administered. With respect to modified cells, the cells with which the subject is contacted may be derived from the subject's own cells (autologous cells) or from another donor (allogeneic cells).

[0084] Those skilled in the art will understand that the differential sensitivity of the modified cells of the present disclosure to CSF1R antagonists may be most clearly manifested in the presence of the CSF1R antagonist.Thus, although the subject may be contacted with the CSF1R antagonist at any time (for example, but not limited to, as a pretreatment or pretreatment before contacting the subject with the modified cells of the present disclosure), some embodiments of the present disclosure include administering the CSF1R antagonist to the subject simultaneously with and / or after contacting the subject with the modified cells or vector.The CSF1R antagonist may be administered continuously or discontinuously, and the start or stop of administration may be scheduled or preprogrammed, or may be responsive to physiological readouts from the subject, such as biomarker concentrations, serum or tissue concentrations of the biomarker or its small molecule active components or metabolites, imaging signals or symptoms, etc.

[0085] As one non-limiting example, in the case of modified cells that exhibit reduced sensitivity (i.e., partial or complete resistance) to a CSF1R antagonist, the CSF1R antagonist may be administered to a subject to reduce the proliferation or survival of native cells within the niche that such cells occupy, thereby conferring a selective advantage to the modified cells and optionally increasing the rate of engraftment or other behavior of the modified cells compared to that observed in the absence of the CSF1R antagonist. This administration may be initiated, and optionally terminated, prior to, concurrent with, or after contacting the subject with the modified cells or vector. Without limiting the above, where the modified cells are microglial cells or microglial progenitor cells, a CSF1R antagonist may be administered to the subject prior to, concomitantly with, and / or after transplantation of the modified microglial lineage cells to limit the proliferation and / or survival of endogenous microglia, thereby conferring an advantage to the modified microglia that may result in a higher degree of engraftment and / or a greater spatial distribution of the modified microglia than would otherwise be observed.

[0086] Alternatively or additionally, the modified cells of the present disclosure may exhibit increased sensitivity to CSF1R antagonists compared to unmodified cells. In this case, the CSF1R antagonist is generally (but not necessarily) withheld before and during the step of contacting the subject with the modified cells, and instead is administered after contacting the subject with the modified cells or vectors to limit the proliferation or survival of the modified cells, for example, at the end of a course of treatment, in response to signals from physiological or pharmacological measurements, and / or in response to adverse events or reduced effectiveness of a therapeutic intervention utilizing the modified cells.

[0087] The modified cells of the present disclosure may optionally include one or more additional modifications compared to the same type of natural cell. These modifications may include, but are not limited to, one or more transgenes, or one or more genetic modifications that correct mutations or reduce or increase expression of gene products. Without being limited to the above, the present disclosure includes microglial cells engineered to express CSF1R alleles with reduced sensitivity to CSF1R antagonists, and secreted transgenes such as enzymes or binding proteins, RNA such as antisense, miRNA or siRNA, cell surface proteins such as chimeric antigen receptors, membrane-bound immunoglobulins or fragments thereof, aptamers, and the like. The present disclosure also includes vectors and methods of making cells substantially as described above, as well as methods of treating a subject, using the cells or vectors to treat the subject.

[0088] Without wishing to limit the present disclosure to any theory or mechanism, it is believed that various methods can be used to achieve the presently claimed cells with differential resistance to CSF1R antagonists. Non-limiting methods / examples of achieving this goal are described herein. Equivalents or substitutes are within the scope of the present disclosure.

