Overexpression of LEMD2, LEMD3, or CHMP7 as a therapeutic mode for tauopathy

Abstract

Provided herein is a method of inhibiting tau aggregates in a cell or subject, comprising administering to the cell or subject a nucleic acid encoding LEM domain-containing protein 2 (LEMD2), charged multivesicular body protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or LEMD2, CHMP7, or LEMD3. Also provided herein is a method of treating or preventing a tauopathy in a subject, comprising administering to the subject a nucleic acid encoding LEMD2, CHMP7, or LEMD3, wherein LEMD2, CHMP7, or LEMD3 inhibits tau aggregates in cells of the subject. Also provided is a nucleic acid encoding LEMD2, CHMP7, or LEMD3 (e.g., in an expression construct and operably linked to a heterologous promoter).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Patent Application No. 63 / 271,839, filed on 26 October 2021, and U.S. Patent Application No. 63 / 369,557, filed on 27 July 2022, each of which is incorporated herein by reference in whole. Submitted as a text file via EFS Web

[0002] Reference to sequence listings The sequence listing described in file 057766-586530.xml is 452 kilobytes in size, was created on October 26, 2022, and is incorporated herein by reference. [Background technology]

[0003] Tau is a member of a large group of microtubule-related proteins that are concentrated in the brain. Tau is primarily found in neurons of the central nervous system, confined to axons, where it functions to enhance microtubule stability. There are six major isoforms of tau, ranging in length from 352 to 441 amino acids, produced by alternative splicing of transcripts from the MAPT gene located on human chromosome 17. Like other microtubule-related proteins such as MAP2, each tau isoform contains a series of three or four tandem repeat units (3RD and 4RD) involved in microtubule binding. While most interest in tau has focused on its role in cytoskeletal microtubule dynamics, recent evidence points to nuclear functions in RNA metabolism and pre-mRNA splicing. Despite decades of research, many questions remain regarding the normal function of tau in neurons and how its dysfunction promotes neurodegenerative diseases. [Overview of the Initiative]

[0004] This specification provides compositions and methods for inhibiting tau aggregates in cells or subjects, methods for reducing phosphorylation in cells or subjects, compositions and methods for treating or preventing tauopathy in subjects, LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), and nucleic acids encoding expression constructs encoding LEMD2, CHMP7, or LEMD3. This specification also provides compositions and methods for reducing or preventing the accumulation of serum neurofilament light chains (sNfL) in subjects.

[0005] In one embodiment, methods are provided for inhibiting or reducing tau aggregates in cells or subjects, for inhibiting or reducing phosphorylation, or for inhibiting or reducing the accumulation of insoluble tau. Similarly, methods are provided for reducing serum neuronal filament light chains (sNfLs) in subjects or for preventing the accumulation of serum neuronal filament light chains (sNfLs). Some such methods are for inhibiting or reducing tau aggregates in cells or subjects. Some such methods are for inhibiting tau aggregates in cells or subjects. Some such methods are for inhibiting tau aggregates in cells or subjects. Some such methods are for inhibiting or reducing phosphorylation in cells or subjects. Some such methods are for inhibiting phosphorylation in cells or subjects. Some such methods are for reducing phosphorylation in cells or subjects. In some such methods, the phosphorylation inhibited or reduced is the phosphorylation of tau on serine 356. In some such methods, the level of phosphorylated tau (e.g., phosphorylated-tau-Ser356) is reduced in the cell body, in the perinuclear region, and / or in the nucleocytoplasm. In some such methods, the level of phosphorylated tau (e.g., phosphorylated-tau-Ser356) is reduced in the cell body. In some such methods, the level of phosphorylated tau (e.g., phosphorylated-tau-Ser356) is reduced in the perinuclear region. In some such methods, the level of phosphorylated tau (e.g., phosphorylated-tau-Ser356) is reduced in the nucleocytoplasm. Some such methods are for inhibiting or reducing the accumulation of insoluble tau in cells or subjects. Some such methods are for inhibiting the accumulation of insoluble tau in cells or subjects. Some such methods are for reducing the accumulation of insoluble tau in cells or subjects.Some such methods involve administering to cells or subjects a LEM domain-containing protein 2 (LEMD2), charged multivesicular body protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or nucleic acids encoding LEMD2, CHMP7, or LEMD3.

[0006] In one embodiment, a method is provided for inhibiting tau aggregates or reducing phosphorylation in cells or subjects. Some such methods are for inhibiting tau aggregates in cells or subjects. Some such methods are for reducing phosphorylation in cells or subjects. Some such methods include administering to cells or subjects LEM domain-containing protein 2 (LEMD2), charged multivesicular protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or nucleic acids encoding LEMD2, CHMP7, or LEMD3.

[0007] Some such methods involve administering LEMD2 or a nucleic acid encoding LEMD2 to cells or a subject. In some such methods, LEMD2 is human LEMD2. Optionally, LEMD2 includes SEQ ID NO: 1 or SEQ ID NO: 5. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7. In some such methods, LEMD2 is mouse LEMD2. Optionally, LEMD2 includes SEQ ID NO: 10. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 255.

[0008] Some such methods involve administering CHMP7 or a nucleic acid encoding CHMP7 to cells or a subject. In some such methods, CHMP7 is human CHMP7. Optionally, CHMP7 includes SEQ ID NO: 15. Optionally, the nucleic acid encoding CHMP7 includes SEQ ID NO: 16 or SEQ ID NO: 17. In some such methods, CHMP7 is mouse CHMP7. Optionally, CHMP7 includes SEQ ID NO: 19. Optionally, the nucleic acid encoding CHMP7 includes SEQ ID NO: 20 or SEQ ID NO: 21.

[0009] Some such methods involve administering LEMD3 or a nucleic acid encoding LEMD3 to cells or a subject. In some such methods, LEMD3 is human LEMD3. Optionally, LEMD3 includes SEQ ID NO: 23. Optionally, the nucleic acid encoding LEMD3 includes SEQ ID NO: 24 or SEQ ID NO: 25. In some such methods, LEMD3 is mouse LEMD3. Optionally, LEMD3 includes SEQ ID NO: 27 or SEQ ID NO: 29. Optionally, the nucleic acid encoding LEMD3 includes SEQ ID NO: 29 or SEQ ID NO: 30.

[0010] In some such methods, a nucleic acid encoding LEMD2, CHMP7, or LEMD3 is administered to cells or a subject. Optionally, the nucleic acid is codon-optimized for expression in human or mouse cells. In some such methods, the nucleic acid includes complementary DNA encoding LEMD2, CHMP7, or LEMD3. In some such methods, the nucleic acid includes messenger RNA encoding LEMD2, CHMP7, or LEMD3.

[0011] In some of such methods, the method comprises administering an expression construct comprising a nucleic acid encoding LEMD2, CHMP7, or LEMD3 operably linked to a promoter. In some of such methods, the promoter is a heterologous promoter. In some of such methods, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In some of such methods, the promoter is a neuron-specific promoter. Optionally, the promoter is a synapsin-1 promoter. Optionally, the promoter is a human synapsin-1 promoter.

[0012] In some of such methods, the nucleic acid is present in a vector. In some of such methods, the vector is a viral vector. In some of such methods, the viral vector is a lentiviral vector or an Adeno-Associated Virus (AAV) vector. In some of such methods, the vector is an AAV vector. Optionally, the AAV vector is an AAV-PHP.eB vector.

[0013] In some of such methods, the cell is a mammalian cell, or the subject is a mammal. In some of such methods, the mammalian cell is a human cell, a rodent cell, a mouse cell, or a rat cell, or the subject is a human, a rodent, a mouse, or a rat. In some of such methods, the cell is a human cell, or the subject is a human.

[0014] In some such methods, the cell is a nerve cell. In some such methods, the cell is present in vivo in a subject. In some such methods, the cell is a nerve cell in the brain of the subject. In some such methods, LEMD2, CHMP7, or LEMD3, or a nucleic acid encoding LEMD2, CHMP7, or LEMD3 is administered to the subject by intracerebroventricular injection, intracranial injection, or intrathecal injection. In some such methods, LEMD2, CHMP7, or LEMD3, or a nucleic acid encoding LEMD2, CHMP7, or LEMD3 is administered to the subject by intraperitoneal injection.

[0015] Some such methods further comprise assessing one or more signs or symptoms of tauopathy or tau aggregates in the cell or the subject. Some such methods further comprise assessing phosphorylated-tau levels in the cell or the subject. Some such methods further comprise assessing serum neurofilament light chain (sNfL) levels in the subject.

[0016] Some such methods reduce the amount of new tau aggregate formation in the cell or the subject. Some such methods reduce the amount of existing tau aggregate formation in the cell or the subject.

[0017] In another aspect, a method of treating or preventing tauopathy in a subject is provided. Some such methods are for treating tauopathy in the subject. Some such methods are for preventing tauopathy in the subject. Some such methods comprise administering to the subject LEM domain-containing protein 2 (LEMD2), charged multivesicular body protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or a nucleic acid encoding LEMD2, CHMP7, or LEMD3 for the subject, wherein LEMD2, CHMP7, or LEMD3 inhibits tau aggregates in cells of the subject.

[0018] Some such methods involve administering LEMD2, or a nucleic acid encoding LEMD2, to a subject. In some such methods, LEMD2 is human LEMD2. Optionally, LEMD2 includes SEQ ID NO: 1 or SEQ ID NO: 5. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7. In some such methods, LEMD2 is mouse LEMD2. Optionally, LEMD2 includes SEQ ID NO: 10. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 255.

[0019] Some such methods involve administering CHMP7, or a nucleic acid encoding CHMP7, to a subject. In some such methods, CHMP7 is human CHMP7. Optionally, CHMP7 includes SEQ ID NO: 15. Optionally, the nucleic acid encoding CHMP7 includes SEQ ID NO: 16 or SEQ ID NO: 17. In some such methods, CHMP7 is mouse CHMP7. Optionally, CHMP7 includes SEQ ID NO: 19. Optionally, the nucleic acid encoding CHMP7 includes SEQ ID NO: 20 or SEQ ID NO: 21.

[0020] Some such methods involve administering LEMD3, or a nucleic acid encoding LEMD3, to a subject. In some such methods, LEMD3 is human LEMD3. Optionally, LEMD3 includes SEQ ID NO: 23. Optionally, the nucleic acid encoding LEMD3 includes SEQ ID NO: 24 or SEQ ID NO: 25. In some such methods, LEMD3 is mouse LEMD3. Optionally, LEMD3 includes SEQ ID NO: 27 or SEQ ID NO: 29. Optionally, the nucleic acid encoding LEMD3 includes SEQ ID NO: 29 or SEQ ID NO: 30.

[0021] In some such methods, a nucleic acid encoding LEMD2, CHMP7, or LEMD3 is administered to a subject. Optionally, the nucleic acid is codon-optimized for expression in human or mouse cells. In some such methods, the nucleic acid includes complementary DNA encoding LEMD2, CHMP7, or LEMD3. In some such methods, the nucleic acid includes messenger RNA encoding LEMD2, CHMP7, or LEMD3.

[0022] In some such methods, the method involves administering an expression construct containing a nucleic acid encoding LEMD2, CHMP7, or LEMD3 operably linked to a promoter. In some such methods, the promoter is a heterologous promoter. In some such methods, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In some such methods, the promoter is a neuron-specific promoter. Optionally, the promoter is a synapsin-1 promoter. Optionally, the promoter is a human synapsin-1 promoter.

[0023] In some such cases, the nucleic acid is present in the vector. In some such cases, the vector is a viral vector. In some such cases, the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector. In some such cases, the vector is an AAV vector. Optionally, the AAV vector is an AAV-PHP.eB vector.

[0024] In some such methods, the subject is a mammal. In some such methods, the subject is a human, rodent, mouse, or rat. In some such methods, the subject is a human.

[0025] In some such methods, the cells are nerve cells. In some such methods, the nerve cells are located in the subject's brain. In some such methods, LEMD2, CHMP7, or LEMD3, or the nucleic acid encoding LEMD2, CHMP7, or LEMD3, is administered to the subject by intracerebroventricular injection, intracranial injection, or intrathecal injection. In some such methods, LEMD2, CHMP7, or LEMD3, or the nucleic acid encoding LEMD2, CHMP7, or LEMD3, is administered to the subject by intraperitoneal injection.

[0026] Some such methods further include evaluating one or more signs or symptoms of tauopathy or tau aggregates in cells or subjects. Some such methods further include evaluating phosphorylated tau levels in cells or subjects. Some such methods further include evaluating serum neuronal filament light chain (sNfL) levels in subjects.

[0027] Some such methods reduce the amount of new tau aggregate formation in cells or subjects. Some such methods reduce the amount of existing tau aggregate formation in cells or subjects.

[0028] In another embodiment, an expression construct is provided comprising a nucleic acid encoding LEM domain-containing protein 2 (LEMD2), charged multivesicular protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), which is operably linked to a heterologous promoter.

[0029] Some such expression constructs include the nucleic acid encoding LEMD2. In some such expression constructs, LEMD2 is human LEMD2. Optionally, LEMD2 includes SEQ ID NO: 1 or SEQ ID NO: 5. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7. In some such expression constructs, LEMD2 is mouse LEMD2. Optionally, LEMD2 includes SEQ ID NO: 10. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. Optionally, the nucleic acid encoding LEMD2 includes SEQ ID NO: 255.

[0030] Some such expression constructs include the nucleic acid encoding CHMP7. In some such expression constructs, CHMP7 is human CHMP7. Optionally, CHMP7 includes SEQ ID NO: 15. Optionally, the nucleic acid encoding CHMP7 includes SEQ ID NO: 16 or SEQ ID NO: 17. In some such expression constructs, CHMP7 is mouse CHMP7. Optionally, CHMP7 includes SEQ ID NO: 19. Optionally, the nucleic acid encoding CHMP7 includes SEQ ID NO: 20 or SEQ ID NO: 21.

[0031] Some such expression constructs include the nucleic acid encoding LEMD3. In some such expression constructs, LEMD3 is human LEMD3. Optionally, LEMD3 includes SEQ ID NO: 23. Optionally, the nucleic acid encoding LEMD3 includes SEQ ID NO: 24 or SEQ ID NO: 25. In some such expression constructs, LEMD3 is mouse LEMD3. Optionally, LEMD3 includes SEQ ID NO: 27 or SEQ ID NO: 29. Optionally, the nucleic acid encoding LEMD3 includes SEQ ID NO: 29 or SEQ ID NO: 30.

[0032] In some such expression constructs, the nucleic acid encoding LEMD2, CHMP7, or LEMD3 is codon-optimized for expression in human or mouse cells. In some such expression constructs, the nucleic acid includes complementary DNA encoding LEMD2, CHMP7, or LEMD3.

[0033] In some such expression constructs, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In some such expression constructs, the promoter is a neuron-specific promoter. Optionally, the promoter is a synapsin-1 promoter. Optionally, the promoter is a human synapsin-1 promoter.

[0034] In some such expression constructs, the nucleic acid is present in the vector. In some such expression constructs, the vector is a viral vector. In some such expression constructs, the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector. In some such expression constructs, the vector is an AAV vector. Optionally, the AAV vector is an AAV-PHP.eB vector. [Brief explanation of the drawing]

[0035] [Figure 1A] Figures 1A to 1D show a screening of mutations that enhance tau aggregates. Figure 1A shows a schematic diagram illustrating the detection of tau aggregates by induction of FRET. Tau biosensor cells treated with a conditioned medium derived from cells lacking tau aggregates and tau-YFP Agg- (IF image in the upper left) do not produce a FRET signal after 3 days of culture (flow cytometry plot in the upper right). Biosensor cells treated with a conditioned medium derived from cells containing tau aggregates and tau-YFP Agg+ (IF image in the lower left) induce a detectable FRET signal in 0.1% of the cells (flow cytometry plot in the lower right). [Figure 1B] Figures 1A–1D show the screening for mutations that enhance tau aggregates. Figure 1B shows a schematic diagram of the CRISPR screening timeline. Lentiviral-packaged CRISPR gRNA libraries were transduced into Cas9-expressing biosensor cells on day 0. Different colored nuclei indicate that each cell received a different gRNA transgene. Cells were sampled on days 3 and 6, and FRET+ cells were sorted and collected by FACS on day 10. gRNA expression was determined by PCR amplification and sequencing of unit replication sequences. [Figure 1C] Figures 1A–1D show a screening of mutations that enhance tau aggregates. Figure 1C shows a plot of gene center enrichment against p-values ​​in FRET+ cells at day 10 for gRNAs targeting the same gene. BANF1 is indicated by circles labeled "X", and PPP2CA is indicated by white circles. [Figure 1D] Figures 1A–1D show a screening of mutations that enhance tau aggregates. Figure 1D shows a secondary screening of 14 primary screening candidate genes for tau aggregate enhancement in biosensor cells seeded in tau-YFP Agg+ conditioned medium, measured as FRET induction (calculated as integrated FRET density) after inactivation with individual lentiviral gRNA expression vectors (four gRNAs for BANF1 and two gRNAs for each of the 13 other genes). The bar heights represent the mean ± SEM for all gRNAs analyzed. Genes targeted by gRNAs are shown on the X-axis. Large FRET induction enhancements for gRNA-targeted BANF1 or PPP2CA confirm these genes as hits in the screening for enhanced tau aggregates. Controls (first two bars) were pseudotransduction without virus and transduction with lentiviral vectors expressing untargeted control gRNAs. See also Figures 5A–5E and 6A–6G.

[0036] [Figure 2A] Figures 2A–2E demonstrate that targeted regulation of protein-coding genes in the nuclear membrane-related BANF1 interaction network can promote or inhibit tau aggregate formation. Figure 2A shows that a string database query for BANF1 returns a functional interaction network of proteins involved in maintaining nuclear membrane integrity. [Figure 2B] Figures 2A–2E demonstrate that targeted regulation of protein-encoding genes in the nuclear membrane-related BANF1 interaction network can promote or inhibit tau aggregate formation. Figure 2B reveals that ANKLE2, a member of the BANF1 interaction network, is identified as a gene whose gRNA-targeted inactivation enhances tau aggregate formation in biosensor cells with tau-YFP Agg+ conditioned medium, and that this is measured as FRET induction (calculated as integrated FRET density, mean ± standard deviation). Cas9-expressing biosensor cells were transduced with lentiviral vectors expressing targeted gRNA and LV-gRNA. Controls (first two bars) were pseudotransduced without virus and transduced with lentiviral vectors expressing control gRNA. Inactivation of ANKLE2, rather than other BANF1 interacting proteins, enhances FRET induction. [Figure 2C] Figures 2A–2E demonstrate that targeted regulation of protein-coding genes in the nuclear membrane-associated BANF1 interaction network can promote or inhibit tau aggregate formation. Figure 2C confirms that inactivation of BANF1, PPP2CA, or ANKLE2 (but not LEMD2 or LEMD3) by individual LV-gRNAs enhances tau aggregate formation, as measured by FRET induction using three different sources of tau seeding activity (sonicated whole cell lysates from tau-YFP Agg+ cells, purified recombinant tau fibrils, and spinal cord lysates from 9-month-old tau P301S transgenic mice). Cas9-expressing biosensor cells were transduced with individual LV-gRNAs. Controls were transduced with control gRNAs (first three bars) and gRNAs targeting LEMD2 and LEMD3, two genes not identified in the network member screening (last six bars). [Figure 2D] Figures 2A–2E show that targeted regulation of protein-encoding genes in the nuclear membrane-related BANF1 interaction network can promote or inhibit tau aggregate formation. Figure 2D shows a diagram of the nuclear membrane with the relevant LEM domain-containing proteins ANKLE2, LEMD2, the short isoform LEMD2iso2, and LEMD3. The repair factor CHMP7 is also shown. These include the ER, endoplasmic reticulum; NPC, pore complex; INM, inner nuclear membrane; ONM; and outer nuclear membrane. [Figure 2E] Figures 2A–2E demonstrate that targeted regulation of protein-encoding genes in the nuclear membrane-associated BANF1 interaction network can promote or inhibit tau aggregates. Figure 2E shows that expression of cDNA encoding LEMD2, LEMD2i2, LEMD3, and CHMP7 proteins in biosensor cells reduces FRET induced by a potent seeding agent (whole cell lysate containing tau-YFP Agg + LIPOFECTAMINE®) compared to control cells expressing firefly luciferase. Biosensor cells were transduced with lentiviral vectors expressing cDNA and LV-cDNA. Bars represent the mean integrated FRET density ± standard deviation for four replicated samples. Significance was determined using unpaired two-tailed t-tests (*p<0.01, **p<0.0001).

[0037] [Figure 3A]Figures 3A to 3H show the identification of genes whose overexpression rescues tau aggregates. Figures 3A and 3B show the rescue of the FRET-induced phenotype by overexpression of LEMD2, LEMD2i2, LEMD3, and CHMP7 nuclear membrane components. dCas9-KRAB expressing biosensor cells were transduced with LV-gRNA-targeted BANF1 (pink) or ANKLE2 (blue), or control gRNA (gray), which transcriptionally represses these gene targets, as well as LV-cDNA encoding LEMD2, LEMD2i2, LEMD3, and CHMP7 proteins. FRET was induced by seeding with spinal cord lysates (Figure 3A) or tau-YFP Agg+ whole cell lysates (Figure 3B) from 9-month-old tau P301S transgenic mice. Bars represent the average integrated FRET density for three replicated samples. Graphs show mean ± SEM. [Figure 3B] Figures 3A to 3H show the identification of genes whose overexpression rescues tau aggregates. Figures 3A and 3B show the rescue of the FRET-induced phenotype by overexpression of LEMD2, LEMD2i2, LEMD3, and CHMP7 nuclear membrane components. dCas9-KRAB expressing biosensor cells were transduced with LV-gRNA-targeted BANF1 (pink) or ANKLE2 (blue), or control gRNA (gray), which transcriptionally represses these gene targets, as well as LV-cDNA encoding LEMD2, LEMD2i2, LEMD3, and CHMP7 proteins. FRET was induced by seeding with spinal cord lysates (Figure 3A) or tau-YFP Agg+ whole cell lysates (Figure 3B) from 9-month-old tau P301S transgenic mice. Bars represent the average integrated FRET density for three replicated samples. Graphs show mean ± SEM. [Figure 3C]Figures 3A–3H show the identification of genes whose overexpression rescues tau aggregates. Figures 3C and 3D show Western blots detecting total tau protein or tau phosphorylated on serine 356. dCas9-KRAB-expressing tau biosensor cells transduced with gRNA-targeted BANF1 or ANKLE2 were treated with whole cell lysates from tau-YFP Agg- cells (left) or tau-YFP Agg+ cells (right), and co-transduced with LV-cDNA for luciferase (Figure 3C) or LEMD2 (Figure 3D). Only cells treated with tau-YFP Agg+ whole cell lysates showed an increase in total tau and P-tau-Ser356 in the insoluble fraction (Figure 3C, red box). This increase is prevented by transduction with LEMD2 cDNA (Figure 3D, red box). Since these Western blots were performed on denatured gels, it is not possible to distinguish between monomeric and polymeric forms of tau. [Figure 3D] Figures 3A–3H show the identification of genes whose overexpression rescues tau aggregates. Figures 3C and 3D show Western blots detecting total tau protein or tau phosphorylated on serine 356. dCas9-KRAB-expressing tau biosensor cells transduced with gRNA-targeted BANF1 or ANKLE2 were treated with whole cell lysates from tau-YFP Agg- cells (left) or tau-YFP Agg+ cells (right), and co-transduced with LV-cDNA for luciferase (Figure 3C) or LEMD2 (Figure 3D). Only cells treated with tau-YFP Agg+ whole cell lysates showed an increase in total tau and P-tau-Ser356 in the insoluble fraction (Figure 3C, red box). This increase is prevented by transduction with LEMD2 cDNA (Figure 3D, red box). Since these Western blots were performed on denatured gels, it is not possible to distinguish between monomeric and polymeric forms of tau. [Figure 3E]Figures 3A–3H show the identification of genes whose overexpression rescues tau aggregates. Figure 3E shows representative confocal microscopy images of immunofluorescence detection of SRRM2 (yellow) and tau phosphorylated on serine 356 (magenta). Biosensor cells were treated with tau-YFP Agg+ whole cell lysate. In cells with tau aggregates, SRRM2 mislocalizes from the nucleus (shown by blue DAPI staining) and colocalizes with cytoplasmic tau aggregates positive for P-tau-Ser356. [Figure 3F] Figures 3A-3H show the identification of genes whose overexpression rescues tau aggregates. Figure 3F shows representative confocal microscopy images of immunofluorescence detection of SRRM2 (yellow) in dCas9-KRAB-expressing biosensor cells transduced with control gRNA or gRNA targeting BANF1 or ANKLE2, and treated with tau-YFP Agg+ whole cell lysate. In cells depleted of BANF1 and ANKLE2, some SRRM2 is detected as cytoplasmic lesions near the nucleus. DAPI staining (blue) identifies the nucleus. Scale bar = 20 μm. [Figure 3G] Figures 3A to 3H show the identification of genes whose overexpression rescues tau aggregates. Figure 3G shows a histogram plot of cells with nuclear SRRM2 only (monochromatic) and cells with nuclear and mislocalized cytoplasmic SRRM2 (oblique lines). [Figure 3H] Figures 3A–3H show the identification of genes whose overexpression rescues tau aggregates. Figure 3H shows the quantification of the percentage of total SRRM2 immunofluorescence intensity in the nucleus of each cell. Quantification for BANF1-depleted biosensor cells is shown in pink, ANKLE2 in blue, and control cells in gray. Fluorescence images of individual cells were quantified using Harmony software (Perkin-Elmer). Bar heights represent the mean ± SEM of all analyzed cells (n=113). Significance was determined using an unpaired two-sided t-test (**p<0.0001).

[0038] [Figure 4A] Figures 4A–4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figure 4A shows representative confocal microscopy images of P-tau-Ser356 (yellow) and microtubule-associated protein 2 (MAP2, red) in wild-type primary mouse cortical neurons 14 days after transduction with a lentiviral vector co-expressing Cas9 with either control gRNA or gRNA-targeted Banf1. DAPI staining (blue) identifies the nucleus. Scale bar = 50 μm. [Figure 4B] Figures 4A–4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figure 4B shows the quantification of DAPI+ cells per culture well. [Figure 4C] Figures 4A to 4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figure 4C shows the quantification of P-tau-Ser356 in the cell body. [Figure 4D] Figures 4A to 4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figure 4D shows the quantification of P-tau-Ser356 in the nucleocytoplasm. [Figure 4E] Figures 4A–4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of tau phosphorylated on serine 356 and impairs nuclear membrane integrity. Figure 4E shows the quantification of P-tau-Ser356 in the perinuclear domains of control and Ankle2, Banf1, and Ppp2ca mutant cells. Primary mouse cortical neurons transduced with LV-Cas9-gRNA were immunostained with antibodies against MAP2 or P-tau-Ser356. [Figure 4F]Figures 4A–4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figure 4F shows representative microscopic images of live primary mouse cortical neurons expressing EF1a-nls:mCherry, treated with gymnosis-delivered ASO for 8 days. Merged images of bright-field and mCherry views (left, red) and mCherry alone (right, white) are shown. Scale bar = 10 μm. [Figure 4G] Figures 4A–4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figures 4G and 4H show the relative expression of Banf1 and Ankle2, respectively, as assessed by TaqMan qRT-PCR and normalized to control treatment. Each value represents the mean ± STDEV of two copies. Gapdh expression was used as a reference gene. [Figure 4H] Figures 4A–4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figures 4G and 4H show the relative expression of Banf1 and Ankle2, respectively, as assessed by TaqMan qRT-PCR and normalized to control treatment. Each value represents the mean ± STDEV of two copies. Gapdh expression was used as a reference gene. [Figure 4I]Figures 4A–4I show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons enhances the production of phosphorylated tau on serine 356 and impairs nuclear membrane integrity. Figure 4I shows the quantification of mCherry fluorescence intensity. Three equivalent areas were measured per cell body. Graphs show mean ± SEM (n=12 for Figures 4B–4E and n=7 for Figure 4I), analyzed by the Mann-Whitney U test, two-sided exact significance, *p<0.01, **p<0.007, ***p<0.0001, ns=not significant). See also Figures 8A–8K and 9A–9G.

[0039] [Figure 5A] Figures 5A to 5H illustrate the development of a tau biosensor cell-based screening platform. Figure 5A shows a negatively stained transmission electron microscopy (TEM) image of recombinant tau Q244-E372;P301L,V337M (tau 244-372LM tau) fibrils. [Figure 5B] Figures 5A to 5H illustrate the development of a tau biosensor cell-based screening platform. Figure 5B shows the static thioflavin T (ThT) fluorescence of tau 244-372LM monomers and fibrils, demonstrating that fibrils bind to the amyloid-specific dye ThT, while monomers do not. [Figure 5C]Figures 5A–5H illustrate the development of a tau biosensor cell-based screening platform. Figures 5C and 5D show the fibrillation dynamics of tau 244–372LM (Figure 5C) with or without heparin, monitored by ThT fluorescence (Figure 5D). Tau 244–372LM were cultured at 37°C with or without heparin at a 4:1 tau-to-heparin ratio using dual-orbit shaking at 700 rpm. With heparin, all three concentrations tested (50, 25, and 10 μM) showed a rapid increase in ThT intensity corresponding to T1 / 2–0.5 hours. Tau 244-372LM cultured without heparin showed a concentration-dependent increase in ThT intensity; 50 μM tau 244-372LM showed a T1 / 2 ~14 hours, and 25 μM tau 244-372LM showed a T1 / 2 ~31 hours, while 10 μM tau 244-372LM showed no increase in ThT intensity within the measured 120 hours. [Figure 5D] Figures 5A–5H illustrate the development of a tau biosensor cell-based screening platform. Figures 5C and 5D show the fibrillation dynamics of tau 244–372LM (Figure 5C) with or without heparin, monitored by ThT fluorescence (Figure 5D). Tau 244–372LM were cultured at 37°C with or without heparin at a 4:1 tau-to-heparin ratio using dual-orbit shaking at 700 rpm. With heparin, all three concentrations tested (50, 25, and 10 μM) showed a rapid increase in ThT intensity corresponding to T1 / 2–0.5 hours. Tau 244-372LM cultured without heparin showed a concentration-dependent increase in ThT intensity; 50 μM tau 244-372LM showed a T1 / 2 ~14 hours, and 25 μM tau 244-372LM showed a T1 / 2 ~31 hours, while 10 μM tau 244-372LM showed no increase in ThT intensity within the measured 120 hours. [Figure 5E]Figures 5A–5H illustrate the development of a tau biosensor cell-based screening platform. Figure 5E shows that the tau biosensor cells are HEK293T cells containing two transgenes that express the 4-repeat domain (4RD) of human protein tau fused to a CFP or YFP fluorescent reporter and contain the P301S pathogenic mutation. Upon treatment with a source of tau seeding activity, the biosensor cells form visible tau aggregates phosphorylated at Ser356. The biosensor cells generate a FRET signal during tau aggregation. [Figure 5F] Figures 5A–5H illustrate the development of a tau biosensor cell-based screening platform. Figure 5F shows representative confocal microscope images of biosensor cells seeded with whole cell lysates from tau-YFP Agg+ cells, demonstrating immunofluorescence detection of P-tau-Ser356 (magenta) and aggregated tau protein (tau-YFP, visualized in yellow). DAPI staining (blue) indicates the nucleus. Scale bar = 20 μm. [Figure 5G] Figures 5A–5H illustrate the development of a tau biosensor cell-based screening platform. Figure 5G shows the evaluation of the tau biosensor Cas9 clone. Cas9 transgene expression was evaluated by TaqMan qRT-PCR 3 and 7 days after transduction with gRNA-targeted PERK, and cleavage efficiency was evaluated by digital PCR (dPCR). [Figure 5H] Figures 5A–5H illustrate the development of a tau biosensor cell-based screening platform. Figure 5H shows a plot of gRNA enrichment against p-values ​​in FRET+ cells at day 10 compared to cells at day 6. gRNA-targeted BANF1 is indicated by circles labeled with 'X'.

[0040] [Figure 6A]Figures 6A–6G show that regulation of nuclear membrane-related genes affects tau aggregates and associated phenotypes. Figure 6A shows that treatment with tau-YFP Agg+ conditioned medium is necessary to generate the FRET signal and to detect the enhancement of the FRET signal resulting from the disruption of tau modifying factor genes. Treatment with fresh medium did not generate the FRET signal, even after the disruption of BANF1 or PPP2CA. [Figure 6B] Figures 6A to 6G show that regulation of nuclear membrane-related genes affects tau aggregates and associated phenotypes. Figure 6B shows a Western blot illustrating a specific decrease in protein levels in Cas9-expressing biosensor cells after transduction with LV-gRNA-targeted BANF1 or PPP2CA. [Figure 6C] Figures 6A–6G show that regulation of nuclear membrane-related genes affects tau aggregates and associated phenotypes. Figure 6C shows that single-cell knockdown clones were isolated using Cas9-expressing biosensor cells transduced with LV-gRNA-targeted BANF1 or PPP2CA. These clones show increased FRET induction only after treatment with tau-YFP Agg+ conditioned medium, which correlates with the percentage of gene editing at the target locus, as assessed by NGS (Next Generation Sequencing). In Figure 6C, FRET induction is calculated as integrated FRET density, where each value represents the mean ± STDEV of at least two replications. [Figure 6D] Figures 6A–6G show that regulation of nuclear membrane-related genes affects tau aggregates and associated phenotypes. Figure 6D shows co-transduction of multiple LV-gRNAs into biosensor cells at high MOI (Multiplicity of Infection). Co-transduction of LV-gRNAs targeting BANF1, ANKLE2, and PPP2CA in different combinations does not result in increased FRET induction compared to targeting ANKLE2 alone. [Figure 6E]Figures 6A to 6G show that regulation of nuclear membrane-related genes affects tau aggregates and associated phenotypes. Figure 6E shows verification of the CRISPRa SAM activation system in biosensor cells. Tau biosensor cells expressing dCas9-SAM were transduced with LV-gRNAs targeting one of 11 genes. TaqMan expression analysis revealed that SAM-mediated transcriptional activation was inversely correlated with basal transcript levels (RPKM, the normalization unit of transcript expression). [Figure 6F] Figures 6A–6G show that regulation of nuclear membrane-related genes affects tau aggregates and associated phenotypes. Figures 6F and 6G show cDNA expression analysis of potential tau modifier genes. Biosensor cells were transduced with individual lentiviral packaging cDNAs, LV-cDNAs, which induce high expression of the encoded proteins. Transduced cells were selected and collected for expression analysis using TaqMan assays designed to specifically amplify these codon-optimized cDNAs (Figure 6F) and MAPT-4RD cDNAs (Figure 6G). ΔCt values ​​are shown for each specific cDNA transduced sample. Each value represents the mean ± STDEV of four replications (shown individually as symbols). [Figure 6G] Figures 6A–6G show that regulation of nuclear membrane-related genes affects tau aggregates and associated phenotypes. Figures 6F and 6G show cDNA expression analysis of potential tau modifier genes. Biosensor cells were transduced with individual lentiviral packaging cDNAs, LV-cDNAs, which induce high expression of the encoded proteins. Transduced cells were selected and collected for expression analysis using TaqMan assays designed to specifically amplify these codon-optimized cDNAs (Figure 6F) and MAPT-4RD cDNAs (Figure 6G). ΔCt values ​​are shown for each specific cDNA transduced sample. Each value represents the mean ± STDEV of four replications (shown individually as symbols).

[0041] [Figure 7A]Figures 7A–7F illustrate the genetic interactions of nuclear membrane components that modify tau aggregates. Figure 7A shows a recently discovered role of BANF1 and LEMD2 in promoting nuclear membrane sealing after damage. Mechanical stress on the nucleus can lead to nuclear membrane disruption, which is repaired by the recruitment of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III complex). At the site of disruption, cytoplasmic BANF1 coats the exposed chromatin and recruits the membrane through interaction with LEMD2. Locally increased concentrations of LEMD2 enable the recruitment of CHMP7 to the granular membrane, which promotes ESCRT-III nucleation. Finally, the ESCRT-III complex with the ATPase VPS4 promotes disruption and nuclear membrane sealing, as discussed by Zhen et al., (2021) EMBO J..40:e106922, whose entire work is incorporated herein by reference. [Figure 7B] Figures 7A to 7F show the genetic interactions of nuclear membrane components that modify tau aggregates. Figure 7B shows the relative expression of BANF1. [Figure 7C] Figures 7A to 7F show the genetic interactions of nuclear membrane components that modify tau aggregates. Figure 7C shows the relative expression of ANKLE2. [Figure 7D] Figures 7A–7F show the genetic interactions of nuclear membrane components that modify tau aggregates. Figure 7D shows the relative expression of MAPT-4RD in dCas9-KRAB and cDNA-expressing biosensor cells. Relative expression was evaluated by TaqMan qRT-PCR and normalized to control treatment. Each value represents the mean ± STDEV of at least two replications. GAPDH expression was used as the reference gene. [Figure 7E]Figures 7A to 7F show the genetic interactions of nuclear membrane components that modify tau aggregates. Figure 7E shows the rescue of the FRET-induced phenotype by overexpression of nuclear membrane components. Overexpression of BANF1 cDNA specifically eliminates the increase in FRET induction by tau-YFP Agg+ cell lysates that occurs from BANF1 knockdown, not from ANKLE2 knockdown, in dCas9-KRAB-expressing biosensor cells. [Figure 7F] Figures 7A to 7F show the genetic interactions of nuclear membrane components that modify tau aggregates. Figure 7F shows a Western blot for detecting BANF1 protein in dCas9-KRAB-expressing biosensor cells that were transduced with LV-gRNA targeting BANF1 or ANKLE2, seeded with whole cell lysates from tau-YFP Agg- cells (left) or tau-YFP Agg+ cells (right), and simultaneously transduced with cDNA expressing luciferase (top) or BANF1 (bottom). Disruption of ANKLE2 causes mislocalization of BANF1 protein from the chromatin-binding fraction to the cytoplasmic fraction, which is not rescued by overexpression of BANF1 cDNA.

[0042] [Figure 8A] Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figure 8A shows a confocal image of primary mouse cortical neurons highlighting the target compartment (nucleocytoplasm (left), perinuclear domain (center), and cell body (right)) segmented by Harmony software. [Figure 8B] Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figure 8B shows the percentage of gene editing at the target cleavage site, as assessed by NGS. [Figure 8C]Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figure 8C shows the relative gene expression of primary mouse cortical neurons transduced with LV-Cas9-gRNA, as evaluated by the TaqMan qRT-PCR assay. Data are normalized to control cells. Each value represents the mean ± STDEV of four copies. Gapdh expression was used as the reference gene. [Figure 8D] Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau proteins. Figure 8D shows representative confocal microscopy images of immunofluorescence staining for P-tau-Ser356 (yellow) and microtubule-associated protein 2 (MAP2, red) in primary cortical neurons 14 days after transduction with LV-Cas9-gRNA. DAPI staining (blue) identifies the nucleus. Scale bar = 50 μm. [Figure 8E] Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figure 8E shows quantification of MAP2 protein in the cell body, revealing significant reductions in signaling associated with the disruption of Ankle2, Banf1, and Ppp2ca, with the latter being the most severe. [Figure 8F] Figures 8A to 8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figure 8F shows representative confocal images of primary mouse cortical neurons immunostained for MAP2 (red) and total tau protein (yellow) after disruption of Ankle2, Banf1, or Ppp2ca. [Figure 8G]Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figure 8G shows quantification of DAPI+ cells per culture well, demonstrating clear toxicity associated with Ppp2ca loss. [Figure 8H] Figures 8A to 8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figure 8H shows the quantification of MAP2 protein in the cell body of primary mouse cortical neurons. [Figure 8I] Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figures 8I–8K show the quantification of total tau in the cell body, perinuclear domain, and nucleocytoplasm of primary mouse cortical neurons transduced with LV-Cas9-gRNA and immunostained with antibodies against MAPP2 or total tau. The graphs show mean ± SEM (n=12), two-sided exact significance, *p<0.01, ns=not significant, analyzed by the Mann-Whitney U test. [Figure 8J] Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figures 8I–8K show the quantification of total tau in the cell body, perinuclear domain, and nucleocytoplasm of primary mouse cortical neurons transduced with LV-Cas9-gRNA and immunostained with antibodies against MAPP2 or total tau. The graphs show mean ± SEM (n=12), two-sided exact significance, *p<0.01, ns=not significant, analyzed by the Mann-Whitney U test. [Figure 8K]Figures 8A–8K show that disruption of Ankle2, Banf1, or Ppp2ca in primary mouse cortical neurons affects phosphorylation and intracellular localization, but does not strongly affect total tau protein. Figures 8I–8K show the quantification of total tau in the cell body, perinuclear domain, and nucleocytoplasm of primary mouse cortical neurons transduced with LV-Cas9-gRNA and immunostained with antibodies against MAPP2 or total tau. The graphs show mean ± SEM (n=12), two-sided exact significance, *p<0.01, ns=not significant, analyzed by the Mann-Whitney U test.