[0089] example

[0090] The following are non-limiting examples of the present invention. It should be understood that the above examples are not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0091] Several approaches can be used independently or in combination to promote competitive engraftment of human microglia, human macrophages and human monocytes and / or their precursor cells (hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs), erythromyeloid progenitor cells (EMPs) or primitive macrophage progenitor cells (PMPs): 1) using the previously published crystal structure of the CSF1R receptor bound to an antagonist (PMID:31434879) to predict amino acid substitutions that may sterically interfere with the binding of CSF1R antagonists without compromising the normal binding of ATP to the same binding pocket; 2) using PCR-mediated random mutagenesis to introduce amino acid changes, followed by exposing iPS-microglia to a CSF1R antagonist and then sequencing the surviving mutants to identify mutations that confer resistance to the CSF1R antagonist but do not simultaneously induce constitutively active CSF1R signaling. 3) Modifying genes downstream of CSF1R, e.g., deleting or transiently reducing expression of SHIP1 (INPP5D) to enhance CSF1R signaling. 4) Pretreating transplanted microglia with agonists of CSF1R signaling can also be used to modulate and transiently increase CSF1R signaling in transplanted cells to promote resistance to CSF1R antagonist treatment, using siRNA or microRNA gene delivery approaches. Additional details and data related to the first two strategies are provided below.

[0092] Strategy 1: Crystal structure of CSF1R receptor bound to antagonist (PMID: 31434879) to predict amino acid substitutions that may sterically hinder the binding of CSF1R antagonists without disrupting the normal binding of ATP to the same binding pocket. A crystal structure of PLX5622 bound to human CSF1R was obtained and the ATP binding pocket was investigated (Figure 7A). Through molecular modeling, single amino acid changes were predicted that would likely impair the binding of PLX5622 or PLX3397 or other CSF1R antagonists without disrupting the normal ATP binding to CSF1R. Specifically, the modeling suggests that substitution of amino acid G795 with either alanine (A), valine (V) or cysteine ​​(C) increases the steric hindrance of PLX5622 and / or PLX3397 binding without disrupting the normal binding ability of ATP (Figure 7A and Figure 7B). Other mutations that prevent binding could also be investigated (Figure 7D). A CRISPR targeting strategy was designed to generate human iPSCs with each of these single amino acid substitutions, i.e., G795A, G795V, G795C, and the CRISPR targeting strategy successfully produced these modified human iPSC lines (Sanger sequencing chromatograms showing the edited sequences are shown in Figure 8). The resulting iPSCs were then differentiated into microglia and exposed to various concentrations of either PLX3397 or PLX5622. A caspase-3 / 7 fluorogenic reporter was used to detect microglial cell death (apoptosis). Figure 4A shows the dose-dependent induction of cell death in wild-type (WT) unedited human iPSC-microglia versus G795A and G795C CSF1R mutant lines in response to PLX3397, and Figure 4B shows the response to PLX5622. Within 24 h of PLX compound treatment, WT human microglia show robust dose-dependent induction of apoptosis in response to either compound. In contrast, both G795A and G795C mutant strains show complete resistance to cell death. Figure 4F provides representative images of cell density (phase contrast microscopy, first row) and fluorescent caspase-3 / 7 activity (second row) across each dose of PLX3397 and PLX5622.

[0093] Further investigation of the crystal structure also suggests other positions that may lead to reduced binding of PLX5622 and / or PLX3397 when mutated. Examples include L785, M637, E633 and V647 (Figure 4D). All of these positions are in close contact with the binding site of PLX5622 and / or PLX3397 and may prevent binding when mutated.

[0094] Strategy 2: Use PCR-mediated random mutagenesis to introduce amino acid changes, followed by exposure of iPS-microglia to CSF1R antagonists, and then sequence the surviving mutants to identify mutations that confer resistance to CSF1R antagonists but do not simultaneously induce constitutively active or suppressed CSF1R signaling. Generate a pool of iPSC or monocyte cell lines with random mutations in the CSF1R gene. This pool is then differentiated into microglia and monitor cell survival in response to either CSF1 / IL-34 withdrawal or CSF1R antagonist treatment. Examine cells that survive longer under these conditions by PCR and sequencing to identify CSF1R mutations that may confer resistance to CSF1R antagonists. Counterscreening is then performed to identify CSF1R mutations that do not result in hyperproliferation or significant loss of proliferation. Once optimal mutations are identified, iPSC lines harboring these mutations will be expanded and microglia derived from these lines will be investigated in vivo in chimeric models to determine resistance to CSF1R antagonists in vivo.