[0043] [Figure 9A] Figures 9A–9G show that Lemd2, Lemd3, and Chmp7 cDNAs can rescue increased phosphorylated tau-Ser356 in Banf1 or Ankle2 targeted knockdown in primary cortical neurons. Neurons were treated with individual ASOs for 10 days via gymnotic delivery. ASOs were replenished with each medium change. Four days after the initial ASO treatment, LV-hSyn1-cDNA was transduced. Quantification of DAPI+ cells per replication well, MAP2 protein in the neuronal cell body, and P-tau-Ser356 in the cell body, perinuclear domain, and nucleocytoplasmic domain is shown in Figures 9A–9D. Neurons were transduced with luciferase-encoding LV-hSyn1-cDNA (Figure 9A), Lemd2 (Figure 9B), Lemd3 (Figure 9C), and Chmp7 (Figure 9D). On day 14, the relative expression of Banf1 (Figure 9E) or Ankle2 (Figure 9F) in ASO-treated neurons was evaluated by target-specific TaqMan qRT-PCR assays and codon-optimized cDNA-specific TaqMan assays (Figure 9G). Graphs show mean ± SEM (n=12), analyzed by the Mann-Whitney U test, with two-sided exact significance, *p<0.05, **p<0.005, ***p<0.0001, ns=not significant. [Figure 9B]Figures 9A–9G show that Lemd2, Lemd3, and Chmp7 cDNAs can rescue increased phosphorylated tau-Ser356 in Banf1 or Ankle2 targeted knockdown in primary cortical neurons. Neurons were treated with individual ASOs for 10 days via gymnotic delivery. ASOs were replenished with each medium change. Four days after the initial ASO treatment, LV-hSyn1-cDNA was transduced. Quantification of DAPI+ cells per replication well, MAP2 protein in the neuronal cell body, and P-tau-Ser356 in the cell body, perinuclear domain, and nucleocytoplasmic domain is shown in Figures 9A–9D. Neurons were transduced with luciferase-encoding LV-hSyn1-cDNA (Figure 9A), Lemd2 (Figure 9B), Lemd3 (Figure 9C), and Chmp7 (Figure 9D). On day 14, the relative expression of Banf1 (Figure 9E) or Ankle2 (Figure 9F) in ASO-treated neurons was evaluated by target-specific TaqMan qRT-PCR assays and codon-optimized cDNA-specific TaqMan assays (Figure 9G). Graphs show mean ± SEM (n=12), analyzed by the Mann-Whitney U test, with two-sided exact significance, *p<0.05, **p<0.005, ***p<0.0001, ns=not significant. [Figure 9C]Figures 9A–9G show that Lemd2, Lemd3, and Chmp7 cDNAs can rescue increased phosphorylated tau-Ser356 in Banf1 or Ankle2 targeted knockdown in primary cortical neurons. Neurons were treated with individual ASOs for 10 days via gymnotic delivery. ASOs were replenished with each medium change. Four days after the initial ASO treatment, LV-hSyn1-cDNA was transduced. Quantification of DAPI+ cells per replication well, MAP2 protein in the neuronal cell body, and P-tau-Ser356 in the cell body, perinuclear domain, and nucleocytoplasmic domain is shown in Figures 9A–9D. Neurons were transduced with luciferase-encoding LV-hSyn1-cDNA (Figure 9A), Lemd2 (Figure 9B), Lemd3 (Figure 9C), and Chmp7 (Figure 9D). On day 14, the relative expression of Banf1 (Figure 9E) or Ankle2 (Figure 9F) in ASO-treated neurons was evaluated by target-specific TaqMan qRT-PCR assays and codon-optimized cDNA-specific TaqMan assays (Figure 9G). Graphs show mean ± SEM (n=12), analyzed by the Mann-Whitney U test, with two-sided exact significance, *p<0.05, **p<0.005, ***p<0.0001, ns=not significant. [Figure 9D]Figures 9A–9G show that Lemd2, Lemd3, and Chmp7 cDNAs can rescue increased phosphorylated tau-Ser356 in Banf1 or Ankle2 targeted knockdown in primary cortical neurons. Neurons were treated with individual ASOs for 10 days via gymnotic delivery. ASOs were replenished with each medium change. Four days after the initial ASO treatment, LV-hSyn1-cDNA was transduced. Quantification of DAPI+ cells per replication well, MAP2 protein in the neuronal cell body, and P-tau-Ser356 in the cell body, perinuclear domain, and nucleocytoplasmic domain is shown in Figures 9A–9D. Neurons were transduced with luciferase-encoding LV-hSyn1-cDNA (Figure 9A), Lemd2 (Figure 9B), Lemd3 (Figure 9C), and Chmp7 (Figure 9D). On day 14, the relative expression of Banf1 (Figure 9E) or Ankle2 (Figure 9F) in ASO-treated neurons was evaluated by target-specific TaqMan qRT-PCR assays and codon-optimized cDNA-specific TaqMan assays (Figure 9G). Graphs show mean ± SEM (n=12), analyzed by the Mann-Whitney U test, with two-sided exact significance, *p<0.05, **p<0.005, ***p<0.0001, ns=not significant. [Figure 9E]Figures 9A–9G show that Lemd2, Lemd3, and Chmp7 cDNAs can rescue increased phosphorylated tau-Ser356 in Banf1 or Ankle2 targeted knockdown in primary cortical neurons. Neurons were treated with individual ASOs for 10 days via gymnotic delivery. ASOs were replenished with each medium change. Four days after the initial ASO treatment, LV-hSyn1-cDNA was transduced. Quantification of DAPI+ cells per replication well, MAP2 protein in the neuronal cell body, and P-tau-Ser356 in the cell body, perinuclear domain, and nucleocytoplasmic domain is shown in Figures 9A–9D. Neurons were transduced with luciferase-encoding LV-hSyn1-cDNA (Figure 9A), Lemd2 (Figure 9B), Lemd3 (Figure 9C), and Chmp7 (Figure 9D). On day 14, the relative expression of Banf1 (Figure 9E) or Ankle2 (Figure 9F) in ASO-treated neurons was evaluated by target-specific TaqMan qRT-PCR assays and codon-optimized cDNA-specific TaqMan assays (Figure 9G). Graphs show mean ± SEM (n=12), analyzed by the Mann-Whitney U test, with two-sided exact significance, *p<0.05, **p<0.005, ***p<0.0001, ns=not significant. [Figure 9F]Figures 9A–9G show that Lemd2, Lemd3, and Chmp7 cDNAs can rescue increased phosphorylated tau-Ser356 in Banf1 or Ankle2 targeted knockdown in primary cortical neurons. Neurons were treated with individual ASOs for 10 days via gymnotic delivery. ASOs were replenished with each medium change. Four days after the initial ASO treatment, LV-hSyn1-cDNA was transduced. Quantification of DAPI+ cells per replication well, MAP2 protein in the neuronal cell body, and P-tau-Ser356 in the cell body, perinuclear domain, and nucleocytoplasmic domain is shown in Figures 9A–9D. Neurons were transduced with luciferase-encoding LV-hSyn1-cDNA (Figure 9A), Lemd2 (Figure 9B), Lemd3 (Figure 9C), and Chmp7 (Figure 9D). On day 14, the relative expression of Banf1 (Figure 9E) or Ankle2 (Figure 9F) in ASO-treated neurons was evaluated by target-specific TaqMan qRT-PCR assays and codon-optimized cDNA-specific TaqMan assays (Figure 9G). Graphs show mean ± SEM (n=12), analyzed by the Mann-Whitney U test, with two-sided exact significance, *p<0.05, **p<0.005, ***p<0.0001, ns=not significant. [Figure 9G]Figures 9A–9G show that Lemd2, Lemd3, and Chmp7 cDNAs can rescue increased phosphorylated tau-Ser356 in Banf1 or Ankle2 targeted knockdown in primary cortical neurons. Neurons were treated with individual ASOs for 10 days via gymnotic delivery. ASOs were replenished with each medium change. Four days after the initial ASO treatment, LV-hSyn1-cDNA was transduced. Quantification of DAPI+ cells per replication well, MAP2 protein in the neuronal cell body, and P-tau-Ser356 in the cell body, perinuclear domain, and nucleocytoplasmic domain is shown in Figures 9A–9D. Neurons were transduced with luciferase-encoding LV-hSyn1-cDNA (Figure 9A), Lemd2 (Figure 9B), Lemd3 (Figure 9C), and Chmp7 (Figure 9D). On day 14, the relative expression of Banf1 (Figure 9E) or Ankle2 (Figure 9F) in ASO-treated neurons was evaluated by target-specific TaqMan qRT-PCR assays and codon-optimized cDNA-specific TaqMan assays (Figure 9G). Graphs show mean ± SEM (n=12), analyzed by the Mann-Whitney U test, with two-sided exact significance, *p<0.05, **p<0.005, ***p<0.0001, ns=not significant. [Modes for carrying out the invention]

[0044] definition As used interchangeably herein, the terms “protein,” “polypeptide,” and “peptide” include polymeric forms of amino acids of any length, including coding and non-coding amino acids, as well as chemically or biochemically modified or derivatized amino acids. These terms also include modified polymers, such as polypeptides having a modified peptide backbone. The term “domain” refers to any portion of a protein or polypeptide having a particular function or structure.

[0045] As used interchangeably herein, the terms “nucleic acid” and “polynucleotide” include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogues or modified versions thereof. These include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, unnatural, or derivatized nucleotide bases.

[0046] The terms "expression vector," "expression construct," or "expression cassette" refer to recombinant nucleic acids containing a desired coding sequence operably ligated to appropriate nucleic acid sequences necessary for the expression of an operably ligated coding sequence in a particular host cell or organism. Nucleic acid sequences required for expression in prokaryotes typically include promoters, operators (activating genes) (optional), ribosome binding sites, and other sequences. Eukaryotic cells are known to generally utilize promoters, enhancers (transcriptional enhancers), termination, and polyadenylation signals, and some elements may be removed and others added without sacrificing necessary expression.

[0047] The term "viral vector" refers to recombinant nucleic acid containing at least one element of viral origin and containing elements sufficient for, or that allow for, packaging into a viral vector particle. Vectors and / or particles can be used to transfer DNA, RNA, or other nucleic acids into cells, either ex vivo or in vivo. Many forms of viral vectors are well known.

[0048] With respect to proteins, nucleic acids, and cells, the term “isolated” typically includes proteins, nucleic acids, and cells that are relatively purified with respect to other cells or biological components that may be present in situ, and includes substantially pure preparations of proteins, nucleic acids, or cells, and substantially pure preparations thereof. The term “isolated” may also include proteins and nucleic acids that have no naturally occurring counterparts, or proteins or nucleic acids that are chemically synthesized and therefore not substantially contaminated by other proteins or nucleic acids. The term “isolated” may also include proteins, nucleic acids, or cells that have been separated or purified from most other cellular or biological components that naturally accompany them (e.g., other cellular proteins, nucleic acids, or cellular or extracellular components).

[0049] The term "wild type" includes entities that possess the structure and / or activity found in a normal state or context (as opposed to mutants, diseased, or modified). Wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0050] The term "endogenous sequence" refers to a nucleic acid sequence that is naturally present within a cell or animal. For example, an endogenous LEMD2 sequence in an animal refers to a naturally occurring LEMD2 sequence at the animal's LEMD2 locus.

[0051] Exogenous molecules or sequences include those that are not normally present in the cell in their original form, or those that are introduced into the cell from an external source. Normal presence refers to their presence in relation to specific developmental stages and environmental conditions of the cell. Exogenous molecules or sequences may include, for example, humanized versions of endogenous sequences, variant versions of corresponding endogenous sequences within the cell, or sequences that are intracellular but in a different form (i.e., not within a chromosome) corresponding to endogenous sequences. In contrast, endogenous molecules or sequences include those that are normally present in their original form in specific cells, at specific developmental stages, and under specific environmental conditions.

[0052] When used in the context of nucleic acids or proteins, the term “heterologous” indicates that the nucleic acid or protein contains at least two segments that do not naturally exist together within the same molecule. For example, when the term “heterologous” is used in reference to a nucleic acid segment or a protein segment, it indicates that the nucleic acid or protein contains two or more subsequences that are not found in nature in the same relationship to each other (e.g., bound together). As an example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid that is not found in nature in relation to other molecules, and is located within or bound to another nucleic acid molecule. For example, a heterologous region of a nucleic acid vector may contain a coding sequence adjacent to a heterologous promoter that is not found in relation to a coding sequence in nature. Similarly, a “heterologous” region of a protein is a segment of amino acids that is located within or bound to another peptide molecule that is not found in relation to other peptide molecules in nature (e.g., fusion proteins or tagged proteins). Similarly, nucleic acids or proteins may contain heterologous labeling, heterologous secretion, or heterologous localization sequences.

[0053] Codon optimization is a process that utilizes codon degeneracy, as indicated by the diversity of three-base-pair codon combinations that specify amino acids, to modify nucleic acid sequences for enhanced expression in specific host cells, generally by maintaining the natural amino acid sequence while replacing at least one codon in the natural sequence with a codon that is more or most frequently used in the host cell's gene. For example, the nucleic acid encoding the Cas9 protein may be modified to a more frequently used alternative codon in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell, compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, in a Codon Usage Database. These tables can be applied in various ways. See Nakamura et al., (2000), Nucleic Acids Research 28:292, which is incorporated herein by reference in its entirety. Computer algorithms (see, for example, Gene Forge) are also available for codon optimization of specific sequences for expression in a particular host.

[0054] A “promoter” is a regulatory region of DNA, typically containing a TATA box, which can instruct RNA polymerase II to initiate RNA synthesis at an appropriate transcription start site for a particular polynucleotide sequence. A promoter may further include other regions that affect the transcription initiation rate. The promoter sequences disclosed herein regulate the transcription of operably linked polynucleotides. Promoters may be active in one or more cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). Promoters may be, for example, constitutively active promoters, conditional promoters, inductive promoters, temporally restricted promoters (e.g., developmentally regulated promoters), or spatially restricted promoters (e.g., cell-specific or tissue-specific promoters). Examples of promoters can be found, for example, in PCT International Publication WO2013 / 176772, which is incorporated herein by reference in its entirety.

[0055] Constitutive promoters are those that are active in all tissues or in specific tissues at all developmental stages. Examples of constitutive promoters include human cytomegalovirus immediate early (hCMV), mouse cytomegalovirus immediate early (mCMV), human elongation factor 1 alpha (hEF1α), mouse elongation factor 1 alpha (mEF1α), mouse phosphoglycerate kinase (PGK), chicken beta actin hybrid (CAG or CBh), SV40 early, and beta-2 tubulin promoters.

[0056] Examples of inductive promoters include chemically controlled promoters and physically controlled promoters. Chemically controlled promoters include, for example, alcohol-controlled promoters (e.g., alcohol dehydrogenase (alcA) gene promoters), tetracycline-controlled promoters (e.g., tetracycline-responsive promoters, tetracycline operator sequence (tetO), tet-On promoters, or tet-Off promoters), steroid-controlled promoters (e.g., rat glucocorticoid receptor, estrogen receptor, or ecdysone receptor promoters), or metal-controlled promoters (e.g., metalloprotein promoters). Physically controlled promoters include, for example, temperature-controlled promoters (e.g., heat shock promoters) and light-controlled promoters (e.g., photo-inducible promoters or photo-inhibiting promoters).

[0057] Tissue-specific promoters can be, for example, neuronal cell-specific promoters.

[0058] Examples of developmentally controlled promoters include those that are active only during the embryonic stage of development or only in adult cells.

[0059] "Operatable linkage" or "operably linked" includes the parallel arrangement of two or more components (e.g., a promoter and another sequence element) such that both components function normally and that at least one of the components may mediate a function affecting at least one of the other components. For example, if a promoter controls the level of transcription of a coding sequence depending on the presence or absence of one or more transcription regulators, the promoter can be operably linked to the coding sequence. An operable linkage may include such sequences being in close proximity to each other or acting in trans (e.g., a regulatory sequence may act separately to control the transcription of a coding sequence).

[0060] The term "in vitro" includes artificial environments and processes or reactions occurring within artificial environments (e.g., test tubes or isolated cells or cell lines). The term "in vivo" includes natural environments (e.g., cells or organisms or bodies) and processes or reactions occurring within natural environments. The term "ex vivo" includes cells removed from the body of an individual and processes or reactions occurring within such cells.

[0061] A composition or method that “comprising” or “including” one or more of the enumerated elements may include other elements not specifically enumerated. For example, a composition that “comprises” or “includes” a protein may include the protein alone or in combination with other components. The transitional phrase “consisting essentially of” means that the scope of the claim is to be interpreted as encompassing the specific elements described in the claim and elements that do not substantially affect the basic and novel characteristics of the claimed invention. Therefore, the term “consisting essentially of” as used in the claims of the present invention is not intended to be interpreted as equivalent to “including.”

[0062] "Optional" or "optionally" means that the event or situation described thereafter may or may not occur, and that the description includes examples of cases in which the event or situation may occur and examples of cases in which it may not occur.

[0063] Specifying a range of values ​​includes all integers within or defining that range, and all subranges defined by integers within that range.

[0064] Unless otherwise specified in the context, the term "about" encompasses values ​​that are ±5% of the stated value.

[0065] The term "and / or" encompasses any and all possible combinations of one or more of the related enumerated items, and the absence of any combination when interpreted as an alternative ("or").

[0066] The term "or" refers to any one member of a given list, and also includes any combination of members of that list.

[0067] The singular articles "a," "an," and "the" include plural references unless the context explicitly specifies otherwise. For example, the terms "protein" or "at least one protein" can include multiple proteins, including mixtures thereof.

[0068] Statistically significant means p ≤ 0.05. (Modes for carrying out the invention)

[0069] I. Overview Microtubule-associated protein tau is an abundant component of neurons in the central nervous system and functions to maintain microtubule stability and promote axonal growth. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found to be hyperphosphorylated and aggregated in neurofibrillary hives. To gain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 gene screening for mutations that enhance tau aggregates. This screening yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promoted the accumulation of tau in phosphorylated and insoluble forms. In complementary screening, we identified three further genes, LEMD2, LEMD3, and CHMP7, which provide protection against tau aggregates when overexpressed. The proteins encoded by these identified genes are involved in the maintenance and repair of the nuclear membrane. These results suggest disruption of nuclear membrane integrity as a possible initiating event in tauopathy, revealing a new target for therapeutic intervention.

[0070] This specification provides a method for inhibiting tau aggregates in cells or subjects, comprising administering to cells or subjects a LEM domain-containing protein 2 (LEMD2), charged multivesicular protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or a nucleic acid encoding LEMD2, CHMP7, or LEMD3. This specification also provides a method for reducing phosphorylation (e.g., phosphorylation on serine 356) in cells or subjects, comprising administering to cells or subjects LEMD2, CHMP7, or LEMD3, or a nucleic acid encoding LEMD2, CHMP7, or LEMD3. This specification also provides a method for reducing serum neuronal filament light chains (sNfL) in subjects or preventing the accumulation of serum neuronal filament light chains (sNfL), comprising administering to subjects LEMD2, CHMP7, or LEMD3, or a nucleic acid encoding LEMD2, CHMP7, or LEMD3. This specification also provides a method for treating tauopathy in a subject, comprising administering to the subject LEMD2, CHMP7, or LEMD3, or a nucleic acid encoding LEMD2, CHMP7, or LEMD3, wherein LEMD2, CHMP7, or LEMD3 inhibits tau aggregates in the subject's cells. Also provided are nucleic acids encoding LEMD2, CHMP7, or LEMD3 (e.g., present in an expression construct and operably linked to a heterologous promoter), constructs containing nucleic acids, vectors containing nucleic acids or constructs, lipid nanoparticles containing nucleic acids, constructs, or vectors, and cells or subjects (e.g., animals) containing nucleic acids, constructs, vectors, or lipid nanoparticles.

[0071] II. Methods for inhibiting tau aggregates and methods for treating or preventing tauopathy This specification provides methods for inhibiting tau aggregates in cells or subjects. Such methods may include administering LEM domain-containing protein 2 (LEMD2), charged multivesicular protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3) (i.e., exogenous LEMD2, CHMP7, or LEMD3) to cells, or such methods may include administering nucleic acids encoding LEMD2, CHMP7, or LEMD3 (i.e., exogenous nucleic acids encoding LEMD2, CHMP7, or LEMD3) to cells or subjects so that LEMD2, CHMP7, or LEMD3 is expressed. This specification also provides a method for reducing phosphorylation (e.g., phosphorylation on serine 356) in cells or subjects, comprising administering LEMD2, CHMP7, or LEMD3, or a nucleic acid encoding LEMD2, CHMP7, or LEMD3 (i.e., an exogenous nucleic acid encoding LEMD2, CHMP7, or LEMD3) to cells or subjects. Phosphorylated tau may be, for example, phosphorylated tau (S356) or phosphorylated tau AT8 (S202, T205). Methods for reducing or preventing the accumulation of serum neuronal filament light chains (sNfL) in subjects are also provided. Such methods may involve administering LEMD2, CHMP7, or LEMD3, or a nucleic acid encoding LEMD2, CHMP7, or LEMD3, to subjects. Methods for treating tauopathy in subjects are also provided. Such a method may include administering LEMD2, CHMP7, or LEMD3 (i.e., exogenous LEMD2, CHMP7, or LEMD3) to a subject, or such a method may include administering a nucleic acid encoding LEMD2, CHMP7, or LEMD3 (i.e., an exogenous nucleic acid encoding LEMD2, CHMP7, or LEMD3) to a subject so that LEMD2, CHMP7, or LEMD3 is expressed. LEMD2, CHMP7, or LEMD3 then inhibits tau aggregates in the subject (e.g., in the subject's cells or in one or more cells of the subject).Methods for preventing tauopathy in a subject are also provided. Such methods may include administering LEMD2, CHMP7, or LEMD3 (i.e., exogenous LEMD2, CHMP7, or LEMD3) to the subject, or such methods may include administering a nucleic acid encoding LEMD2, CHMP7, or LEMD3 (i.e., an exogenous nucleic acid encoding LEMD2, CHMP7, or LEMD3) to the subject so that LEMD2, CHMP7, or LEMD3 is expressed. LEMD2, CHMP7, or LEMD3 then inhibits tau aggregates in the subject (e.g., in the subject's cells or in one or more cells of the subject).

[0072] Microtubule-associated protein tau (neurofibrillary tangle protein, paired helical filament-tau (PHF-tau), or simply tau) is a protein that promotes microtubule assembly and stability. It is primarily expressed in nerve cells and preferentially localizes to axonal compartments. Tau is encoded by the MAPT gene (also known as MAPTL, MTBT1, TAU, or MTAPT). Tau plays a role in stabilizing microtubules in nerve cells and thus promoting axon elongation. In humans, it is represented as a set of six isoforms, specifically spliced ​​from the transcript of a single gene located on chromosome 17. Each tau isoform contains a series of 3 / 4 tandem repeat units (which vary by isoform) that bind to and stabilize microtubules. The microtubule-binding repeat region of tau can be phosphorylated by various kinases and is adjacent to serine / threonine-rich regions associated with tau hyperphosphorylation in a family of related neurodegenerative diseases called tauopathies.

[0073] Tau protein, known in humans as MAPT (microtubule-associated protein tau), is a product of alternative splicing from a single gene. The tau repeat domain carries sequence motifs involved in aggregation (i.e., it is the agglutinative domain from tau). Depending on the splicing, the repeat domain of the tau protein has either three or four repeat regions that constitute the protein's agglutinative core, often referred to as the Repeat Domain (RD). Specifically, the tau repeat domain represents the microtubule-binding core and contains the R2 and R3 hexapeptide motifs involved in tau aggregates. The human brain contains six tau isoforms ranging in length from 352 to 441 amino acids. These isoforms vary at the carboxyl terminus depending on the presence or absence of one or two insertion domains at the amino terminus, as well as the presence of either three or four repeat domains (R1-R4). The repeating domain located on the carboxyl terminal half of tau is thought to be important for microtubule binding and for the pathological aggregation of tau to paired helical filaments (PHFs), which are core components of the neurofibrillary condensation seen in tauopathy.

[0074] In some of the above methods for inhibiting tau aggregates, reducing phosphorylation, treating tauopathy, or preventing tauopathy, the method comprises administering LEMD2 or a nucleic acid encoding LEMD2 to cells or a subject. In some of the above methods for reducing serum neuronal filament light chains (sNfLs) or preventing the accumulation of serum neuronal filament light chains (sNfLs), the method comprises administering LEMD2 or a nucleic acid encoding LEMD2 to a subject. For example, LEMD2 may be wild-type LEMD2. LEMD2 is involved in organizing nuclear structures and is necessary to maintain the integrity of the nuclear membrane.

[0075] For example, LEMD2 is human LEMD2. Human LEMD2 (also called LEM domain-containing protein 2, hLEM2, or LEM domain nuclear membrane protein 2) is assigned UniProt reference number Q8NC56. The human gene encoding LEMD2 (LEMD2, or LEM domain nuclear membrane protein 2) is assigned NCBI GeneID221496 and is found at position 6p21.31 on chromosome 6 (construct: GRCh38.p13 (GCF_000001405.39); position: NC_000006.12 (33771213.33794274, complement)). At least two isoforms of human LEMD2 are known. The first isoform is 503 amino acids and is assigned UniProt reference number Q8NC56-1 and NCBI reference number NP_851853.1 (Sequence ID 1). An exemplary coding sequence is assigned the reference number CCDS4785.1 (SEQ ID NO: 2), and one exemplary mRNA (cDNA) sequence is assigned the reference number NM_181336.4 (SEQ ID NO: 4). A codon-optimized coding sequence to distinguish it from natural CDS is described in SEQ ID NO: 3. The second isoform is 201 amino acids and is assigned the UniProt reference number Q8NC56-2 and the NCBI reference numbers NP_001137416.1 and NP_001335638.1 (SEQ ID NO: 5). An exemplary coding sequence is assigned the reference number CCDS47411.1 (SEQ ID NO: 6), and exemplary mRNA (cDNA) sequences are assigned the reference numbers NM_001143944.1 and NM_001348709.2 (SEQ ID NO: 8 and SEQ ID NO: 9, respectively). A codon-optimized coding sequence to distinguish it from natural CDS is described in SEQ ID NO: 7.

[0076] In another example, LEMD2 is mouse LEMD2. Mouse LEMD2 is assigned UniProt reference number Q6DVA0. The mouse gene encoding LEMD2 (LEMD2) is assigned NCBI GeneID224640 and is found on chromosome 17 at position 17;17 A3.3 (construct: GRCm39 (GCF_000001635.27); position: NC_000083.7 (27408574.27426228, complement)). The exemplary mouse LEMD2 is 511 amino acids and is assigned UniProt reference number Q6DVA0-1 and NCBI reference number NP_666187.2 (sequence number 10). An exemplary coding sequence is assigned the reference number CCDS50043.1 (sequence number 11), and one exemplary mRNA (cDNA) sequence is assigned the reference number NM_146075.2 (sequence number 14). Codon-optimized coding sequences to distinguish them from natural CDS are described in sequence numbers 12 and 13. Another codon-optimized coding sequence to distinguish them from natural CDS is described in sequence number 255.

[0077] In some of the above methods for inhibiting tau aggregates, reducing phosphorylation, treating tauopathy, or preventing tauopathy, the method comprises administering CHMP7 or a nucleic acid encoding CHMP7 to cells or a subject. In some of the above methods for reducing serum neuronal filament light chains (sNfLs) or preventing the accumulation of serum neuronal filament light chains (sNfLs), the method comprises administering CHMP7 or a nucleic acid encoding CHMP7 to a subject. For example, CHMP7 may be wild-type CHMP7. CHMP7 is an ESCRT-III-like protein required to recruit the ESCRT-III complex to the nuclear membrane in late epoch. Together with SPAST, the ESCRT-III complex promotes nuclear membrane sealing and mitotic spindle degradation during late epoch. CHMP7 also plays a role in the endosomal sorting pathway.

[0078] For example, CHMP7 is human CHMP7. Human CHMP7 (also known as charged polyspheric protein 7 or chromatin-modified protein 7) is assigned UniProt reference number Q8WUX9. The human gene encoding CHMP7 (CHMP7) is assigned NCBI GeneID91782 and is found at position 8p21.3 on chromosome 8 (construct: GRCh38.p13 (GCF_000001405.39); position: NC_000008:11 (23243637..23262000)). The exemplary CHMP7 protein consists of 453 amino acids and is assigned UniProt reference number Q8WUX9-1 and NCBI reference number NP_689485.1 (sequence number 15). An exemplary coding sequence is assigned the reference number CCDS6040.1 (sequence number 16), and one exemplary mRNA (cDNA) sequence is assigned the reference number NM_152272.5 (sequence number 18). A coding sequence with optimized codons to distinguish it from natural CDS is described in sequence number 17.

[0079] In another example, CHMP7 is mouse CHMP7. Mouse CHMP7 is assigned UniProt reference number Q8R1T1. The mouse gene encoding CHMP7 (CHMP7) is assigned NCBI GeneID105513 and is found on chromosome 14 at position 14;14 D2 (construct: GRCm39 (GCF_000001635.27); position: NC_000080.7 (69954428.69970019, complement)). The exemplary mouse CHMP7 is 451 amino acids and is assigned UniProt reference number Q8R1T1-1 and NCBI reference number NP_598839.2 (sequence number 19). An exemplary coding sequence is assigned the reference number CCDS27242.1 (sequence number 20), and one exemplary mRNA (cDNA) sequence is assigned the reference number NM_134078.4 (sequence number 22). A coding sequence with optimized codons to distinguish it from natural CDS is described in sequence number 21.

[0080] In some of the above methods for inhibiting tau aggregates, reducing phosphorylation, treating tauopathy, or preventing tauopathy, the method comprises administering LEMD3 or a nucleic acid encoding LEMD3 to cells or a subject. In some of the above methods for reducing serum neuronal filament light chains (sNfLs) or preventing the accumulation of serum neuronal filament light chains (sNfLs), the method comprises administering LEMD3 or a nucleic acid encoding LEMD3 to a subject. For example, LEMD3 may be wild-type LEMD3.

[0081] For example, LEMD3 is human LEMD3. Human LEMD3 (also called the inner nuclear membrane protein Man1 or LEM domain-containing protein 3) is assigned UniProt reference number Q9Y2U8. The human gene encoding LEMD3 (LEMD3, MAN1, or LEM domain-containing protein 3) is assigned NCBI GeneID23592 and is found at position 12q14.3 on chromosome 12 (construct: GRCh38.p13 (GCF_000001405.39); position: NC_000012.12 (65169583..65248355)). The exemplary LEMD3 protein consists of 911 amino acids and is assigned UniProt reference number Q9Y2U8-1 and NCBI reference number NP-055134.2 (SEQ ID NO: 23). An example coding sequence is assigned the reference number CCDS8972.1 (sequence number 24), and one example mRNA (cDNA) sequence is assigned the reference number NM_014319.5 (sequence number 26). A coding sequence with optimized codons to distinguish it from natural CDS is described in sequence number 25.

[0082] In another example, LEMD3 is mouse LEMD3. Mouse LEMD3 is assigned UniProt reference number Q9WU40. The mouse gene encoding LEMD3 (Lemd3) is assigned NCBI GeneID380664 and is found on chromosome 10 at position 10;10 D2 (construct: GRCm39(GCF_000001635.27); position: NC_000076.7(120759316..120815491, complement)). The exemplary mouse LEMD3 is 921 amino acids and is assigned UniProt reference number Q9WU40-1 (sequence number 27). The coding sequence with codons optimized to distinguish it from natural CDS is described in sequence number 28. Another exemplary mouse sequence, LEMD3, is 918 amino acids long and has been assigned NCBI reference number NP_001074662.2 (sequence number 29). An exemplary coding sequence has been assigned reference number CCDS48703.1 (sequence number 30), and one exemplary mRNA (cDNA) sequence has been assigned reference number NM_001081193.2 (sequence number 31).

[0083] Some such methods involve administering a nucleic acid encoding LEMD2, a nucleic acid encoding CHMP7, or a nucleic acid encoding LEMD3 to a cell or subject. The nucleic acid may be a nucleic acid construct, which is described in more detail elsewhere herein. In some cases, the nucleic acid encoding LEMD2, CHMP7, or LEMD3 may be a native coding sequence. In other cases, it may be codon-optimized (e.g., codon-optimized for expression in humans or mice). For example, the nucleic acid may be modified to alternative codons that are more frequently used in human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cells of interest.

[0084] The nucleic acid encoding LEMD2, CHMP7, or LEMD3 may be DNA or RNA. The nucleic acid may, in some cases, be messenger RNA (mRNA) encoding LEMD2, CHMP7, or LEMD3. The nucleic acid may, in some cases, be complementary DNA (cDNA) encoding LEMD2, CHMP7, or LEMD3. For example, such a nucleic acid may consist only of a coding sequence without any intervening introns. In other cases, the nucleic acid may contain one or more introns that separate the exons in the LEMD2, CHMP7, or LEMD3 coding sequence. For example, the nucleic acid may contain a LEMD2, CHMP7, or LEMD3 genomic sequence containing both exons and introns.

[0085] In some cases, the nucleic acid is present in an expression construct containing a nucleic acid encoding LEMD2, CHMP7, or LEMD3 operably ligated to a promoter. The promoter can be any suitable promoter for in vivo expression in animals or in vitro expression in isolated cells. The promoter can be a constitutively active promoter (e.g., a CAG promoter or a U6 promoter), a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Such promoters are well known and discussed elsewhere in this specification. In certain cases, the promoter is active in nerve cells. In some cases, the promoter is a heterologous promoter (i.e., a promoter in which the LEMD2, CHMP7, or LEMD3 nucleic acid is not operably ligated in nature). In other cases, the promoter may be an endogenous promoter (i.e., LEMD2 nucleic acid operably ligated to the LEMD2 promoter, CHMP7 nucleic acid operably ligated to the CHMP7 promoter, or LEMD3 nucleic acid operably ligated to the LEMD3 promoter). Heterogeneous promoters may be any type of promoter disclosed elsewhere in this specification. For example, the promoter may be a constitutive promoter, such as the EF1α promoter. Alternatively, the promoter may be a tissue-specific promoter or an inducible promoter. For example, as another example, the promoter may be a neuron-specific promoter. An example of a suitable neuron-specific promoter that is highly specific at low levels of expression is the synapsin-1 promoter (e.g., a human synapsin-1 promoter, such as the promoter described in SEQ ID NO: 44). In some embodiments, the synapsin-1 promoter may be used in conjunction with hemoglobin subunit beta (HBB) intron 2 (e.g., downstream of the synapsin-1 promoter), such as the one described in SEQ ID NO: 254. The inclusion of this element can enhance gene expression.

[0086] The nucleic acids and expression constructs disclosed herein may also include post-transcriptional regulatory elements, such as the post-transcriptional regulatory elements of woodchuck hepatitis virus.

[0087] Nucleic acids and expression constructs may further include one or more polyadenylation signal sequences. For example, a nucleic acid construct may include a polyadenylation signal sequence located at 3' of the LEMD2, CHMP7, or LEMD3 coding sequence. Any suitable polyadenylation signal sequence may be used. The term polyadenylation signal sequence refers to any sequence that directs the termination of transcription and the addition of a poly(A) tail to the mRNA transcript. In eukaryotes, the transcription terminator is recognized by a protein factor, and after termination, polyadenylation follows, which is the process of adding a poly(A) tail to the mRNA transcript in the presence of poly(A) polymerase. Mammalian poly(A) signals typically consist of a core sequence about 45 nucleotides long, flanked by a variety of auxiliary sequences that help to enhance the efficiency of cleavage and polyadenylation. The core sequence, referred to as the poly-A recognition motif or poly-A recognition sequence, consists of a highly conserved upstream element (AATAAA or AAUAAA) within mRNA that is recognized by a cleavage and polyadenylation-specificity factor (CPSF), and a downstream region (U or G and U-rich) that is poorly defined and bound by a cleavage stimulation factor (CstF). Examples of transcription terminators that can be used include, for example, the human growth hormone (HGH) polyadenylation signal, the Simian virus 40 (SV40) late polyadenylation signal, the rabbit betaglobin polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, the phosphoglycerate kinase (PGK) polyadenylation signal, the AOX1 transcription termination sequence, the CYC1 transcription termination sequence, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells. Examples of suitable polyadenylation signals include, for example, those described in SEQ ID NOs. 252 and 253.

[0088] Nucleic acids and expression constructs may also optionally include a polyadenylation signal sequence upstream of the LEMD2, CHMP7, or LEMD3 coding sequence. The polyadenylation signal sequence upstream of the LEMD2, CHMP7, or LEMD3 coding sequence may be adjacent to a recombinase recognition site recognized by a site-specific recombinase. In some constructs, the recombinase recognition site is also adjacent to a selection cassette containing, for example, the coding sequence for a drug-resistant protein. In some constructs, the recombinase recognition site is not adjacent to a selection cassette. The polyadenylation signal sequence interferes with the transcription and expression of the protein or RNA encoded by the coding sequence. However, upon exposure to a site-specific recombinase, the polyadenylation signal sequence may be excised, allowing for the expression of the protein or RNA.

[0089] Such a configuration may enable tissue-specific or developmental-stage-specific expression when the polyadenylation signal sequence is excised in a tissue-specific or developmental-stage-specific manner. Tissue-specific or developmental-stage-specific excision of the polyadenylation signal sequence can be achieved if the animal containing the nucleic acid or expression construct further includes a coding sequence for a site-specific recombinase operably linked to the tissue-specific or developmental-stage-specific promoter. The polyadenylation signal sequence is then excised only in those tissues or developmental stages, enabling tissue-specific or developmental-stage-specific expression. In one embodiment, LEMD2, CHMP7, or LEMD3 encoded by the nucleic acid or expression construct may be expressed in a neuronal-cell-specific manner.

[0090] Site-specific recombinases include enzymes that can promote recombination between recombinase recognition sites where two recombination sites are physically separated within a single nucleic acid or on separate nucleic acids. Examples of recombinases include Cre, Flop, and Dre recombinases. An example of a Cre recombinase gene is Crei, in which the two exons encoding Cre recombinase are separated by an intron, preventing expression in prokaryotic cells. Such recombinases may further include nuclear localization signals to promote nuclear localization (e.g., NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by site-specific recombinases and can function as substrates for recombination events. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2, and lox5171.

[0091] The nucleic acids disclosed herein may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single-stranded or double-stranded, and may be linear or circular in form. Nucleic acid constructs may be naked nucleic acids, as described elsewhere herein, or may be delivered by vectors such as AAV vectors. In the linear form, the ends of the nucleic acid may be protected by known methods (e.g., from exonuclease degradation). For example, one or more dideoxynucleotide residues may be added to the 3' end of a linear molecule, and / or self-complementary oligonucleotides may be attached to one or both ends. For example, see Chang et al., (1987) "Proc. Natl. Acad. Sci. USA" 84:4959-4963, and Nehls et al., (1996) "Science" 272:886-889, each of which is incorporated herein by reference in whole. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified nucleotide-nucleotide bonds such as phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues. A nucleic acid or expression construct may, in some cases, contain one or more of the following terminal structures: hairpins, loops, inverted terminal repeats (ITRs), or toroids. For example, a nucleic acid or expression construct may contain an ITR.

[0092] Nucleic acid or expression constructs may include modifications or sequences that provide additional desirable features (e.g., modified or regulated stability, tracking or detection by fluorescent labeling, or binding sites for proteins or protein complexes). A nucleic acid construct may include one or more fluorescent labels, purification tags, epitope tags, or combinations thereof. For example, a nucleic acid construct may include one or more fluorescent labels (e.g., fluorescent proteins or other fluorophores or dyes), such as at least one, at least two, at least three, at least four, or at least five fluorescent labels. Exemplary fluorescent labels include fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, 5-(and-6)-carboxytetramethylrhodamine (TAMRA), and fluorophores such as Cy7. A wide range of fluorescent dyes for labeling oligonucleotides are commercially available (e.g., from Integrated DNA Technologies). The label or tag may be located at the 5' end, 3' end, or inside the nucleic acid construct. For example, the nucleic acid construct may be conjugated at the 5' end having an IR700 fluorophore (5'IRDYE® 700) from Integrated DNA Technologies.

[0093] Nucleic acids and expression constructs may also include conditional alleles. Conditional alleles may be multifunctional alleles, as described in U.S. Patent No. 2011 / 0104799, which is incorporated herein by reference in its entirety. For example, a conditional allele may include (a) a sense-oriented working sequence with respect to the transcription of a target gene, (b) a sense or antisense-oriented drug selection cassette (DSC), (c) a nucleotide sequence of interest (NSI) for antisense orientation, and (d) a conditional by inversion module (COIN, utilizing an exon-splitting intron and an invertable gene-trap-like module). See, for example, U.S. Patent No. 2011 / 0104799. The conditional allele may further include a recombinable unit that recombines upon exposure to a first recombinase to form a conditional allele that (i) lacks an activator sequence and a DSC, and (ii) contains a sense-directed NSI and an antisense-directed COIN. See, for example, U.S. Patent No. 2011 / 0104799.

[0094] Nucleic acids and expression constructs may also contain polynucleotides encoding a selection marker. Alternatively, nucleic acids and expression constructs may lack polynucleotides encoding a selection marker. The selection marker may be contained in a selection cassette. Optionally, the selection cassette may be a self-deletion cassette. For example, in this application, each of these is referenced to U.S. Patents 8,697,851 and 2013 / 0312129, which are incorporated herein by reference in their entirety. As an example, a self-deletion cassette may include a Crei gene (containing two exons encoding Cre recombinase separated by an intron) operably linked to a mouse Prm1 promoter and a neomycin resistance gene operably linked to a human ubiquitin promoter. By using the Prm1 promoter, the self-deletion cassette can be specifically deleted in the germ cells of male F0 animals. An exemplary selection marker is neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puro r ), blasticidine S deaminase (bsr r Examples include xanthine / guanine phosphoribosyltransferase (GPT), herpes simplex virus thymidine kinase (HSV-K), or combinations thereof. The polynucleotide encoding the selection marker can be operably ligated to an active promoter in the target cell. Examples of promoters are described elsewhere in this specification.

[0095] Nucleic acids or expression constructs may also include reporter genes. Exemplary reporter genes include those encoding luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, and alkaline phosphatase. Such reporter genes can be operably linked to active promoters in the target cells. Examples of promoters are described elsewhere in this specification.

[0096] Nucleic acids or expression constructs may be present in vectors, such as viral vectors. Vectors may contain additional sequences, such as origins of replication, promoters, and genes encoding antibiotic resistance.

[0097] Some vectors may be circular, while others may be linear. Vectors may be packaged to be delivered via lipid nanoparticles, liposomes, non-lipid nanoparticles, or viral capsids. Non-exclusive exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0098] The nucleic acid or expression construct may be in a vector such as a viral vector. The viral vector may be, for example, an adeno-associated virus (AAV) vector or a lentivirus (LV) vector (i.e., a recombinant AAV vector or a recombinant LV vector). Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. The virus can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. The virus can integrate into the host genome, or alternatively, does not integrate into the host genome. Such viruses can also be engineered to have reduced immunogenicity. The virus can be replication-competent or replication-defective (e.g., defective in one or more genes necessary for additional rounds of virion replication and / or packaging). The virus can cause transient expression, long-term expression (e.g., for at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or persistent expression. Exemplary viral titers (e.g., AAV titers) include about 10 12 , about 10 13 , about 10 14 , about 10 15 , and about 10 16 vector genomes / mL, which are mentioned as examples. Other exemplary viral titers (e.g., AAV titers) include about 10 12 , about 10 13 , about 10 14 , about 10 15 , and about 10 16 vector genomes (vg) / kg body weight, which are mentioned as examples.

[0099] In one example, the nucleic acid or expression construct is present in the AAV vector. AAV can be any suitable serotype and may be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). The ssDNA AAV genome consists of two open reading frames, Rep and Cap, with two adjacent terminal inversion sequences that enable the synthesis of complementary DNA strands. When constructing an AAV transfer plasmid, the transgene is placed between two ITRs, and Rep and Cap may be supplied trans. In addition to Rep and Cap, AAV may require a helper plasmid containing adenovirus-derived genes. These genes (E4, E2a, and VA) mediate AAV replication. For example, the transfer plasmid, Rep / Cap, and helper plasmid can be transfected into HEK293 cells containing the adenovirus gene E1+ to generate infectious AAV particles. Alternatively, the Rep, Cap, and adenovirus helper genes may be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses, such as retroviruses.

[0100] Multiple serotypes of AAV have been identified. These serotypes differ in the types of cells they infect (i.e., their targeting), enabling preferential transduction of specific cell types. Examples of serotypes for CNS tissues include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. For the selectivity of AAV serotypes for gene delivery in nerve cells, see, for example, Hammond et al., (2017) "PLoS One" 12(12):e0188830, which is incorporated in its entirety herein by reference. In specific examples, the AAV-PHP.eB vector is used. The AAV-PHP.eB vector exhibits a high ability to cross the blood-brain barrier, increasing its CNS transduction efficiency. In specific examples, the AAV9 vector is used.

[0101] Directionality can be further refined by pseudotyping, which is a mixture of capsids and genomes from different viral serotypes. For example, AAV2 / 5 represents a virus containing a serotype 2 genome packaged in a serotype 5 capsid. The use of pseudotyped viruses can not only improve transduction efficiency but also alter directionality. Furthermore, viral directionality can be modified using hybrid capsids derived from different serotypes. For example, AAV-DJ contains a hybrid capsid derived from eight serotypes and exhibits high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that exhibits the characteristics of AAV-DJ but with enhanced uptake into the brain. AAV serotypes can also be modified by mutation. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V. Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of AAV6 mutations include S663V and T492V. Other pseudotypes / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0102] To accelerate the expression of a transgene, self-complementary AAV (scAAV) variants may be used. Because AAV relies on the cell's DNA replication mechanism to synthesize the complementary strand of its single-stranded DNA genome, transgene expression may be delayed. To address this delay, scAAVs containing complementary sequences that can spontaneously anneal upon infection may be used, eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors may also be used.

[0103] To increase packaging capacity, longer transgenes may be split into two AAV transfer plasmids, the first being a 3' splice donor and the second a 5' splice acceptor. During cell co-infection, these viruses can form concatemers and splice together to express the full-length transgene. This allows for the expression of longer transgenes, but at a reduced efficiency. A similar method for increasing capacity utilizes homologous recombination. For example, the transgene may be split into two transfer plasmids, but with substantial sequence overlap so that co-expression induces homologous recombination and expression of the full-length transgene.

[0104] In some cases, LEMD2, CHMP7, or LEMD3, or nucleic acids encoding LEMD2, CHMP7, or LEMD3, associate with lipid nanoparticles. Lipid formulations can protect biomolecules from degradation while simultaneously improving their uptake into cells. Lipid nanoparticles are particles containing multiple lipid molecules physically bound to each other by intermolecular forces. These include microspheres (monolayer and multilayer vesicles, e.g., liposomes), dispersed phases in emulsions, micelles, or inner phases in suspensions. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or amphoteric lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the time nanoparticles can remain in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in PCT International Publication WO2016 / 010840A1, which is incorporated in its entirety herein by reference. Exemplary lipid nanoparticles may comprise a cationic lipid and one or more other components. In one example, the other components may comprise a helper lipid such as cholesterol. In another example, the other components may comprise a helper lipid such as cholesterol and a neutral lipid such as DSPC. In yet another example, the other components may comprise a helper lipid such as cholesterol, any neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033.

[0105] LNPs may contain one or more or all of the following: (i) lipids for encapsulation and endosomal escape, (ii) neutral lipids for stabilization, (iii) helper lipids for stabilization, and (iv) stealth lipids. See, for example, Farnleitner et al., (2018) "Cell Rep." 22(9):2227-2235, and PCT International Publication No. WO2017 / 173054A1, each of which is incorporated herein by reference in whole. Specific examples of the use of LNPs for delivery to the brain are disclosed in Nabhan et al., (2016), "Sci.Rep." 6:20019, which is incorporated herein by reference in whole.

[0106] LEMD2, CHMP7, LEMD3, or nucleic acids encoding LEMD2, CHMP7, or LEMD3 may be administered to cells or subjects by any suitable means. Various methods and compositions are provided herein for enabling the introduction of molecules (e.g., nucleic acids or proteins) into cells or subjects.