[0095] Resistance to each of the CSF1R inhibitors tested was quantified using a fluorogenic caspase 3 / 7 detector (Essen BioScience) and time-lapse imaging over 24 hours. WT microglia showed significantly increased levels of caspases to increasing concentrations of PLX3397 (MedChemExpress), PLX5622 (MedChemExpress), edicotinib and BLZ945 compared to DMSO control treatment over 24 hours of imaging, indicating an increased cell death response to inhibition of macrophage colony-stimulating factor / colony-stimulating factor 1 receptor (CSF1R). On the other hand, G795A iPSC-microglia and G795C iPSC-microglia did not show a significant increase in caspase levels to increasing concentrations of either PLX3397, PLX5622, edicotinib or BLZ945 compared to DMSO control treatment. These results indicate acquired resistance to PLX treatment as a result of the genetically altered CSF1 receptor (see Figures 4A, 4B, 4C, 4D and 4E).

[0096] Cell Death Assay: iPS-microglia were seeded at 70K cells per 96-well plate (6 wells / strain / condition). At time 0, all microglia were treated with IncuCyte Caspase-3 / 7 Green Apoptosis Assay Reagent 1:1000. Cells were maintained in the indicated media: fresh complete medium + 0.1% DMSO, complete medium + 250 nM PLX3397, complete medium + 500 nM PLX3397, complete medium + 1 μm PLX3397, complete medium + 250 nM PLX5622, complete medium + 500 nM PLX5622, complete medium + 1 μm PLX5622, complete medium + 250 nM edicotinib, complete medium + 500 nM edicotinib, complete medium + 1 μm edicotinib, complete medium + 250 nM BLZ945, complete medium + 500 nM BLZ945, complete medium + 1 μm BLZ945. Four 20× images per well were collected every hour for 24 hours. IncuCyte 2020B software was used to generate image masks for phase confluence (visually gated out apoptotic cells) and caspase 3 / 7 signal (green). Graphs show caspases normalized to phase confluence with three strains: WT, G795A, G795C (see Figure 4A, Figure 4B, Figure 4C, Figure 4D, and Figure 4E).

[0097] Embodiment

[0098] The following embodiments are intended to be illustrative only and not limiting in any way.

[0099] Embodiment 1: Engineered human cells exhibiting differential resistance to CSF1R antagonists.

[0100] Embodiment 2: The cell of embodiment 1, comprising a nucleic acid encoding a modified CSF1R protein that exhibits differential resistance to a CSF1R antagonist.

[0101] Embodiment 3: A modified human cell comprising a nucleic acid encoding a modified CSF1R protein that exhibits differential resistance to a CSF1R antagonist.

[0102] Embodiment 4: The cell of any one of embodiments 1 to 3, comprising one or more genetic modifications in the CSF1R protein.

[0103] Embodiment 5: The cell of embodiment 4, wherein the one or more genetic modifications result in a change in the amino acid sequence of CSF1R, and the change optionally includes a substitution of an amino acid residue selected from G795, L785, M637, E633 and V647.

[0104] Embodiment 6: The cell of any one of embodiments 1 to 5, wherein the one or more genetic modifications result in an altered ATP-binding pocket.

[0105] Embodiment 7: The cell of embodiment 6, wherein the modified ATP-binding pocket has a reduced binding space.

[0106] Embodiment 8: The cell of embodiment 7, wherein the modified ATP-binding pocket is incapable of binding to a CSF1R antagonist.

[0107] Embodiment 9: The cell of embodiment 6, wherein the modified ATP-binding pocket is capable of binding ATP.

[0108] Embodiment 10: The cell of any one of embodiments 1 to 9, wherein the one or more genetic modifications of the CSF1R protein do not interfere with the ATP binding activity of the CSF1R protein.