[0107] The methods provided herein do not depend on any specific method for introducing nucleic acids or proteins into cells, but only on the ability of the nucleic acids or proteins to access the inside of the cell. Methods for introducing nucleic acids and proteins into various cell types are well known in the art and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0108] Transfection protocols, as well as protocols for introducing molecules (e.g., nucleic acids or proteins) into cells, may vary. Non-limiting transfection methods include chemical transfection methods using liposomes; nanoparticles; calcium phosphate (Graham et al., (1973) "Virology" 52(2):456-67; Bacchetti et al., (1977) "Proc. Natl. Acad. Sci. USA" 74(4):1590-4; and Kriegler, M (1991). Transcription and Expression: A Laboratory Manual. New York: WH Freeman and Company. pp9697, each of which is incorporated herein by reference in whole); dendrimers; or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation, and phototransfection. Examples of particle-based transfection include the use of gene guns or magnet-assisted transfection (Bertram (2006) "Current Pharmaceutical Biotechnology 7", 277-28, the entire text of which is incorporated herein by reference). Viral methods may also be used for transfection.

[0109] The introduction of molecules (e.g., nucleic acids or proteins) into cells can also be mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection. Nucleofection is an improved electroporation technique that allows delivery of nucleic acid substrates not only to the cytoplasm but also to the nucleus across the nuclear membrane. The use of nucleofection in the methods disclosed herein typically requires far fewer cells than conventional electroporation (e.g., only about 2 million compared to 7 million for conventional electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR® system.

[0110] The introduction of molecules (e.g., nucleic acids or proteins) into cells can also be achieved by microinjection. Microinjection of mRNA is preferably into the cytoplasm (e.g., to deliver mRNA directly to the translation mechanism), while microinjection of proteins or protein-coding DNA is preferably into the nucleus. Alternatively, microinjection can be performed by injection into both the nucleus and the cytoplasm, where the needle is first introduced into the nucleus and the first volume is injected, and then the second volume is injected into the cytoplasm while the needle is removed from the cell. Methods for performing microinjection are well known. For example, see Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, "Manipulating the Mouse Embryo," Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), Meyer et al. (2010) "Proc. Natl. Acad. Sci. USA" 107:15022-15026, and Meyer et al. (2012) "Proc. Natl. Acad. Sci. USA" 109:9354-9359, which are incorporated herein by reference in their entirety.

[0111] Other methods for introducing molecules (e.g., nucleic acids or proteins) into cells include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-permeable peptide-mediated delivery, or implantable device-mediated delivery. Methods for administering nucleic acids or proteins to subjects for in vivo cell modification are disclosed elsewhere in this specification. Specifically, molecules (e.g., nucleic acids or proteins) may be introduced into cells or non-human animals in carriers such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid spirals, or lipid microtubules. Some specific examples of delivery to non-human animals include hydrodynamic delivery, viral delivery (e.g., lentiviral delivery, or adeno-associated virus (AAV) delivery), and lipid nanoparticle-mediated delivery.

[0112] In one example, LEMD2, CHMP7, LEMD3, or nucleic acids encoding LEMD2, CHMP7, or LEMD3 may be administered via viral transduction, such as lentiviral transduction or adeno-associated virus transduction. In another example, LEMD2, CHMP7, LEMD3, or nucleic acids encoding LEMD2, CHMP7, or LEMD3 may be administered via lipid nanoparticle (LNP) mediated delivery.

[0113] In vivo administration may be via any preferred route that allows LEMD2, CHMP7, LEMD3, or the nucleic acid encoding LEMD2, CHMP7, or LEMD3 to reach the intended target cell(s) (e.g., nerve cells in the subject's brain) or target tissue (e.g., the brain). Examples of administration routes include parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Systemic administration modes include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intra-arterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. A specific example is intravenous infusion. Intranasal infusion and intravitreous injection are other specific examples. Local administration routes include, for example, intrathecal, intraventricular, intraparenchymal (local intraparenchymal delivery to the striatum (e.g., caudate nucleus or putamen), cerebral cortex, anterior central gyrus, hippocampus (e.g., dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, thalamus, tectum, putamen, or nigra parenchyma), intraocular, intraorbital, subconjunctival, intravitreous, subretinal, and scleral pathways. Significantly smaller amounts of components (compared to systemic approaches) may be more effective when administered locally (e.g., intraparenchymal or intravitreous) than when administered systemically (e.g., intravenously). Local administration routes may also reduce or eliminate the incidence of potentially toxic side effects that may occur when a therapeutically effective dose of the component is administered systemically. For example, LEMD2, CHMP7, LEMD3, or nucleic acids encoding LEMD2, CHMP7, or LEMD3 may be administered directly to the subject's brain or nerve cells within the subject's brain. In certain cases, administration to the subject is by intrathecal or intracranial injection (e.g., stereotactic surgery for injection into the hippocampus and other brain regions, or intraventricular injection). In certain cases, administration to the subject is by intraventricular injection. In another specific case, administration to the subject is by intracranial injection. In yet another specific case, administration to the subject is by intrathecal injection.

[0114] The frequency and number of administrations may depend, among other factors, particularly on the half-life of the administered composition and the route of administration. The introduction of nucleic acids or proteins into cells or non-human animals may be carried out once or multiple times over a period of time. For example, the introduction may be carried out at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, at least sixteen times, at least seventeen times, at least eighteen times, at least nineteen times, or at least twenty times over a period of time.

[0115] The cells or subjects in this method may be, for example, mammals, non-human mammals, and humans. Mammals may be, for example, non-human mammals, humans, rodents, rats, mice, or hamsters. Other non-human mammals include, for example, non-human primates, monkeys, apes, cats, dogs, rabbits, horses, bulls, deer, bison, and domestic animals (e.g., cattle breeds such as cattle and steers, sheep breeds such as sheep and goats, and pig breeds such as pigs and wild boars). The term "non-human" excludes humans. In specific examples, the cells or subjects are human.

[0116] The cells may be isolated cells (e.g., in vitro) or in vivo within a subject (e.g., an animal or mammal). The cells may also be in any type of undifferentiated or differentiated state. For example, the cells may be nerve cells.

[0117] The cells provided herein may be normal and healthy cells, or they may be diseased cells containing tau aggregates. For example, cells may have a tendency towards tau aggregates, or they may have pre-existing tau aggregates.

[0118] In one example, the cells are human cells, rodent cells, mouse cells, or rat cells, e.g., human nerve cells, rodent nerve cells, mouse nerve cells, or rat cells. In a specific example, the cells are human nerve cells. In a particular example, the cells are present in the subject in vivo (e.g., nerve cells in the subject's brain). For example, such a method could be a method of inhibiting tau aggregates or a method of reducing phosphorylation in the subject's cells (e.g., nerve cells in the subject's brain).

[0119] Such methods may further include screening cells or subjects to confirm the presence of LEMD2, CHMP7, LEMD3, or nucleic acids encoding LEMD2, CHMP7, or LEMD3. Screening cells or subjects for LEMD2, CHMP7, LEMD3, or nucleic acids encoding LEMD2, CHMP7, or LEMD3 can be carried out by any well-known means. Such methods may further include screening cells or subjects to confirm the expression of nucleic acids encoding LEMD2, CHMP7, or LEMD3. Screening cells or subjects for the expression of nucleic acids encoding LEMD2, CHMP7, or LEMD3 can be carried out by any well-known means. For example, methods for measuring protein expression and methods for measuring the expression of mRNA encoded by coding sequences are well-known.

[0120] One example of an assay that can be used is the BASESCOPE® RNA In Situ Hybridization (ISH) assay, a method that can quantify cell-specific edited transcripts, including single-nucleotide alterations, in relation to intact fixed tissue. The BASESCOPE® RNA ISH assay can complement NGS and qPCR in the characterization of gene editing. While NGS / qPCR can provide quantitative mean values ​​for wild-type and edited sequences, it does not provide information on the heterogeneity or percentage of edited cells within the tissue. The BASESCOPE® ISH assay can provide a landscape view of the entire tissue and quantification of wild-type versus edited transcripts at single-cell resolution, which can quantify the actual number of cells in the target tissue containing edited mRNA transcripts. The BASESCOPE® assay uses paired oligo ("ZZ") probes to amplify the signal without nonspecific background and achieve single-molecule RNA detection. However, the design of the BASESCOPE® probe and signal amplification system enables the detection of single RNA molecules using 1ZZ probes, allowing for differential detection of single nucleotide edits and mutations in intact fixed tissue.

[0121] As another example, reporter genes can be used for screening. For instance, nucleic acids encoding LEMD2, CHMP7, or LEMD3 may encode LEMD2, CHMP7, or LEMD3 fused to a reporter gene such as a fluorescent protein. Exemplary reporter genes include those encoding luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, and alkaline phosphatase.

[0122] As another example, a selection marker can be used to screen for cells having nucleic acids encoding LEMD2, CHMP7, LEMD3, or LEMD2, CHMP7, or LEMD3. An example selection marker is neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puro r ), blastocydin S deaminase (bsr r Examples include xanthine / guanine phosphoribosyltransferase (GPT), or herpes simplex virus thymidine kinase (HSV-K).

[0123] The method may further include evaluating one or more signs or symptoms of tauopathy or tau aggregates by any preferred means. Examples of such signs and symptoms are discussed in more detail elsewhere herein and include, for example, tau hyperphosphorylation or tau aggregation. Other signs and symptoms may include, for example, increased tau and / or phosphorylated tau in the insoluble fraction after cell fractionation, increased phosphorylated tau, increased phosphorylated tau in the cell body dendritic compartment of neurons, increased phosphorylated tau in the perinuclear region of neurons, decreased nucleus-to-cytoplasmic ratio of nuclear pore complex proteins Nup98-Nup96 (Nup98) in neurons, decreased nucleus-to-cytoplasmic ratio of GTP-binding nucleoprotein Ran (Ran) in neurons, or decreased nucleus-to-cytoplasmic ratio of Ran GTPase-activated protein 1 (RanGAP1) in neurons. Phosphorylated tau may be, for example, phosphorylated tau (S356) or phosphorylated tau AT8 (S202, T205). Other signs and symptoms may include, for example, serum neuronal filament light chains (sNfL). This may be performed approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or longer after introduction of, for example, LEMD2, CHMP7, LEMD3, or the nucleic acid encoding LEMD2, CHMP7, or LEMD3. For example, evaluation may be performed approximately 2 to 6 weeks, or approximately 3 to 5 weeks, after introduction of LEMD2, CHMP7, LEMD3, or the nucleic acid encoding LEMD2, CHMP7, or LEMD3.

[0124] The methods described herein can, for example, reduce the amount of new tau aggregate formation (new tau aggregation) in cells or subjects, and / or reduce the amount of existing tau aggregate formation (existing tau aggregation) in cells or subjects. For example, the methods described herein can prevent new tau aggregate formation, and / or reverse existing tau aggregate formation. The methods described herein can also, for example, reduce the level of phosphorylated tau (e.g., phosphorylated tau (S356) or phosphorylated tau AT8 (S202, T205)) in cells or subjects.

[0125] The methods described herein can, for example, reduce the amount of new serum neuronal filament light chain (sNfL) accumulation in a subject and / or reduce the amount of existing serum neuronal filament light chain (sNfL) levels in a subject. For example, the methods described herein can prevent new tau serum neuronal filament light chain (sNfL) accumulation and / or reverse existing serum neuronal filament light chain (sNfL) accumulation.

[0126] Some of the methods described herein are for the treatment or prevention of tauopathy in a subject. In some methods (e.g., methods for treatment), the subject has one or more signs or symptoms of tauopathy. For example, the subject may have pre-existing tau aggregate formation in one or more cells. Tauopathy is a class of diseases caused by misfolding of the tau protein. They are a group of progressive neurodegenerative disorders pathologically defined by the presence of tau protein aggregates in the brain. Tauopathy is a group of heterogeneous neurodegenerative states characterized by abnormal tau deposition in the brain. These include, for example, Alzheimer's disease, Down syndrome, Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In AD and other tauopathies, tau proteins are abnormally hyperphosphorylated and aggregate into bundles of filaments (paired spiral filaments) that appear as neurofibrillary junctions.

[0127] Some of the methods described herein are for the treatment or prevention of primary tauopathy. Primary tauopathy is a group of neurodegenerative diseases in which tau is considered to be a major contributor to the neurodegenerative process. In primary tauopathy, a dissociation exists between tau, a microtubule-associated protein, and microtubules as a result of tau hyperphosphorylation. This dissociation between tau and microtubules leads to tau fibrillation and inclusion body formation, as well as microtubule dysfunction. All diseases considered to be primary tauopathy share the common characteristic of abnormal deposition of aggregated tau in the brain. Other diseases in which tau deposition may be observed exist, but for some reason tau coexists with other proteins or tau is not considered a major neurodegenerative process. Diseases in this latter category include: Alzheimer's disease also contains β-amyloid; Lewy body dementia also contains α-synuclein; myotonic dystrophy; subacute sclerosing panencephalitis; Down syndrome; and Niemann-Pick disease type C.

[0128] Six isoforms of tau gene exist that are expressed in the adult brain. These six isoforms originate from alternative splicing of three N-terminal exons in the tau gene: exon 2, exon 3, and exon 10. Three of the six isoforms result from splicing-in of exon 10, while the other three result from splicing-out of exon 10. Splicing-in of exon 10 produces an isoform with four repeat microtubule-binding domains, while splicing-out of exon 10 produces an isoform with three repeat microtubule-binding domains. This is important because while a healthy human brain consists of equal amounts of tau with three and four repeat-binding domains, some primary tauopathies are characterized by a predominance of isoforms with four repeat-binding domains (4R tauopathies), some by a predominance of isoforms with three repeat-binding domains (3R tauopathies), and some by an almost equal mixture of isoforms with three and four repeat-binding domains (3R+4R tauopathies). See Table 1.

[0129] [Table 1]

[0130] Several tau pathogenic mutations exist that are associated with (e.g., segregation) or cause tauopathy, such as pro-aggregation mutations. Pathogenic tau mutations, which can be either exon or intronic, generally alter the relative production of tau isoforms, potentially leading to changes in the tendency of microtubule constructs and / or tau to aggregate. For example, a mutation may be an aggregation-sensitizing mutation that sensitizes tau to seeding but does not cause tau to aggregate readily on its own. For instance, a mutation may be a disease-associated P301S mutation. A P301S mutation refers to a human tau P301S mutation, or a corresponding mutation in another tau protein when optimally aligned with the human tau protein. Other pathogenic tau mutations include, for example, A152T, G272V, K280del, P301L, S320F, V337M, R406W, P301L / V337M, K280del / I227P / I308P, G272V / P301L / R406W, and A152T / P301L / S320F. See alzforum.org / mutations / mapt, Brandt et al. (2005) "Biochim. Biophys. Acta", 1739:331-354, and Wolfe (2009) "J. Biol. Chem.", 284(10):6021-6025, which are incorporated herein by reference in their entirety.

[0131] The methods described herein may alleviate one or more signs and symptoms of tauopathy in cells or subjects. Some examples of signs and symptoms of tauopathy at the cellular level include tau hyperphosphorylation (e.g., in the dendritic compartment of the cell body of nerve cells, because tau is generally considered an axonal protein, but is found in the dendritic compartment of degenerating nerve cells, and this redistribution is thought to trigger neurodegeneration in Alzheimer's disease), tau aggregation, abnormal shape of the nuclear lamina, and impaired nucleocytoplasmic transport. Other signs and symptoms at the biological level may include neurofibrillary hierarchies (e.g., neocortex, amygdala, hippocampus, brainstem, or spinal cord), neuronal loss (e.g., hippocampus, amygdala, or neocortex), microgliosis, synaptic loss, cognitive impairment, or motor impairment. Other signs and symptoms may include, for example, increased tau and / or phosphorylated tau in the insoluble fraction after cell fractionation, increased phosphorylated tau in the dendritic compartment of the cell body of neurons, increased phosphorylated tau in the perinuclear region of neurons, decreased nucleus-to-cytoplasmic ratio of nuclear pore complex proteins Nup98-Nup96 (Nup98) in neurons, decreased nucleus-to-cytoplasmic ratio of GTP-binding nucleoprotein Ran (Ran) in neurons, or decreased nucleus-to-cytoplasmic ratio of Ran GTPase-activating protein 1 (RanGAP1) in neurons. Phosphorylated tau may be, for example, phosphorylated tau (S356) or phosphorylated tau AT8 (S202, T205). Other signs and symptoms may include, for example, serum neuronal filament light chains (sNfL), biomarkers of neuronal damage, which are strong indicators of neurodegenerative processes. Nerve filament light chains (NF1s) are cytoskeletal protein components whose release into the bloodstream indicates neuronal damage and are well-known biomarkers for many neurodegenerative diseases. See, for example, Rubsamen et al., (2021), "BMC Medicine," 19:38, and Loeffler et al., (2020), "Front. Neurosci." 14:295-579, which are incorporated herein by reference in their entirety.Neurofilament light (NF-L) is a 68 kDa cytoskeletal intermediate filament protein expressed in nerve cells. It associates with 125 kDa neurofilament medium (NF-M) and 200 kDa neurofilament heavy (NF-H) to form neurofilaments. These are major components of the neuronal cytoskeleton and are thought to primarily function in providing structural support to axons and regulating axonal diameter. Neurofilaments may be released in significant amounts after axonal injury or neurodegeneration. NF-L has been shown to be associated with traumatic brain injury, multiple sclerosis, frontotemporal dementia, and other neurodegenerative diseases.

[0132] III. Nucleic acids encoding LEMD2, CHMP7, or LEMD3 This specification provides nucleic acids or nucleic acid constructs encoding LEMD2, CHMP7, or LEMD3 (i.e., exogenous nucleic acids encoding LEMD2, CHMP7, or LEMD3). The nucleic acids or nucleic acid constructs may be isolated nucleic acid constructs.

[0133] Some nucleic acids or nucleic acid constructs described herein include nucleic acids encoding LEMD2. For example, LEMD2 may be wild-type LEMD2. In one example, LEMD2 is human LEMD2. Human LEMD2 (also called LEM domain-containing protein 2, hLEM2, or LEM domain nuclear membrane protein 2) is assigned UniProt reference number Q8NC56. The human gene encoding LEMD2 (LEMD2, or LEM domain nuclear membrane protein 2) is assigned NCBI GeneID221496 and is found at position 6p21.31 on chromosome 6 (construct: GRCh38.p13 (GCF_000001405.39); position: NC_000006.12 (33771213.33794274, complement)). At least two isoforms of human LEMD2 are known. The first isoform has 503 amino acids and is assigned UniProt reference number Q8NC56-1 and NCBI reference number NP_851853.1 (Sequence ID 1). An exemplary coding sequence is assigned reference number CCDS4785.1 (Sequence ID 2), and one exemplary mRNA (cDNA) sequence is assigned reference number NM_181336.4 (Sequence ID 4). A coding sequence with optimized codons to distinguish it from natural CDS is described in Sequence ID 3. The second isoform has 201 amino acids and is assigned UniProt reference number Q8NC56-2 and NCBI reference numbers NP_001137416.1 and NP_001335638.1 (Sequence ID 5). The exemplary coding sequence is assigned reference number CCDS47411.1 (sequence number 6), and the exemplary mRNA (cDNA) sequences are assigned reference numbers NM_001143944.1 and NM_001348709.2 (sequence number 8 and sequence number 9, respectively). A coding sequence with optimized codons to distinguish it from natural CDS is described in sequence number 7.

[0134] In another example, LEMD2 is mouse LEMD2. Mouse LEMD2 is assigned UniProt reference number Q6DVA0. The mouse gene encoding LEMD2 (LEMD2) is assigned NCBI GeneID224640 and is found on chromosome 17 at position 17;17 A3.3 (construct: GRCm39 (GCF_000001635.27); position: NC_000083.7 (27408574.27426228, complement)). The exemplary mouse LEMD2 is 511 amino acids and is assigned UniProt reference number Q6DVA0-1 and NCBI reference number NP_666187.2 (sequence number 10). An exemplary coding sequence is assigned the reference number CCDS50043.1 (sequence number 11), and one exemplary mRNA (cDNA) sequence is assigned the reference number NM_146075.2 (sequence number 14). Codon-optimized coding sequences to distinguish them from natural CDS are described in sequence numbers 12 and 13. Another codon-optimized coding sequence to distinguish them from natural CDS is described in sequence number 255.

[0135] Some nucleic acids or nucleic acid constructs described herein include nucleic acids encoding CHMP7. For example, CHMP7 may be wild-type CHMP7. In one example, CHMP7 is human CHMP7. Human CHMP7 (also called charged polyspheric protein 7 or chromatin-modified protein 7) is assigned UniProt reference number Q8WUX9. The human gene encoding CHMP7 (CHMP7) is assigned NCBI GeneID91782 and is found at position 8p21.3 on chromosome 8 (construct: GRCh38.p13 (GCF_000001405.39); position: NC_000008:11 (23243637..23262000)). An exemplary CHMP7 protein is 453 amino acids and is assigned UniProt reference number Q8WUX9-1 and NCBI reference number NP_689485.1 (SEQ ID NO: 15). An exemplary coding sequence is assigned the reference number CCDS6040.1 (sequence number 16), and one exemplary mRNA (cDNA) sequence is assigned the reference number NM_152272.5 (sequence number 18). A coding sequence with optimized codons to distinguish it from natural CDS is described in sequence number 17.

[0136] In another example, CHMP7 is mouse CHMP7. Mouse CHMP7 is assigned UniProt reference number Q8R1T1. The mouse gene encoding CHMP7 (CHMP7) is assigned NCBI GeneID105513 and is found on chromosome 14 at position 14;14 D2 (construct: GRCm39 (GCF_000001635.27); position: NC_000080.7 (69954428.69970019, complement)). The exemplary mouse CHMP7 is 451 amino acids and is assigned UniProt reference number Q8R1T1-1 and NCBI reference number NP_598839.2 (sequence number 19). An exemplary coding sequence is assigned the reference number CCDS27242.1 (sequence number 20), and one exemplary mRNA (cDNA) sequence is assigned the reference number NM_134078.4 (sequence number 22). A coding sequence with optimized codons to distinguish it from natural CDS is described in sequence number 21.

[0137] Some nucleic acids or nucleic acid constructs described herein include nucleic acids encoding LEMD3. For example, LEMD3 may be wild-type LEMD3. In one example, LEMD3 is human LEMD3. Human LEMD3 (also called the inner nuclear membrane protein Man1 or LEM domain-containing protein 3) is assigned UniProt reference number Q9Y2U8. The human gene encoding LEMD3 (LEMD3, MAN1, or LEM domain-containing protein 3) is assigned NCBI GeneID23592 and is found at position 12q14.3 on chromosome 12 (construction: GRCh38.p13(GCF_000001405.39); position: NC_000012.12(65169583..65248355)). The exemplary LEMD3 protein consists of 911 amino acids and is assigned UniProt reference number Q9Y2U8-1 and NCBI reference number NP-055134.2 (SEQ ID NO: 23). The exemplary coding sequence is assigned reference number CCDS8972.1 (SEQ ID NO: 24), and one exemplary mRNA (cDNA) sequence is assigned reference number NM_014319.5 (SEQ ID NO: 26). A coding sequence with optimized codons to distinguish it from natural CDS is described in SEQ ID NO: 25.

[0138] In another example, LEMD3 is mouse LEMD3. Mouse LEMD3 is assigned UniProt reference number Q9WU40. The mouse gene encoding LEMD3 (Lemd3) is assigned NCBI GeneID380664 and is found on chromosome 10 at position 10;10 D2 (construct: GRCm39(GCF_000001635.27); position: NC_000076.7(120759316..120815491, complement)). The exemplary mouse LEMD3 is 921 amino acids and is assigned UniProt reference number Q9WU40-1 (sequence number 27). The coding sequence with codons optimized to distinguish it from natural CDS is described in sequence number 28. Another exemplary mouse sequence, LEMD3, is 918 amino acids long and has been assigned NCBI reference number NP_001074662.2 (sequence number 29). An exemplary coding sequence has been assigned reference number CCDS48703.1 (sequence number 30), and one exemplary mRNA (cDNA) sequence has been assigned reference number NM_001081193.2 (sequence number 31).

[0139] In some cases, nucleic acids encoding LEMD2, CHMP7, or LEMD3 may be native coding sequences. In other cases, they may be codon-optimized (e.g., codon-optimized for human or mouse expression). For example, nucleic acids may be modified to alternative codons that are more frequently used in human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest.

[0140] The nucleic acid encoding LEMD2, CHMP7, or LEMD3 may be DNA or RNA. The nucleic acid may, in some cases, be messenger RNA (mRNA) encoding LEMD2, CHMP7, or LEMD3. The nucleic acid may, in some cases, be complementary DNA (cDNA) encoding LEMD2, CHMP7, or LEMD3. For example, such a nucleic acid may consist only of a coding sequence without any intervening introns. In other cases, the nucleic acid may contain one or more introns that separate the exons in the LEMD2, CHMP7, or LEMD3 coding sequence. For example, the nucleic acid may contain a genomic sequence containing both exons and introns.

[0141] In some cases, the nucleic acid is present in an expression construct containing a nucleic acid encoding LEMD2, CHMP7, or LEMD3 operably ligated to a promoter. The promoter can be any suitable promoter for in vivo expression in animals or in vitro expression in isolated cells. The promoter can be a constitutively active promoter (e.g., a CAG promoter or a U6 promoter), a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Such promoters are well known and discussed elsewhere in this specification. In certain examples, the promoter is active in nerve cells. In some cases, the promoter is a heterologous promoter (i.e., a promoter in which the LEMD2, CHMP7, or LEMD3 nucleic acid is not operably ligated in nature). In other cases, the promoter may be an endogenous promoter (i.e., LEMD2 nucleic acid operably ligated to the LEMD2 promoter, CHMP7 nucleic acid operably ligated to the CHMP7 promoter, or LEMD3 nucleic acid operably ligated to the LEMD3 promoter). Heterogeneous promoters may be any type of promoter disclosed elsewhere in this specification. For example, the promoter may be a constitutive promoter, such as the EF1α promoter. Alternatively, the promoter may be a tissue-specific promoter or an inducible promoter. For example, as another example, the promoter may be a neuron-specific promoter. A suitable example of a neuron-specific promoter is the synapsin-1 promoter (e.g., a human synapsin-1 promoter, such as the promoter described in SEQ ID NO: 44). In some embodiments, the synapsin-1 promoter may be used in conjunction with hemoglobin subunit beta (HBB) intron 2 (e.g., downstream of the synapsin-1 promoter), such as the one described in SEQ ID NO: 254. Inclusion of this element may enhance gene expression.

[0142] The nucleic acids and expression constructs disclosed herein may also include post-transcriptional regulatory elements, such as the post-transcriptional regulatory elements of woodchuck hepatitis virus.

[0143] Nucleic acids and expression constructs may further include one or more polyadenylation signal sequences. For example, a nucleic acid construct may include a polyadenylation signal sequence located at 3' of the LEMD2, CHMP7, or LEMD3 coding sequence. Any suitable polyadenylation signal sequence may be used. The term polyadenylation signal sequence refers to any sequence that directs the termination of transcription and the addition of a poly(A) tail to the mRNA transcript. In eukaryotes, the transcription terminator is recognized by a protein factor, and after termination, polyadenylation is the process of adding a poly(A) tail to the mRNA transcript in the presence of poly(A) polymerase. Mammalian poly(A) signals typically consist of a core sequence about 45 nucleotides long, flanked by a variety of auxiliary sequences that help to enhance the efficiency of cleavage and polyadenylation. The core sequence, referred to as the poly-A recognition motif or poly-A recognition sequence, consists of a highly conserved upstream element (AATAAA or AAUAAA) within mRNA, recognized by a cleavage and polyadenylation specificity factor (CPSF), and a downstream region (U or G and U-rich) that is poorly defined and bound by a cleavage stimulator (CstF). Examples of transcriptional terminators that can be used include, for example, the human growth hormone (HGH) polyadenylation signal, the Simian virus 40 (SV40) late polyadenylation signal, the rabbit betaglobin polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, the phosphoglycerate kinase (PGK) polyadenylation signal, the AOX1 transcription termination sequence, the CYC1 transcription termination sequence, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells. Examples of suitable polyadenylation signals include, for example, those described in SEQ ID NOs. 252 and 253.

[0144] Nucleic acids and expression constructs may also include a polyadenylation signal sequence upstream of the LEMD2, CHMP7, or LEMD3 coding sequence. The polyadenylation signal sequence upstream of the LEMD2, CHMP7, or LEMD3 coding sequence may be adjacent to a recombinase recognition site recognized by a site-specific recombinase. In some constructs, the recombinase recognition site is also adjacent to a selection cassette containing, for example, the coding sequence for a drug-resistant protein. In some constructs, the recombinase recognition site is not adjacent to a selection cassette. The polyadenylation signal sequence interferes with the transcription and expression of the protein or RNA encoded by the coding sequence. However, upon exposure to a site-specific recombinase, the polyadenylation signal sequence may be excised, allowing for the expression of the protein or RNA.

[0145] Such a configuration may enable tissue-specific or developmental-stage-specific expression when the polyadenylation signal sequence is excised in a tissue-specific or developmental-stage-specific manner. Tissue-specific or developmental-stage-specific excision of the polyadenylation signal sequence can be achieved if the animal containing the nucleic acid or expression construct further includes a coding sequence for a site-specific recombinase operably linked to the tissue-specific or developmental-stage-specific promoter. The polyadenylation signal sequence is then excised only in those tissues or developmental stages, enabling tissue-specific or developmental-stage-specific expression. In one embodiment, LEMD2, CHMP7, or LEMD3 encoded by the nucleic acid or expression construct can be expressed in a neuronal-cell-specific manner.

[0146] Site-specific recombinases include enzymes that can promote recombination between recombinase recognition sites where two recombination sites are physically separated within a single nucleic acid or on separate nucleic acids. Examples of recombinases include Cre, Flop, and Dre recombinases. An example of a Cre recombinase gene is Crei, in which the two exons encoding Cre recombinase are separated by an intron, preventing expression in prokaryotic cells. Such recombinases may further include nuclear localization signals to promote nuclear localization (e.g., NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by site-specific recombinases and can function as substrates for recombination events. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox, and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2, and lox5171.

[0147] The nucleic acids disclosed herein may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single-stranded or double-stranded, and may be linear or circular in form. Nucleic acid constructs may be naked nucleic acids, as described elsewhere herein, or may be delivered by vectors such as AAV vectors. In the linear form, the ends of the nucleic acid may be protected by known methods (e.g., from exonuclease degradation). For example, one or more dideoxynucleotide residues may be added to the 3' end of a linear molecule, and / or self-complementary oligonucleotides may be attached to one or both ends. See, for example, Chang et al., (1987) "Proc. Natl. Acad. Sci. USA" 84:4959-4963, and Nehls et al., (1996) "Science" 272:886-889, each of which is incorporated herein by reference in whole. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified nucleotide bonds such as phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues. A nucleic acid or expression construct may, in some cases, contain one or more of the following terminal structures: hairpins, loops, terminal inversion sequences (ITRs), or toroids. For example, a nucleic acid or expression construct may contain an ITR.

[0148] Nucleic acid or expression constructs may include modifications or sequences that provide additional desirable features (e.g., modified or regulated stability, tracking or detection by fluorescent labeling, or binding sites for proteins or protein complexes). A nucleic acid construct may include one or more fluorescent labels, purification tags, epitope tags, or combinations thereof. For example, a nucleic acid construct may include one or more fluorescent labels (e.g., fluorescent proteins or other fluorophores or dyes), such as at least one, at least two, at least three, at least four, or at least five fluorescent labels. Exemplary fluorescent labels include fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, 5-(and-6)-carboxytetramethylrhodamine (TAMRA), and fluorophores such as Cy7. A wide range of fluorescent dyes for labeling oligonucleotides are commercially available (e.g., from Integrated DNA Technologies). The label or tag may be located at the 5' end, 3' end, or inside the nucleic acid construct. For example, the nucleic acid construct may be conjugated at the 5' end having an IR700 fluorophore (5'IRDYE® 700) from Integrated DNA Technologies.

[0149] Nucleic acids and expression constructs may also include conditional alleles. Conditional alleles may be multifunctional alleles, as described in U.S. Patent No. 2011 / 0104799, which is incorporated herein by reference in whole. For example, a conditional allele may include (a) a sense-oriented working sequence with respect to the transcription of a target gene, (b) a sense or antisense-oriented drug selection cassette (DSC), (c) a nucleotide sequence of interest for antisense orientation (NSI), and (d) a reverse-oriented conditional inversion module (COIN, utilizing an exon-splitting intron and an invertible gene-trap-like module). See, for example, U.S. Patent No. 2011 / 0104799. A conditional allele may further include a recombinable unit that recombines upon exposure to a first recombinase to form a conditional allele that (i) lacks the working sequence and DSC, and (ii) includes a sense-oriented NSI and an antisense-oriented COIN. For example, refer to U.S. Patent No. 2011 / 0104799.

[0150] Nucleic acids and expression constructs may also contain polynucleotides encoding a selection marker. Alternatively, nucleic acids and expression constructs may lack polynucleotides encoding a selection marker. The selection marker may be contained in a selection cassette. Optionally, the selection cassette may be a self-deletion cassette. For example, in this application, each of these is referenced to U.S. Patents 8,697,851 and 2013 / 0312129, which are incorporated herein by reference in their entirety. As an example, a self-deletion cassette may include a Crei gene (containing two exons encoding Cre recombinase separated by an intron) operably linked to a mouse Prm1 promoter and a neomycin resistance gene operably linked to a human ubiquitin promoter. By using the Prm1 promoter, the self-deletion cassette can be specifically deleted in the germ cells of male F0 animals. An exemplary selection marker is neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r), puromycin-N-acetyltransferase (puro r ), blasticidine S deaminase (bsr r Examples include xanthine / guanine phosphoribosyltransferase (GPT), herpes simplex virus thymidine kinase (HSV-K), or combinations thereof. The polynucleotide encoding the selection marker can be operably ligated to an active promoter in the target cell. Examples of promoters are described elsewhere in this specification.

[0151] Nucleic acids or expression constructs may also include reporter genes. Exemplary reporter genes include those encoding luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, and alkaline phosphatase. Such reporter genes can be operably linked to active promoters in the target cells. Examples of promoters are described elsewhere in this specification.

[0152] IV. Vectors This specification also provides vectors comprising nucleic acids, nucleic acid constructs, or expression constructs encoding LEMD2, CHMP7, or LEMD3. The vectors may include additional sequences, such as, for example, a replication origin, a promoter, and a gene encoding antibiotic resistance.

[0153] Some vectors may be circular, while others may be linear. Vectors may be packaged to be delivered via lipid nanoparticles, liposomes, non-lipid nanoparticles, or viral capsids. Non-exclusive exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0154] Nucleic acids or expression constructs may be present in vectors, such as viral vectors. Viral vectors may be, for example, adeno-associated virus (AAV) vectors or lentivirus (LV) vectors (i.e., recombinant AAV vectors or recombinant LV vectors). Other exemplary viruses / viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. Viruses can infect dividing cells, non-dividing cells, or both. Viruses may be integrated into the host genome, or alternatively, not integrated into the host genome. Such viruses may also be engineered to reduce immunity. Viruses may be replicable or replication-deficient (e.g., lacking one or more genes necessary for an additional round of virion replication and / or packaging). Viruses may cause transient expression, prolonged expression (e.g., at least one week, two weeks, one month, two months, or three months), or persistent expression. Viral vectors may be genetically modified from their wild-type counterparts. For example, a viral vector may include one or more nucleotide insertions, deletions, or substitutions to facilitate cloning or to alter one or more properties of the vector. Such properties may include packaging ability, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some examples, a portion of the viral genome may be deleted so that the virus can package larger exogenous sequences. In some examples, the viral vector may have enhanced transduction efficiency. In some examples, the immune response induced by the virus in the host may be reduced. In some examples, viral genes (such as integrases) that facilitate the integration of the viral sequence into the host genome may be mutated so that the virus becomes non-integrating. In some examples, the viral vector may be replication-deficient. In some examples, the viral vector may include exogenous transcriptional or translational regulatory sequences to drive the expression of the coding sequence on the vector. In some examples, the virus may be helper-dependent.For example, a virus may require one or more helper viruses to supply the viral components (such as viral proteins) necessary for amplifying the vector and packaging it into viral particles. In such cases, one or more helper components, including one or more vectors encoding viral components, may be introduced into a host cell or population of host cells together with the vector system described herein. In other examples, the virus may not contain helpers. For example, a virus may be able to amplify and package a vector without helper viruses. In some examples, the vector system described herein may also encode viral components necessary for viral amplification and packaging. An example viral titer (e.g., AAV titer) is about 10. 12 , about 10 13 , about 10 14 , about 10 15 , and about 10 16 One example is vector genome / mL. Other exemplary viral titers (e.g., AAV titer) are approximately 10 12 , about 10 13 , about 10 14 , about 10 15 , and about 10 16 One example is vector genome (vg) / kg body weight. In one example, the viral titer is approximately 10 13 ~about 10 14 The concentration is vg / mL or vg / kg.

[0155] In one example, the nucleic acid or expression construct is present in the AAV vector. AAV can be any suitable serotype and may be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). The ssDNA AAV genome consists of two open reading frames, Rep and Cap, with two adjacent terminal inversion sequences that enable the synthesis of complementary DNA strands. When constructing an AAV transfer plasmid, the transgene is placed between two ITRs, and Rep and Cap may be supplied trans. In addition to Rep and Cap, AAV may require a helper plasmid containing adenovirus-derived genes. These genes (E4, E2a, and VA) mediate AAV replication. For example, the transfer plasmid, Rep / Cap, and helper plasmid can be transfected into HEK293 cells containing the adenovirus gene E1+ to produce infectious AAV particles. Alternatively, Rep, Cap, and the adenovirus helper gene may be combined into a single plasmid. Similar packaging cells and methods can also be used for other viruses, such as retroviruses.

[0156] Adeno-associated viruses (AAVs) are endemic to several species, including humans and non-human primates (NHPs). To date, at least 12 native serotypes and hundreds of native variants have been identified and characterized. See, for example, Li et al., (2020), "Nat. Rev. Genet.", 21:255-272, which is incorporated herein by reference in its entirety. AAV particles are naturally composed of a non-membrane icosahedral protein capsid containing a single-stranded DNA (ssDNA) genome. The DNA genome contains two inverted terminal repeats (ITRs) adjacent to each other, which act as the origin of viral replication and packaging signals. The rep gene encodes four proteins required for viral replication and packaging, while the cap gene encodes three structural capsid subunits that define AAV serotypes, and a constructor-activating protein (AAP) that promotes virion constructs in some serotypes.

[0157] Recombinant AAV (rAAV) is currently one of the most commonly used viral vectors in gene therapy to treat human diseases by delivering therapeutic transgenes in vivo to target cells. In fact, rAAV vectors consist of an icosahedral capsid similar to that of natural AAV, although rAAV virions do not form a capsid containing the AAV protein-coding sequence or AAV replication sequence. These viral vectors are non-replicating. The only viral sequences required in rAAV vectors are two ITRs, which are necessary to induce genome replication and packaging during the production of rAAV vectors. The rAAV genome lacks the AAV rep and cap genes and does not replicate in vivo. rAAV vectors are produced by combining the AAV ITRs with an adjacent transgene cassette of interest, expressing the rep and cap genes in trans along with additional viral helper proteins.

[0158] In therapeutic rAAV genomes, gene expression cassettes are positioned between ITR sequences. Typically, an rAAV genome cassette contains a promoter for driving the expression of a therapeutic transgene, followed by a polyadenylation sequence. The ITRs adjacent to the rAAV expression cassette are usually derived from AAV2, the first serotype isolated and converted into a recombinant viral vector. Since then, most rAAV production methods rely on AAV2Rep-based packaging systems. See, for example, Colella et al. (2017), "Mol.Ther.Methods Clin.Dev." 8:87-104, which is incorporated in its entirety herein by reference.

[0159] Some non-limiting examples of ITRs that may be used include ITRs that contain, essentially consist of, or consist of SEQ ID NO: 245, SEQ ID NO: 246, or SEQ ID NO: 247, or SEQ ID NO: 248. Other examples of ITRs may contain one or more mutations compared to SEQ ID NO: 245, SEQ ID NO: 246, or SEQ ID NO: 247, or SEQ ID NO: 248, and may be identical in at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 245, SEQ ID NO: 246, or SEQ ID NO: 247, or SEQ ID NO: 248. In some rAAV genomes disclosed herein, nucleic acids encoding nuclease agents (or their components) are flanked on both sides by the same ITRs (i.e., an ITR at the 5' end and the reverse complementary strand of the ITR at the 3' end). In one example, each terminal ITR contains, essentially consists of, or may consist of, sequence number 245. In another example, each terminal ITR contains, essentially consists of, or may consist of, sequence number 246. In one example, at least one terminal ITR contains, essentially consists of, or consists of, sequence number 247 or sequence number 248. In one example, the ITR on the 5' terminal contains, essentially consists of, or consists of, sequence number 247 or sequence number 248. In one example, the ITR on the 3' terminal contains, essentially consists of, or consists of, sequence number 247 or sequence number 248. In one example, each terminal ITR contains, essentially consists of, or may consist of, sequence number 247 or sequence number 248. In one example, at least one terminal ITR contains, essentially consists of, or consists of, sequence number 245. In one example, the ITR on the 5' terminal contains, essentially consists of, or consists of, sequence number 245. In one example, the ITR at the 3' end contains, essentially consists of, or comprises SEQ ID NO: 245. In another example, the ITRs at each end contain, essentially consist of, or may comprise SEQ ID NO: 245. In other rAAV genomes disclosed herein, the nucleic acids encoding the nuclease (or its components) are flanked by different ITRs at each end.In one example, one terminal ITR contains, essentially consists of, or comprises sequence number 245, and the other terminal ITR contains, essentially consists of, or comprises sequence number 246. In another example, one terminal ITR contains, essentially consists of, or comprises sequence number 245, and the other terminal ITR contains, essentially consists of, or comprises sequence number 247 or sequence number 248. In another example, one terminal ITR contains, essentially consists of, or comprises sequence number 246, and the other terminal ITR contains, essentially consists of, or comprises sequence number 247 or sequence number 248.

[0160] The specific serotype of a recombinant AAV vector influences its in vivo directivity to specific tissues. The AAV capsid protein mediates attachment and entry into target cells, followed by endosomal escape and transport to the nucleus. Therefore, serotype selection when developing rAAV vectors influences which cell types and tissues the vector is most likely to bind to and transduce when injected in vivo.

[0161] Upon entering the nucleus, the ssDNA genome is released from the virion, and a complementary DNA strand is synthesized to produce a double-stranded DNA (dsDNA) molecule. The double-stranded AAV genome spontaneously circularizes via their ITRs, becoming an episome that persists outside the chromosome within the nucleus. Therefore, for episomal gene therapy programs, rAAV-delivered rAAV episomes provide long-term promoter-driven gene expression in non-dividing cells. However, this rAAV-delivered episomal DNA is diluted as the cell divides. In contrast, the gene therapies described herein are based on gene insertions that enable long-term gene expression.

[0162] Multiple serotypes of AAV have been identified. These serotypes differ in the types of cells they infect (i.e., their targeting), enabling preferential transduction of specific cell types. Serotypes for CNS tissues include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. The selectivity of AAV serotypes for gene delivery in nerve cells is discussed, for example, in Hammond et al., (2017) "PLoS One" 12(12):e0188830, which is incorporated in its entirety herein by reference. In specific examples, the AAV-PHP.eB vector is used. The AAV-PHP.eB vector exhibits a high ability to cross the blood-brain barrier, increasing its CNS transduction efficiency. In specific examples, the AAV9 vector is used.

[0163] Directionality can be further refined by pseudotyping, which is a mixture of capsids and genomes from different viral serotypes. For example, AAV2 / 5 represents a virus containing a serotype 2 genome packaged in a serotype 5 capsid. The use of pseudotyped viruses can not only improve transduction efficiency but also alter directionality. Furthermore, viral directionality can be modified using hybrid capsids derived from different serotypes. For example, AAV-DJ contains a hybrid capsid derived from eight serotypes and exhibits high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that exhibits the characteristics of AAV-DJ but with enhanced uptake into the brain. AAV serotypes can also be modified by mutation. Examples of mutational modifications of AAV2 include Y444F, Y500F, Y730F, and S662V. Examples of mutational modifications of AAV3 include Y705F, Y731F, and T492V. Examples of AAV6 mutations include S663V and T492V. Other pseudotypes / modified AAV variants include AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, AAV8.2, and AAV / SASTG.

[0164] To accelerate the expression of a transgene, self-complementary AAV (scAAV) variants may be used. Because AAV relies on the cell's DNA replication mechanism to synthesize the complementary strand of its single-stranded DNA genome, transgene expression may be delayed. To address this delay, scAAVs containing complementary sequences that can spontaneously anneal upon infection may be used, eliminating the need for host cell DNA synthesis. However, single-stranded AAV (ssAAV) vectors may also be used.