[0109] Embodiment 11: The cell of any one of embodiments 1 to 10, wherein the one or more genetic modifications of the CSF1R protein do not induce constitutively active CSF1R signaling.

[0110] Embodiment 12: The cell of embodiment 11, wherein the modified CSF1R protein is activated by a CSF1 ligand.

[0111] Embodiment 13: The cell of embodiment 12, wherein the CSF1 ligand induces phosphorylation of the modified CSF1R protein.

[0112] Embodiment 14: The cell of embodiment 11, wherein the modified CSF1R protein is activated by an IL-34 ligand.

[0113] Embodiment 15: The cell of embodiment 14, wherein the IL-34 ligand induces phosphorylation of the modified CSF1R protein.

[0114] Embodiment 16: The cell of embodiment 4, wherein the one or more genetic modifications are introduced ex vivo.

[0115] Embodiment 17: The cell of embodiment 4, wherein one or more genetic modifications are induced by transfecting or introducing into the cell a nucleic acid encoding a transgene, or by transducing or introducing a targeted nuclease, nickase, or base editing effector, or a ribonucleoprotein complex.

[0116] Embodiment 18: The cell of embodiment 17, wherein introducing the nucleic acid encoding the transgene into the cell comprises contacting the cell with a viral vector.

[0117] Embodiment 19: The cell of any one of embodiments 1 to 18, wherein the cell comprising the modified CSF1R protein retains the gene expression profile of a cell comprising a wild-type CSF1R protein.

[0118] Embodiment 20: The cell of any one of embodiments 1 to 19, further comprising one or more modifications in another gene.

[0119] Embodiment 21: A modified human cell according to any one of embodiments 1 to 20, expressing CSF1R.

[0120] Embodiment 22: The cell of embodiment 21, which is induced and differentiated to express CSF1R and is selected from the group consisting of pluripotent stem cells, hematopoietic stem cells, erythromyeloid progenitor cells and hematopoietic progenitor cells.

[0121] Embodiment 23: The cell of embodiment 22, which is a CSF1R expressing cell and is selected from the group consisting of microglia, macrophages, monocytes or other phagocytes.

[0122] Embodiment 24: A composition comprising a CSF1R-expressing cell according to any one of embodiments 20 to 23.

[0123] Embodiment 25: The composition of embodiment 24, wherein the CSF1R-expressing cell is a CSF1R-expressing cell.

[0124] Embodiment 26: The composition of embodiment 25, comprising a plurality of cells, wherein at least 0.1%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the cells are CSF1R-expressing cells that have differential resistance to a CSF1R antagonist.

[0125] Embodiment 27: A method of treating a subject comprising: (a) administering to the subject a CSF1R antagonist in an amount sufficient to inhibit CSF1R signaling in cells of the subject; and (b) contacting the subject with modified CSF1R-expressing cells that have differential resistance to the CSF1R antagonist.

[0126] Embodiment 28: A method of treating a subject comprising: (a) contacting the subject with a modified CSF1R-expressing cell having differential resistance to a CSF1R antagonist; and (b) administering to the subject a CSF1R antagonist in an amount sufficient to inhibit CSF1R signaling in the subject's cells.

[0127] Embodiment 29: The method of embodiment 27 and embodiment 28, wherein the CSF1R-expressing cell is a CSF1R-expressing cell.

[0128] Embodiment 30: The method of any one of embodiments 27 to 29, wherein the differential resistance to the CSF1R antagonist is partial resistance or complete resistance to the CSF1R antagonist.

[0129] Embodiment 31: The method of any one of embodiments 27 to 30, wherein the modified CSF1R-expressing cells retain the gene expression profile of wild-type CSF1R-expressing cells.

[0130] Embodiment 32: The method of any one of embodiments 27-31, wherein the modified CSF1R-expressing cells express a gene product or other genetic modification useful for treating, curing, ameliorating, preventing, or alleviating a neurological disease.

[0131] Embodiment 33: The method of embodiment 32, wherein the gene product is a transgene that is not normally expressed by the CSF1R-expressing cell.