[0165] To increase packaging capacity, longer transgenes may be split into two AAV transfer plasmids, the first being a 3' splice donor and the second a 5' splice acceptor. During cell co-infection, these viruses can form concatemers and splice together to express the full-length transgene. This allows for the expression of longer transgenes, but at a reduced efficiency. A similar method for increasing capacity utilizes homologous recombination. For example, the transgene may be split into two transfer plasmids, but with substantial sequence overlap so that co-expression induces homologous recombination and expression of the full-length transgene.

[0166] IV. Lipid Nanoparticles This specification also provides lipid nanoparticles comprising LEMD2, CHMP7, or LEMD3, or nucleic acids, nucleic acid constructs, expression constructs, or vectors encoding LEMD2, CHMP7, or LEMD3.

[0167] Lipid formulations can protect biomolecules from degradation while simultaneously improving their uptake into cells. Lipid nanoparticles are particles containing multiple lipid molecules physically bound to one another by intermolecular forces. These include microspheres (monolayer and multilayer vesicles, e.g., liposomes), dispersed phases in emulsions, micelles, or internal phases in suspensions. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that may be included are neutral lipids (i.e., uncharged or amphoteric lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the time nanoparticles can remain in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in PCT International Publication WO2016 / 010840A1, which is incorporated in its entirety herein by reference. Exemplary lipid nanoparticles may comprise cationic lipids and one or more other components. In one example, the other components may include helper lipids such as cholesterol. In another example, the other components may include helper lipids such as cholesterol and neutral lipids such as DSPC. In yet another example, the other components may include helper lipids such as cholesterol, any neutral lipid such as DSPC, and stealth lipids such as S010, S024, S027, S031, or S033.

[0168] LNPs may contain one or more or all of the following: (i) lipids for encapsulation and endosomal escape, (ii) neutral lipids for stabilization, (iii) helper lipids for stabilization, and (iv) stealth lipids. See, for example, Farnleitner et al., (2018) "Cell Rep." 22(9):2227-2235, and PCT International Publication No. WO2017 / 173054A1, each of which is incorporated herein by reference in whole. Specific examples of the use of LNPs for delivery to the brain are disclosed in Nabhan et al., (2016), "Sci.Rep." 6:20019, which is incorporated herein by reference in whole.

[0169] V. Composition Compositions comprising LEMD2, CHMP7, or LEMD3, or nucleic acids, nucleic acid constructs, expression constructs, vectors, or lipid nanoparticles disclosed herein are also provided herein. Such compositions may be used, for example, for administering LEMD2, CHMP7, or LEMD3 to cells or subjects, or for expressing LEMD2, CHMP7, or LEMD3 in cells or subjects. Such compositions may be used, for example, for inhibiting tau aggregates in cells or subjects. Such compositions may be used, for example, for reducing phosphorylation in cells or subjects. Such compositions may be used, for example, for reducing serum neuronal filament light chains (sNfLs) or preventing the accumulation of serum neuronal filament light chains (sNfLs) in subjects. Such compositions may be used, for example, for treating tauopathy in subjects. Such compositions may be used, for example, for preventing tauopathy in subjects.

[0170] VI. Cells or animals Also provided herein are cells or subjects (e.g., animals) containing LEMD2, CHMP7, or LEMD3, or nucleic acids, nucleic acid constructs, expression constructs, vectors, or lipid nanoparticles. The cells or subjects may express LEMD2, CHMP7, or LEMD3.

[0171] Cells or subjects may be, for example, mammals, non-human mammals, and humans. Mammals may be, for example, non-human mammals, humans, rodents, rats, mice, or hamsters. Other non-human mammals include, for example, non-human primates, monkeys, apes, cats, dogs, rabbits, horses, bulls, deer, bison, and domestic animals (e.g., cattle breeds such as cattle and steers, sheep breeds such as sheep and goats, and pig breeds such as pigs and wild boars). The term "non-human" excludes humans.

[0172] The cells may be isolated cells (e.g., in vitro) or in vivo within a subject (e.g., an animal or mammal). The cells may also be in any type of undifferentiated or differentiated state. For example, the cells may be nerve cells.

[0173] The cells provided herein may be normal and healthy cells, or they may be diseased cells containing tau aggregates. For example, cells may have a tendency towards tau aggregates, or they may have pre-existing tau aggregates.

[0174] In one example, the cells are human cells, rodent cells, mouse cells, or rat cells, such as human nerve cells, rodent nerve cells, mouse nerve cells, or rat cells. In a specific example, the cells are human nerve cells. In a particular example, the cells are present in vivo in the subject (e.g., nerve cells in the subject's brain).

[0175] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated herein by reference in whole to the same extent that each item is specifically and individually indicated to be incorporated by reference. If different versions of an array are associated with an accession number at different times, the version associated with the accession number on the effective filing date of this application is meant. The effective filing date, where applicable, means the filing date of an accession number prior to the actual filing date or of a priority application. Similarly, if different versions of a publication, website, etc., are published at different times, unless otherwise specified, the version published most recently on the effective filing date of this application is meant. Unless otherwise indicated, any feature, process, element, embodiment, or aspect of the present invention may be used in combination with any other. While the present invention is described in detail through diagrams and examples for the purposes of clarity and understanding, it will become apparent that certain changes and modifications can be made within the scope of the appended claims.

[0176] A brief explanation of arrays The nucleotide and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter notation for amino acids. Nucleotide sequences follow the standard rule of starting from the 5' end of the sequence and proceeding forward (i.e., from left to right in each row) to the 3' end. Only one strand of each nucleotide sequence is shown, but complementary strands are understood to be included by arbitrary reference to the shown strand. Where a nucleotide sequence encoding an amino acid sequence is provided, it is understood that its codon-degenerate variant encoding the same amino acid sequence is also provided. Amino acid sequences follow the standard rule of starting from the amino end of the sequence and proceeding forward (i.e., from left to right in each row) to the carboxyl end.

[0177] [Table 2-1] [Table 2-2]

[0178] Example 1. Loss of nuclear membrane integrity promotes the aggregation of microtubule-associated protein tau. In Alzheimer's disease and other tauopathy, tau is abnormally hyperphosphorylated and aggregates into bundles of paired helical filaments, appearing as neurofibrillary hierarchies. Tau fibrillation into insoluble aggregates is not only a characteristic feature of the disease but also a contributing factor to neurotoxicity. Neurodegenerative diseases with tau pathology are characterized by the propagation of tau aggregates through the central nervous system in a typical pattern, a process correlated with disease progression. This progressive pathology is proposed to progress along neuroanatomical circuits and occur through prion-like mechanisms of intercellular transmission of misfolded tau. Mutations in the MAPT gene have been associated with frontotemporal dementia and other tauopathy, providing a mechanism for disease emanating in these cases; however, the pathogenesis of tau pathology in Alzheimer's disease and other tauopathy unrelated to MAPT mutations is poorly understood. While most interest in tau has focused on its role in cytoskeletal microtubule dynamics, recent evidence suggesting that nuclear components may be associated with cytoplasmic tau aggregates could offer insights into the mechanisms by which tau misfolds and aggregates in disease.

[0179] To discover perturbations that can induce tau aggregates and to elucidate normal processes that protect cells from tau pathology, we performed a series of gene screenings for mutations that promote or inhibit tau aggregates in human embryonic kidney (HEK293T) biosensor cell lines. See, for example, Holmes et al., (2014) "Proc. Natl. Acad. Sci. USA", 111:E4376-4385, and Sanders et al., (2014) "Neuron", 82 1271-1288, each of which is incorporated herein by reference in whole. The biosensor cells stably express two transgenes encoding the 4RD complete microtubule-binding domain of tau, each containing a pathogenic mutation from proline 301 to serine, fused to either cyan fluorescent protein (tau-CFP) or yellow fluorescent protein (tau-YFP). Tau fragment aggregates are detected when two tau fusion proteins are in a favorable, close orientation to induce Forster resonance energy transfer (FRET) between CFP and YFP. Tau aggregates can be induced in biosensor cells by treatment with tau seeding agents, such as extracts from cells and tissues containing pathogenic tau aggregates or misfolded fibrils of wild-type or mutant tau. Tau aggregates in biosensor cells can be visualized by fluorescence microscopy, while FRET allows for quantification of the number of cells containing aggregates by flow cytometry and their purification by fluorescence-activated cell sorting (FACS). Tau aggregates can also be visualized by fluorescence microscopy.

[0180] To facilitate gene screening, the inventors expressed Cas9 in tau biosensor cell lines and then introduced a library of lentiviral-expressed CRISPR guide RNAs (gRNAs) that directed specific Cas9 cleavage in almost all protein-coding genes in the genome, creating a large collection of cells each containing a gene-specific mutagen into which it was incorporated. Cells possessing the desired trait (e.g., acquisition or loss of FRET) could be isolated by FACS, and the incorporated gRNAs they possessed could be discovered by DNA sequencing, thereby identifying the genes that, when mutated, produce the desired trait. As a complementary screening strategy, the inventors modified tau biosensor cell lines with a Synergistic Activation Mediator (SAM) CRISPR platform for gRNA-targeted gene activation. From gene screening using these two complementary approaches, the inventors identified genes that promote or enhance tau aggregates when inactivated and prevent or inhibit tau aggregates when overexpressed. These genes shared a common biological function (maintenance of nuclear membrane integrity) (a process that was not involved in tau pathology prior to our research).

[0181] result Use of tau biosensor cell lines for CRISPR-Cas9 gene screening. Tau biosensor cells express tau-CFP and tau-YFP reporter fusion proteins in a stable, soluble state without visible fluorescent aggregates or FRET signaling. When biosensor cells are treated with potent seeding agents, such as purified recombinant tau fibrils complexed with transfection reagents, visible fluorescent aggregates and FRET are induced in the majority of cells (Figures 5A-5F). Intracellular aggregates are detected by antibodies that recognize tau phosphorylated on serine 356 (P-tau-Ser356, Figure 5F), which is a prominent feature of pathological tau aggregates in the human brain, correlated with seeding activity that promotes intercellular propagation. To prepare tau biosensor cell lines for CRISPR-Cas9 gene screening, the inventors transduced them with a lentiviral vector expressing Streptococcus pyogenes Cas9 and selected clones that were evaluated for Cas9 mRNA expression and mutagenesis efficiency using gene-specific gRNA. For biosensor cell lines for CRISPR-Cas9 gene inactivation screening, the inventors selected clones with the lowest Cas9 expression level sufficient to induce maximum gene disruption activity (>80% of alleles mutated) (Clone E, Figure 5G).

[0182] Visible fluorescent tau aggregates can also be induced in HEK293T cells that stably express only one of the biosensor fluorescent fusion proteins (Figure 1A), but these cells do not produce the FRET signal. To establish an agglutination-positive (Agg+) cell line, we treated tau-YFP cells with recombinant tau fibrils complexed with LIPOFECTAMINE® and isolated cell clones that stably proliferated and maintained tau-YFP aggregates through numerous cell divisions. Treatment of tau biosensor cells with cell-free conditioned medium from tau-YFP Agg+ cells induced low levels of tau aggregates indicated by the FRET signal in approximately 0.1% of cells (FRET+ cells) analyzed by flow cytometry (Figure 1A). In contrast, conditioned medium derived from the tau-YFP Agg-parent cell line did not induce FRET. Weak induction of tau aggregates from Agg+ cells using conditioned medium may represent a better model of natural seeding and intercellular propagation in tauopathy than purified tau fibrils or tissue extracts complexed with transfection reagents. For these reasons, we decided to use conditioned medium as the seeding method in our screening for mutations that promote tau aggregate formation.

[0183] Screening for mutations that enhance tau aggregates. Figure 1B shows a screening protocol for discovering mutations that enhance tau aggregates. The inventors transduced Cas9-expressing subclones of tau biosensor cell lines with a human genome-scale CRISPR-Cas9 knockout (GeCKO) library packaged in a lentiviral expression vector. The combined hGeCKO-A and B half libraries contain 111,985 unique single gRNAs (6 gRNAs per gene) targeting 19,050 genes in the human genome, and 1,000 untargeted gRNAs as negative controls. The inventors performed transduction of the libraries at a low MOI to ensure that most cells accept at most one gRNA construct within a cellular applicability range of 300 gRNAs, and selected viral vector integration events in transduced cells by proliferation in the presence of puromycin. Six days after transduction of the GeCKO library, the inventors introduced a tau aggregate seeding agent by transferring the cells to a new medium consisting of a 3:1 mixture of Agg+ conditioned medium and fresh growth medium. The inventors sampled the transduced cell cultures on day 3 after transduction and on day 6 immediately before the addition of the conditioned medium seeding agent. After growing the cells for a further 4 days in the presence of the seeding agent, the cultures were harvested and FRET+ cells were purified by FACS. The inventors performed five replicate-versus-screenings using the GeCKO A library and the GeCKO B library separately, generating samples on day 3 and day 6, and FACS-purified cells on day 10.

[0184] As a control, the inventors performed the same pair of screenings but without the addition of a seeding agent. FACS analysis of these screenings did not detect FRET+ cells and was identical to seeding using conditioned medium from Agg- cell lines (Figure 1A). These results indicate that CRISPR-Cas9 mutagenesis alone, without a seeding agent, cannot induce spontaneous tau aggregates as measured by FRET. Either single-gene mutations are insufficient to induce tau aggregates, or such mutations are too rare to detect in the number of cells analyzed. Even in experimental screening using Agg+ conditioned medium seeding agent, the final FACS analysis at day 10 revealed that CRISPR-Cas9 mutagenesis did not enhance the proportion of FRET+ cells beyond the approximately 0.1% level produced in the absence of mutagenesis (Figure 1A). These results suggest that our CRISPR-Cas9 screening protocol failed to detect any mutations that could enhance seeding-induced tau aggregates, or that such mutations were too rare to result in a discernible increase in the small number of FRET+ cells produced by weak seeding agents.

[0185] To search for evidence of mutations that may have promoted tau aggregate formation, the inventors isolated DNA from a pool of 10 FRET+ cells and performed quantitative ILLUMINA® sequencing on PCR unit replication sequences derived from gRNA expression cassettes. The inventors employed two strategies to identify potential gRNA-specific mutations. First, the inventors used DESeq2 (Love et al., (2014), "Genome"), which is incorporated herein by reference in its entirety. Using Biol., 15:550, we identified gRNAs whose sequence reads were enriched in the FRET+10 day sample compared to the day 3 or day 6 sample, but not in the day 6 sample compared to the day 3 sample. Using enrichment factors of ≥ 1.5 (≧) and negative binomial Wald test p-values ​​< 0.05 (<) as cutoff thresholds, we found that 104 gRNAs associated with 100 genes were significantly enriched in the FRET+10 day sample compared to both the day 3 and day 6 samples. Of the 100 genes identified, only one, barrier-versus-self integration factor 1 (BANF1), was represented by four significantly enriched gRNAs (circles labeled "X" in Figure 5H). The remaining 99 genes were represented by a single significantly enriched gRNA. From CRISPR screening Because the generated read count data may not follow the data distribution assumed by the Wald test used by DESeq2, we have developed an alternative gene-centered enrichment analysis algorithm that does not require any assumptions about the data distribution across samples. Similar to DESeq2, our alternative algorithm has two components: an enrichment factor (referred to as a multiplier change in DESeq2) and an enrichment p-value. The enrichment factor is similar to that used in DESeq2, except that it is summarized at the gene level by averaging the enrichment of all gRNAs targeting the same gene. Unlike existing CRISPR data analysis methods, our method uses a hypergeometric distribution-based p-value calculation that applies only to the enrichment of different gRNAs targeting genes in each sample at FRET+10 days.The final enrichment p-value for a gene is the average of the p-values ​​obtained from each of the 10 FRET+10 day samples. Our alternative algorithm identified BANF1 as the top hit (Figure 1C) and confirmed the DESeq2 results. Based on a visual inspection of data quality between genes ranked by p-value, and enrichment from DESeq2 and our gene-centered method, we selected BANF1 and 13 other genes that showed enrichment with at least two active gRNAs for experimental validation.

[0186] To identify candidate genes, the inventors tested 30 individual GeCKO library lentiviral gRNA expression vectors—four for BANF1 and two each for 13 other genes—for their ability to enhance FRET induced by Agg+ conditioned medium. Transduction of tau biosensor cells by each of the four BANF1-targeted gRNAs (pink bars in Figure 1D), and both gRNAs targeting the PPP2CA gene (blue bars in Figure 1D), and the blue dots in Figure 1C, increased the induction of FRET signaling by 15–20 times compared to no gRNA or control gRNA (Figure 1D). None of the gRNAs for the other candidate genes promoted FRET enhancement. CRISPR-Cas9 disruption of BANF1 and PPP2CA did not induce FRET when fresh medium was used instead of conditioned medium (Figure 6A). Western blot assays confirmed that gRNA-targeted BANF1 and PPP2CA significantly reduced the protein produced from the targeted gene (Figure 6B). Furthermore, individual isolated BANF1 and PPP2CA knockdown clones revealed a positive correlation between the degree of gene editing and the enhancement of FRET induced by Agg+ conditioned medium (Figure 6C). Combined inactivation of BANF1 and PPP2CA resulted in a clear additive effect on FRET induction (Figure 6D), suggesting that these two genes are involved in a common function.

[0187] The validated gene hits from gene knockout screening encode proteins that are part of a functional network of components that maintain the nuclear membrane. To better understand the biology of the hits confirmed from the screening, we used the top hit BANF1 to search its protein-protein association network in String. See Szklarczyk et al., (2019), "Nucleic Acids Res.", 47:D607–D613, which is incorporated herein by reference in its entirety. The network revealed that BANF1 directly interacts with a second validated hit, PPP2CA, in a catalytic relationship (Figure 2A). In multiple functional enrichment analyses of the network, the nuclear membrane stands out as the most important feature. To test whether mutations in other components of the BANF1 interaction network could promote tau aggregate enhancement, we tested gRNAs for ANKLE2, VRK1, PPP2R2A, EMD, LEMD2, LEMD3, and TMPO for their ability to induce enhanced FRET (Figure 2A). We also included CHMP7 for its role in maintaining nuclear membrane integrity. Only gRNA-targeted ANKLE2 was able to induce a significant enhancement of FRET compared to the control (Figure 2B). The inventors confirmed that disruption of BANF1, PPP2CA, and ANKLE2 resulted in enhancement of tau aggregates indicated by FRET using three different seeding methods: sonicated whole cell lysates from tau-YFP Agg+ cells, purified misfolded recombinant tau fibrils (tau 244-372LM, Figures 5A-5D), and spinal cord extracts from tau P301S transgenic mice (Figure 2C). See, for example, Yoshiyama et al., (2007), "Neuron", 53:337-351, which is incorporated herein by reference in its entirety. Inactivation of ANKLE2 induced a stronger FRET signal than the loss of either BANF1 or PPP2CA, and combining ANKLE2 inactivation with inactivation of BANF1 or PPP2CA did not result in an additive effect (Figure 6D).Our primary, secondary, and tertiary screenings of biologically related genes point to an unexpected link between microtubule-associated protein tau and the biological processes that maintain the nuclear membrane (Figure 2D), suggesting that defects in nuclear membrane integrity may initiate pathogenesis or promote its spread in tauopathies.

[0188] Screening for genes that inhibit or prevent tau aggregate formation when overexpressed. Since disruption of BANF1, PPP2CA, or ANKLE2 enhances tau aggregate formation, the normal function of these genes may act to protect cells from tau pathogenesis. To discover further protective genes whose expression can reverse or prevent tau aggregate formation, we reversed the logic of our CRISPR-Cas9 gene disruption screening by using a SAM system for gRNA-targeted gene activation to screen for genes whose activated or enhanced expression neutralizes FRET induction in tau biosensor cells. Similar to gene disruption screening, we first established tau biosensor subclones that stably express components of the SAM system and confirmed that gene expression could be induced or enhanced using specific SAM gRNAs (Figure 6E). Next, as shown in Figure 1B, the inventors performed five repeated screenings of SAM-ready biosensor cell lines by transduction using a human SAM gRNA library, except that they replaced the weakly acclimatized medium seeding agent with sonicated lysates of tau-YFP Agg+ cells conjugated with lipofectamine™, a strong seeding agent. This seeding method routinely produces approximately 60% FRET+ cells compared to 0.1% for acclimatized medium. For overexpression screening, cells were sampled on days 7 and 10 after seeding agent addition. On day 13, FRET+ and FRET- cells were collected by FACS. For gRNA sequence enrichment analysis, the inventors used the DESeq2 algorithm to compare the FRET- population with both FRET+ cells and cells sampled on days 7 and 10. These analyses revealed two gRNAs for the gene LEMD2 that were significantly enriched in FRET cells. String analysis (Figure 2A) identified LEMD2 within the nuclear membrane functional network, where LEMD2 binds to BANF1 and, like ANKLE2, is an essential component of the inner nuclear membrane (Figure 2D). For secondary confirmation of LEMD2, the inventors used cDNA expression rather than dCas9-SAM transcription enhancement.The inventors tested cDNA expressing two isoforms of the LEMD2 and closely related LEMD3 proteins, as well as cDNA of CHMP7, because CHMP7 binds to LEMD2 and is involved in the repair of nuclear membrane disruption, thereby promoting membrane sealing (Figure 7A). The sequences of the lentiviral cDNA constructs are described in SEQ ID NOs: 34, 36, 37, and 39. Expression of all four cDNAs in biosensor cells reduced FRET induction by potent seeding agents (Figures 2E and 6F) and did not affect the expression of the MAPT-4RD transgene (Figure 6G). The LEMD2 isoform and CHMP7 showed stronger rescue than LEMD3.