[0132] Embodiment 34: The method of embodiment 33, wherein the CSF1R signal inhibited by the CSF1R antagonist is the proliferation or survival of CSF1R-expressing cells endogenous to the subject.

[0133] Embodiment 35: The method of any one of embodiments 27 to 34, wherein the CSF1R antagonist is administered to the subject in an amount sufficient to reduce proliferation and / or survival of endogenous CSF1R-expressing cells compared to modified CSF1R-expressing cells.

[0134] Embodiment 36: A method of treating a subject comprising: (a) contacting the subject with modified CSF1R-expressing cells; and (b) differentially altering the proliferation or survival of unmodified CSF1R-expressing cells compared to the modified CSF1R-expressing cells.

[0135] Embodiment 37: The method of embodiment 36, wherein the modified CSF1R-expressing cell is a CSF1R-expressing phagocyte.

[0136] Embodiment 38: The method of embodiment 36 and embodiment 37, wherein the modified CSF1R-expressing cells are partially resistant to a CSF1R antagonist.

[0137] Embodiment 39: The method of embodiment 36, wherein the step of differentially altering the proliferation or survival of unmodified CSF1R-expressing cells comprises administering to the subject a CSF1R antagonist in an amount sufficient to reduce the proliferation and / or survival of unmodified CSF1R-expressing cells compared to modified CSF1R-expressing cells.

[0138] Embodiment 40: A nucleic acid composition encoding a modified CSF1R protein that exhibits differential resistance to CSF1R antagonists.

[0139] Embodiment 41: The composition of embodiment 40, wherein the differential resistance to a CSF1R antagonist is partial or complete resistance to a CSF1R antagonist.

[0140] Embodiment 42: The composition of embodiment 40, wherein the differential resistance to a CSF1R antagonist is increased resistance to a CSF1R antagonist.

[0141] Embodiment 43: The composition of embodiment 40, wherein the differential resistance to a CSF1R antagonist is reduced resistance to a CSF1R antagonist.

[0142] Embodiment 44: The composition of any one of embodiments 40 to 43, wherein the modified CSF1R protein comprises one or more genetic modifications.

[0143] Embodiment 45: The composition of embodiment 44, wherein the one or more genetic modifications result in a change in the amino acid sequence of CSF1R, and the change optionally includes a substitution of an amino acid residue selected from G795, L785, M637, E633 and V647.

[0144] Embodiment 46: The composition of embodiment 44, wherein the one or more genetic modifications result in a modified ATP-binding pocket.

[0145] Embodiment 47: The composition of embodiment 46, wherein the modified ATP-binding pocket has a reduced binding space.

[0146] Embodiment 48: The composition of embodiment 47, wherein the modified ATP-binding pocket is unable to bind to a CSF1R antagonist.

[0147] Embodiment 49: The composition of embodiment 46, wherein the modified ATP-binding pocket is capable of binding to ATP.

[0148] Embodiment 50: The composition of embodiment 44, wherein the one or more genetic modifications of the CSF1R protein do not interfere with the ATP binding activity of the CSF1R protein.

[0149] Embodiment 51: The composition of embodiment 44, wherein the one or more genetic modifications of the CSF1R protein do not induce constitutively active CSF1R signaling.

[0150] Embodiment 52: The composition of embodiment 51, wherein the modified CSF1R protein is activated by a CSF1 ligand.

[0151] Embodiment 53: The composition of embodiment 52, wherein the CSF1 ligand induces phosphorylation of the modified CSF1R protein.

[0152] Embodiment 54: The composition of embodiment 51, wherein the modified CSF1R protein is activated by an IL-34 ligand.

[0153] Embodiment 55: The cell of embodiment 54, wherein the IL-34 ligand induces phosphorylation of the modified CSF1R protein.

[0154] Embodiment 56: An expression vector comprising the nucleic acid composition of any one of embodiments 40 to 55, operably linked to an expression control sequence.

[0155] Embodiment 57: A cultured cell transfected with the vector of embodiment 56, or a progeny of said cell, wherein the cultured cell expresses the polypeptide.

[0156] Embodiment 58: An endogenous cell infected with the vector of embodiment 56, wherein the endogenous cell expresses the polypeptide.

[0157] As used herein, the term "about" refers to ±20% of the referenced number.

[0158] Although preferred embodiments of the present disclosure have been shown and described, it will be readily apparent to one skilled in the art that modifications may be made thereto without exceeding the scope of the appended claims. Thus, the scope of the present disclosure is limited only by the claims. In some embodiments, the figures presented in this patent application are drawn to scale, including angles, dimensional ratios, etc. In some embodiments, the figures are merely representative, and the claims are not limited by the dimensions of the figures. In some embodiments, the description of the present disclosure described herein using the phrase "comprising" includes embodiments that may be described as "consisting essentially of" or "consisting of", and thus satisfies the written description requirement for claiming one or more embodiments of the present disclosure using the phrase "consisting essentially of" or "consisting of". [Sequence List Free Text]

[0159] Sequence Listing 3 <223> where nnn is the GCT Sequence Listing 3 <223> where nnn is GCC Sequence Listing 3 <223> where nnn is the GCA Sequence Listing 3 <223> where nnn is GCG Sequence Listing 5 <223> Where nnn is the TGT Sequence Listing 5 <223> where nnn is the TGC Sequence Listing 7 <223> where NNN is the GTT Sequence Listing 7 <223> where NNN is the GTC Sequence Listing 7 <223> where NNN is the GTA Sequence Listing 7 <223> where NNN is GTG

Claims

1. 1. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a modified CSF1R protein, A recombinant nucleic acid molecule comprising one or more genetic modifications that result in a modified CSF1R protein having a modified ATP-binding pocket and differential resistance to a CSF1R antagonist compared to a CSF1R protein having the amino acid sequence of SEQ ID NO:

2.

2. 2. The recombinant nucleic acid molecule of claim 1, wherein the one or more genetic modifications comprise an amino acid substitution corresponding to: (i) a glycine residue at amino acid position 795 (G795) (ii) a leucine residue at amino acid position 785 (L785) (iii) a methionine residue at amino acid position 637 (M637) (iv) a glutamic acid residue at amino acid position 633 (E633), or (v) A valine residue at amino acid position 647 (V647).

3. 2. The recombinant nucleic acid molecule of claim 1, wherein the one or more genetic modifications comprise a substitution of an amino acid corresponding to a glycine residue at amino acid position 795 (G795).

4. 3. The recombinant nucleic acid molecule of claim 2, wherein the one or more genetic modifications include an amino acid substitution of a glycine residue at amino acid position 795 of SEQ ID NO:2 to a cysteine ​​(G795C).

5. 3. The recombinant nucleic acid molecule of claim 2, wherein the one or more genetic modifications include an amino acid substitution of a glycine residue at amino acid position 795 of SEQ ID NO:2 to alanine (G795A).

6. 2. The recombinant nucleic acid molecule of claim 1, wherein the modified CSF1R protein comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:

8.

7. The recombinant nucleic acid molecule of claim 1 , wherein the modified CSF1R protein comprises the amino acid sequence of SEQ ID NO:

4.

8. The recombinant nucleic acid molecule of claim 1 , wherein the modified CSF1R protein comprises the amino acid sequence of SEQ ID NO:

6.

9. The recombinant nucleic acid molecule of claim 1 , wherein the modified CSF1R protein is a modified human CSF1R protein.

10. The recombinant nucleic acid molecule of claim 1 , further comprising a nucleic acid sequence of a transgene or a reporter gene.

11. The CSF1R antagonists include PLX5622, PLX3397 (pexidartinib), BLZ945, Ki20227, AJ-40346527, JNJ-527 (edicotinib), cFMS receptor inhibitor II, AZ304, ARRY-382, YM-90709, GW2580, PLX108-01, PLX7486, PLX647, ARRY-38 2, JNJ-40346527, emactuzumab (RG7155), AMG820, IMC-CS4 (LY3022855), MCS110, BPR1R024, AZD7507, JTE-952, JNJ-28312141, c-FMS-IN-8, CSF1R-IN-22, and combinations thereof.

12. The recombinant nucleic acid molecule of any one of claims 1 to 11, wherein the modified ATP-binding pocket of the modified CSF1R protein does not bind to the CSF1R antagonist.

13. 12. The recombinant nucleic acid molecule of any one of claims 1-11, wherein the modified ATP-binding pocket of the modified CSF1R protein binds ATP and sterically hinders binding of a CSF1R antagonist.

14. The recombinant nucleic acid molecule of any one of claims 1 to 11, wherein the one or more genetic modifications of the CSF1R protein do not induce constitutively active CSF1R signaling.

15. The modified CSF1R protein is (i) binds to ATP; (ii) is activated by a CSF1R ligand or an IL-34 ligand, which induces phosphorylation of the modified CSF1R protein; (iii) has reduced sensitivity to CSF1R inhibitors compared to the CSF1R protein consisting of the amino acid sequence of SEQ ID NO: 2; A recombinant nucleic acid molecule according to any one of claims 1 to 11.

16. An expression vector comprising the recombinant nucleic acid molecule of claim 1 operably linked to an expression control sequence.

17. The expression vector of claim 16 , wherein the expression vector comprises a DNA vector, an RNA vector, a liposome, or a viral vector.

18. 18. The expression vector of claim 17, wherein the expression vector comprises a lentiviral vector or an adeno-associated viral (AAV) vector.

19. The expression vector of claim 16 , wherein the expression vector is a liposome.

20. 2. A modified human cell, or a progeny thereof, comprising the recombinant nucleic acid molecule of claim 1, wherein the modified human cell expresses a modified CSF1R protein.

21. 21. The modified human cell of claim 20, or a progeny thereof, wherein the modified human cell is an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), an erythromyeloid progenitor cell, or a hematopoietic progenitor cell.

22. 21. The modified human cell of claim 20, wherein the modified human cell is a CSF1R-expressing cell.

23. 21. The modified human cell of claim 20, or a progeny thereof, wherein the modified human cell is a microglial cell, a macrophage or a monocyte.

24. 24. The modified human cell of claim 23, or a progeny thereof, wherein the modified human cell is a microglial cell.

25. 21. The modified human cell of claim 20, or a progeny thereof, wherein the modified human cell is an induced pluripotent stem cell (iPSC)-derived microglial cell or an iPSC-derived hematopoietic progenitor cell.

26. 21. The modified human cell of claim 20, or a progeny thereof, wherein the modified human cell is derived from a cell of a subject.

27. 21. The modified human cell of claim 20, wherein the modified human cell is derived from a cell of a human donor.

28. A pharmaceutical composition comprising a recombinant nucleic acid molecule according to any one of claims 1 to 11, an expression vector according to any one of claims 16 to 19, or a modified human cell according to any one of claims 20 to 27, or a progeny thereof.

29. 29. The pharmaceutical composition according to claim 28, for the treatment of a neurological disorder in a subject in need thereof, wherein the subject is administered a CSF1R antagonist prior to, simultaneously with, or after administration of CSF1R, the CSF1R antagonist being selected from the group consisting of PLX5622, PLX3397 (pexidartinib), BLZ945, Ki20227, AJ-40346527, JNJ-527 (edicotinib), cFMS receptor inhibitor II, AZ304, ARRY-382, YM-90709, GW2580, PLX108-01, PLX7486, PLX647, A pharmaceutical composition selected from the group consisting of ARRY-382, JNJ-40346527, emactuzumab (RG7155), AMG820, IMC-CS4 (LY3022855), MCS110, BPR1R024, AZD7507, JTE-952, JNJ-28312141, c-FMS-IN-8, CSF1R-IN-22, and combinations thereof.

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