[0189] To further investigate the protective properties of LEMD2, LEMD3, and CHMP7, the inventors tested the ability of cDNA overexpression of these proteins to rescue tau aggregates induced by the loss of BANF1 and ANKLE2. To set up these screenings, the inventors first established a tau biosensor subclone that stably expresses a catalytically dead form of Cas9 (dCas9-KRAB) fused to a Kruppel-associated box repression domain. Next, the inventors established stable knockdown of BANF1 and ANKLE2 gene expression by transduction with a promoter-targeted lentiviral gRNA expression vector. The seeding agent induced tau aggregates, as indicated by a strong FRET signal in BANF1 and ANKLE2 knockdown biosensor cells compared to control cells stably expressing the control gRNA, gNT303 (Figures 3A and 3B). As a positive control, BANF1 cDNA expression abolished seeding-induced FRET in BANF1 knockdown cell lines, but not in cells with ANKLE2 knockdown (Figure 7E). Expression of both isoforms of LEMD2, as well as cDNAs of LEMD3 and CHMP7, all abolished or significantly reduced FRET induction by two different seeding agents (Figures 3A and 3B). Expression of all four cDNAs did not reverse dCas9-KRAB transcriptional repression of BANF1 and ANKLE2 (Figures 7B and 7C). Although we did not evaluate tau-4RD protein levels, the expression of these cDNAs did not affect MAPT-4RD cDNA transgene expression as assessed by TAQMAN qRT-PCR (Figure 7D), indicating that the rescue of FRET induction by these cDNAs was a direct effect. The sequences of the lentiviral cDNA construct are described in SEQ ID NOs. 32, 34, 36, 37, and 39.

[0190] To investigate how LEMD2 expression affects the biochemical behavior of tau, the inventors analyzed the intracellular fractions of BANF1 and ANKLE2 knockdown cell lines, both with and without LEMD2 overexpression. In the absence of seeding agents, the inventors found that the majority of tau protein was distributed between the cytoplasmic and soluble nuclear fractions in control dCas9-KRAB biosensor cells expressing the control gRNA (upper left quadrant of Figures 3C and 3D). This pattern did not change with BANF1 or ANKLE2 knockdown, but treatment with tau-YFP Agg+ cell lysates caused tau accumulation in the insoluble fraction in BANF1 or ANKLE2 knockdown cells, but not in control cells (upper right quadrant of Figure 3C). Insoluble tau was phosphorylated on serine 356 (Figure 3C, lower right quadrant), which is a marker of tau aggregates in seeded tau biosensor cells and correlates with seeding activity in tau-YFP Agg+ cells (Figure 5F). Consistent with its ability to prevent tau aggregates as assessed by FRET (Figures 2E, 3A, and 3B), LEMD2 cDNA expression prevented the accumulation of tau in seeding-induced insoluble and phosphorylated biochemical isoforms (Figure 3D).

[0191] Loss of BANF1 or ANKLE2 induces mislocalization of the nuclear speckle component SRRM2. Our results demonstrate that loss of nuclear membrane components BANF1 and ANKLE2 enhances tau aggregates in tau biosensor cells and promotes the accumulation of P-tau-Ser356 in the insoluble intracellular fraction (Figures 3A-3D), but these changes in the biochemical properties of tau require induction by a seeding agent. Our biochemical fractions indicate that tau is an abundant component of the nucleus. Could the reduction of BANF1 and ANKLE2 lead to disruption of nuclear function that promotes susceptibility to tau seeding? Lester et al. showed that micronucleus and nucleolar RNA, as well as components of nuclear speckles and pre-mRNA splicing sites, are associated with tau aggregates induced by potent seeding agents in tau biosensor cells. Lester et al., (2021), "Neuron," 109(10):1675-1691, is incorporated herein by reference in its entirety. In particular, the nuclear speckle component SRRM2 co-localized with tau in large cytoplasmic aggregates. The inventors reproduced this result using tau-YFP Agg+ whole cell lysate as a tau seeding agent (Figure 3E). However, even without tau seeding, the inventors found that biosensor cells with dCas9-KRAB-induced BANF1 or ANKLE2 reduction showed mislocalization of SRRM2 from the nucleus to the cytoplasm (Figure 3F), but these cells still showed no signs of tau aggregates or phosphorylation (see biochemical analysis of similar cells in Figure 3C). In normal tau biosensor cells, immunofluorescence detection of SRRM2 is almost entirely confined to the nucleus. However, in BANF1-deficient and ANKLE2-deficient cells, some SRRM2 is clearly visible in perinuclear lesions (Figure 3F), indicating protein leakage in a form that appears to be locally enriched.Visual counting of immunofluorescence cells revealed that approximately half of the BANF1-deficient cells lost exclusive nuclear retention of SRRM2 and exhibited both nuclear and cytoplasmic localization patterns, while nearly 30% of the ANKLE 2-deficient cells showed both nuclear and cytoplasmic SRRM2 detection (Figure 3G). Using different types of quantitative analysis, the inventors determined the percentage of nucleus-related SRRM2 immunofluorescence in each cell tested. The majority of control cells had approximately 90% of their SRRM2 signaling in the nucleus. In BANF1-deficient and ANKLE 2-deficient cells, the inventors observed two populations: the first group was similar to the control, with 90% of SRRM2 retained in the nucleus; the second group showed a nearly equal distribution (40–60%) of SRRM2 between the nucleus and cytoplasm (Figure 3H). The inventors observed similar effects for BANF1. Decreased ANKLE2 expression leads to the accumulation of BANF1 protein in the cytoplasm and its loss from the chromatin-bound fraction (Figure 7F). These results suggest that loss of nuclear membrane integrity in the absence of apparent tau aggregates may be an initiation event that allows SRRM2, tau, and possibly other nuclear components to leak into the cytoplasm, ultimately inducing their aggregation.

[0192] Decreased expression of Banf1, Ankle2, and Ppp2ca leads to increased tau phosphorylation on serine 356 and impaired nuclear membrane integrity in mouse cortical neurons. Our gene screening was performed in highly proliferative HEK293T cells undergoing a constant cycle of nuclear disruption and reformation. To investigate the consequences of nuclear membrane perturbation in postmittal cells associated with tau pathology, we transduced wild-type primary mouse cortical neurons with lentiviruses in which Cas9 is co-expressed with a control gRNA, or a gRNA targeting Banf1, Ankle2, or Ppp2ca, or with the control gRNA. Gene disruption was confirmed by NGS and TAQMAN qRT-PCR (Figures 8B-8C). Disruption of Banf1, Ankle2, or Ppp2ca resulted in a significant increase in P-tau-Ser356 in the perinuclear domain and nucleocytoplasm (Figures 4A, 4D, 4E, and 8D). P-tau-Ser356 signaling was enhanced in the cell body upon knockdown of Banf1 or Ankle2, but not with Ppp2ca (Figure 4C). Disruption of any of the three genes did not affect total tau in the cell body (Figures 8F and 8I). Disruption of Banf1 resulted in a slight loss of tau detection in the perinuclear domain and nucleocytoplasm (Figures 8J-8K), while knockdown of Ppp2ca resulted in a slight increase in tau detection in the perinuclear domain (Figure 8J). Disruption of Ppp2ca appeared to cause some toxicity (Figures 4B, 8E, 8G, and 8H).

[0193] Next, the inventors sought to evaluate the effect of Banf1 and Ankle2 depletion on nuclear membrane integrity in primary mouse cortical neurons. For this purpose, the inventors used a fluorescent nuclear reporter (NLS: mCherry) and ASO-targeted mouse Banf1, Ankle2, or scrambled ASO sequences as a control. The ASO was introduced into mouse cortical neurons via gymnotic delivery, and qRT-PCR analysis confirmed the specific and significantly reduced expression of Banf1 and Ankle2 (Figures 4G and 4H). Live-cell imaging studies revealed an increased detection of mCherry in the cell bodies of mouse cortical neurons treated with Banf1 or Ankle2 ASO compared to control ASO, as is evident in Figure 4F. Quantification of mCherry fluorescence intensity revealed a significant increase in mCherry in the cell bodies of Ankle2 or Banf1 depleted neurons, likely due to nuclear leakage.

[0194] To further confirm the specific nuclear membrane-related effects of ASO-mediated depletion of Ankle2 and Banf1 in mouse cortical neurons, we replicated rescue experiments performed in biosensor cells (Figures 3A-3D). We treated mouse cortical neurons with control ASOs or ASOs targeting Banf1 or Ankle2, along with LV-cDNA encoding Lemd2, Lemd3, Chmp7, or a control luciferase. Depletion of Ankle2 or Banf1 in mouse cortical neurons increased P-tau-Ser356 in somatic cells, the perinuclear domain, and the nucleocytoplasmic domain (Figure 9A). This effect was significantly reduced or eliminated by co-expression of cDNA encoding Lemd2, Lemd3, or Chmp7 (Figures 9B-9D). Knockdown expression of Banf1 or Ankle2 was confirmed by qRT-PCR and cDNA expression (Figures 9E-9G). In summary, these results provide strong evidence of a link between nuclear membrane leakage and increased tau phosphorylation on serine 356 in both biosensor cells and primary cortical neurons. Although P-tau-Ser356 is a biomarker of tau aggregates, insolubility, and seeding activity (Figures 5D and 3C), the cortical neurons in these experiments were still not treated with a tau seeding agent. Unlike proliferating HEK293T biosensor cells, which require a seeding event to induce tau phosphorylation on serine 356 in response to the loss of Banf1, Ankle2, or Ppp2ca in postmitted cortical neurons, the loss of nuclear membrane components is sufficient to trigger biochemical changes in tau that may be precursors to pathogenesis.

[0195] Consideration Most research on tauopathy focuses on cell and tissue damage caused by widespread misfolding and aggregation of tau. Cellular and mouse models of the disease often utilize strong overexpression of mutant tau protein or application of high doses of misfolded amyloid fibrils derived from mutant forms. See, for example, Allen et al. (2002), "J. Neurosci.", 22:9340-9351, Frank et al. (2008), "Acta Neuropathol.", 115:39-53, and Yoshiyama et al. (2007), 53:337-351, which are incorporated herein by reference in their entirety. While these types of studies may model the impairments associated with the later stages of tauopathy, they do not necessarily address the underlying causes. In this study, our concern was different. The inventors sought to gain a better understanding of the initial events that initiate tau pathology by asking about the types of normal cellular processes, pathways, and functions that may promote tau misfolding and aggregation when disrupted.

[0196] Our initial gene screening, using CRISPR-Cas9 mutagenesis, discovered mutations that enhance tau aggregates in tau biosensor cell lines highly sensitive to tau seeding agents. See, for example, Holmes et al., (2014) "Proc. Natl. Acad. Sci. USA", 111:E4376-4385, and Sanders et al., (2014) "Neuron", 82 1271-1288, each of which is incorporated herein by reference in whole. We found that diluted conditioned medium from cells with stable tau aggregates could induce FRET in a small percentage of cells without the aid of transfection reagents. When biosensor cells were mutated in the absence of seeding agents, we found no FRET-positive cells. These results suggested that mutations in single genes causing spontaneous aggregation were extremely rare or nonexistent, even in systems primed for tau aggregates. However, when the inventors challenged mutagenic cells with a weakened seeding medium, they discovered two genes that promote tau aggregate formation when disrupted, as indicated by enhanced FRET. One gene, PPP2CA, is a major tau phosphatase and encodes a catalytic subunit of the 2A family of serine / threonine protein phosphatases associated with Alzheimer's disease. The other gene, BANF1, encodes a barrier-to-autointegration factor, a small, abundant, and highly conserved DNA-binding protein involved in several key cellular processes. Furthermore, homozygous missense mutations in the BANF1 gene have been found in patients diagnosed with progeria syndrome, in which cells exhibit morphological abnormalities of the nuclear membrane. PPP2CA and BANF1 are part of a functional network involved in the maintenance, circulation, and repair of the nuclear membrane (Figures 2A-2E, 3A-3H, and 7A-7F).Systematic testing by other members of this network identified a third gene, ANKLE2 (ankyrin repeat and LEM domain-containing protein 2), which promotes enhanced FRET in biosensor cells when disrupted.

[0197] BANF1 connects chromatin to the inner membrane of the nuclear membrane and interacts with the nuclear lamina (Figure 2D). It also binds to the LEM (LAP2 / Emerin / MAN1) domain of other protein components of the inner nuclear membrane. In dividing cells, phosphorylation of BANF1 by VRK1 kinase, a member of the BANF1 interaction network (Figure 2A), disrupts its binding to chromatin and LEM proteins, promoting nuclear membrane lysis before entering mitosis. After the completion of mitosis, ANKLE2 has two functions during nuclear membrane reformation: ANKLE2 inhibits VRK1 kinase activity and enhances PPP2CA phosphatase activity, promoting dephosphorylation of BANF1 so that chromatin can be reconnected to the nuclear membrane via its association with LEM proteins. In addition to its demonstrated function, the association of PPP2CA with Alzheimer's disease may be partly due to its involvement in the maintenance and reuse of the nuclear membrane. During interphase, BANF1 promotes the repair of nuclear membrane damage, coating nuclear DNA at the site of damage and preventing activation of the circular GMP-AMP synthetase-interferon gene (cGAS-STING) innate immune pathway.

[0198] The inventors confirmed the link between tau and the nuclear membrane by demonstrating that enhanced seeding induction of tau aggregates upon loss of BANF1 and ANKLE2 can be rescued by overexpression of LEMD2, its related protein LEMD3, or CHMP7, a protein involved in the repair of nuclear membrane damage. The inventors demonstrated that overexpression of LEMD2 not only neutralizes enhanced tau aggregates as measured by FRET, but also prevents the biochemical accumulation of phosphorylated insoluble tau on serine 356. The fact that LEMD2, LEMD3, and CHMP7 did not hit in loss-of-function screening for enhanced tau aggregates suggests that their roles in nuclear membrane maintenance may overlap. Given the role of CHMP7 in nuclear membrane repair in particular, their discovery in overexpression screening indicates a more prominent function of these three proteins in the repair of nuclear membrane damage, which would otherwise promote tau aggregate formation. Previous studies have shown a link between tau misfolding and defects in nuclear function. Pathogenic tau aggregates are associated with disruption of nucleocytoplasmic transport via the nuclear pore complex, which acts as a regulatory gatekeeper for transport into and out of the nucleus. In contrast, genes identified by our CRISPR-Cas9 screening encode proteins that maintain the fundamental barrier function of the nuclear membrane. Our biochemical fractionation and immunofluorescence analyses show that tau is abundant in the nucleus (Figures 3A–3H and 8A–8K), suggesting an important function in this cellular compartment. Lester et al. also showed that tau is an abundant component of the nucleus, and when induced to aggregate, it associates with nuclear and nucleolar small RNAs, as well as components of nuclear speckle (one of which is the RNA-binding protein SRRM2, which has been found to be mislocalized in the cytoplasm). Lester et al., (2021), "Neuron", 109(10):1675–1691, is incorporated herein by reference in its entirety. The inventors reproduced SRRM2 mislocalization by reducing the expression of BANF1 or ANKLE2, but in their case, they did not use a seeding agent and did not detect tau aggregates.Loss of a single nuclear membrane component initiated events associated with tau pathogenesis in Alzheimer's disease. Similarly, in primary mouse cortical neurons, a cell type more associated with tauopathy, we found that disruption of Banf1, Ankle2, or Ppp2ca led to enhanced production of P-tau-Ser356, particularly in the perinuclear and nuclear domains. The induction of this biomarker of tau aggregation, insolubility, and dissemination activity in mouse neurons not treated with a dispersant strongly suggested loss of nuclear membrane integrity as an initiating event for the promotion of tau aggregates and the initiation of tau pathogenesis. Suppression of nuclear envelope function alters the biochemical properties of tau and can disrupt the retention of nuclear components, allowing their appearance in the cytoplasm to form the nuclei of tau aggregates. Postmitral neurons that do not undergo a continuous cycle of nuclear membrane collapse and reconstruction may be more susceptible to events that impair nuclear membrane integrity.

[0199] Much of the interest in tau in neurodegenerative diseases has focused on its role in promoting axonal microtubule stability. What is the connection between axonal microtubules and the nuclear membrane? As axons lengthen during normal neuronal cell growth, this elongation may be transmitted to the nucleus via the cytoskeleton, potentially causing deformation of the nuclear membrane. Structural modifications of the nuclear membrane can alter the binding of BANF1 to chromatin, facilitating adaptive responses in gene expression. This same type of transmission between axonal microtubules and the nucleus can also cause transient disruption of the nuclear membrane, releasing some of the nuclear contents into the cytoplasm. One such component may be tau, which, upon release from its nuclear storage, can bind to unoccupied sites on growing microtubules. Both tau's nucleic acid-binding partners in the nucleus and microtubules in the cytoplasm are polyanions that can be bound by tau's positively charged microtubule-binding domain, implying an exchange of binding partners from the nucleus to the cytoplasm. Alternatively, if tau is released from the nucleus as a result of impaired nuclear membrane integrity in the absence of axonal microtubule synthesis, the tau enters the cytoplasm without available binding partners. This can promote tau misfolding, insolubility, and aggregation. Subsequent axonal microtubule growth may stimulate the release of tau from the nucleus, but this can become the seed of further tau aggregates, which can prevent its binding to newly formed microtubules, thereby compromising their stability, and are filled by previously aggregated forms. Thus, initial damage to the nuclear membrane can tilt the balance between nuclear tau and cytoplasmic tau towards dead-end cytoplasmic aggregate forms, leading to loss of neuronal health and function. Such a scenario can also be imagined for the RNA-binding protein TDP-43, which is primarily a nuclear component and is found in cytoplasmic aggregates at the end of the disease in many cases of amyotrophic lateral sclerosis and frontotemporal dementia not associated with TDP-43 mutations. In these cases, similar to tau, TDP-43 may lack a suitable cytoplasmic RNA binding partner to prevent its misfolding and aggregation.

[0200] Our results highlight nuclear membrane integrity as a promising new research area in efforts to understand the origins of tauopathic diseases and develop novel treatment approaches.

[0201] Materials and methods Tau FRET biosensor cell cultures. HEK293T tau-CFP / tau-YFP (tau) biosensor cells, expressing a transgene encoding the 4RD microtubule-binding domain of tau fused to a fluorescent reporter CFP or YFP, were grown in DMEM culture medium containing DMEM (GIBCO, catalog no. 11971-025) with 10% fetal bovine serum (GIBCO, catalog no. 16000-036) and 1% penicillin / streptomycin (GIBCO, catalog no. 15140-122), and maintained at 37°C with 5% CO2.

[0202] Primary mouse cortical neuron cultures. Wild-type primary mouse cortical neurons (MCNs) were purchased from ThermoFisher (GIBCO, catalog number A15586). Following the manufacturer's user guide, the cells were cultured in Neurobasal Plus Medium (GIBCO, catalog number A35829-01) + 1% B-27 Plus Supplement (GIBCO, catalog number A35828-01) + 1X GlutaMAX Supplement (GIBCO, catalog number 35050-061) in poly-D-lysine-treated 96-well plates (Greiner Bio-One, catalog number 655946) at a density of approximately 20,000 neurons per well, at a volume of 100 μL, and maintained at 37°C and 5% CO2. Three days after seeding, (Cas9+gRNA) constructs were transduced into nerve cells as mouse-targeted gRNAs Banf1_gRNA3, Ankle2_gRNA3, Ppp2ca_gRNA2, or control gRNA-untargeted 303 or gNT303. These constructs were cloned into pLentiCRISPR-v2 Cas9 expression vectors and packaged in lentivirus (GenScript). After 6 hours, half of the Neurobasal medium volume was replaced and replenished every 3-4 days. For ASO treatment, MCNs were treated three days after seeding via gymnotic delivery with ASO-targeted Banf1 or Ankle2 or scrambled control ASO (synthesized by IDT). MCNs were maintained in culture for 2 weeks before fixation for expression analysis and immunofluorescence analysis.

[0203] Lentiviral particle production. Lentiviral particles are produced after standard LIPOFECTAMINE®-mediated co-transfection of HEK293T cells using a transfer plasmid (expression vector, GenScript) encoding a gRNA library or individual gRNAs or Cas9 components, along with a second-generation packaging plasmid encoding the gag, pol, and rev genes, and a third plasmid encoding the VSV-G envelope. HEK293T cells are 10 × 10⁶ hours prior to transfection. 6Cells were placed in 150 mm cell culture dishes in DMEM medium containing DMEM (GIBCO, catalog no. 11971-025) at a cell / plate density, supplemented with 10% fetal bovine serum (GIBCO, catalog no. 16000-036) and 1% penicillin / streptomycin (GIBCO, catalog no. 15140-122). On the day of transfection, the DMEM medium was replaced with Opti-MEM medium (GIBCO, catalog no. 31985-070) supplemented with 25 nM chloroquine (Sigma-Aldrich, catalog no. C6628-25G). DNA mixtures were prepared by mixing 20 μg of transfer DNA and 20 μg of packaging DNA, and by mixing 10 μg of membrane DNA in 1.5 mL of Opti-MEM with 60 μL of PLUS® reagent (GIBCO, catalog no. 11514015). In parallel, 100 μL of LIPOFECTAMINE® LTX (Life Technologies, catalog no. 15338500) was diluted in 1.5 mL of OptiMEM medium. Both mixtures were mixed for 20 minutes and then added to the cells. The culture medium was changed 6 hours after transfection. The cells were cultured at 37°C in an incubator with a 5% CO2 atmosphere. After 48 hours, the culture medium containing lentivirus particles was centrifuged to remove debris and filtered. The supernatant was treated with DNase to remove residual DNA. Lentivirus batches were concentrated by ultracentrifugation and resuspended overnight in phosphate-buffered saline (PBS, GIBCO, catalog no. 14040-133). The virus particles were finally divided into equal portions and stored at -80°C. Lentiviral vectors were titrated using the NucleoSpin RNA virus kit (Takara, catalog no. 740956.250) and the Lenti-X qRT-PCR titration kit (Takara, catalog no. 631235). Lentiviral gRNA library particle titers were determined by limiting dilution (adapted protocol from SIGMA Mission RNAi).

[0204] Expression and purification of recombinant human MAPT (Q244-E372; P301L, V337M) protein. Recombinant human tau (Q244-E372; P301L, V337M) protein (tau244-372LM) was expressed in BL21(DE3)-transformed E. coli. Lysis and purification of tau244-372LM protein were performed in the same manner as in published reports. The supernatant from the cell lysates was passed through HiTrap SP-Sepharose HP resin to isolate tau244-372LM protein. Next, the peak fraction from this process was applied to a Superdex 75 26 / 600 column (Cytiva) and eluted in 50 mM HEPES, pH 7.0, 50 mM NaCl, 1 mM EDTA, 2.5% glycerol, and 10 mM dithiothreitol. SDS-Page analysis showed that the protein was purified to a homogeneity of ≥90%. The concentration of this substance was determined relative to the standard curve in the colorimetric BCA assay kit from Pierce.

[0205] Preparation of recombinant human MAPT (Q244-E372; P301L, V337M) fibrils. A stock concentration of low molecular weight heparin (United States Pharmacopeia, catalog number 1235820; average molecular weight 4370 daltons) was prepared by dissolving enoxaparin sodium in Milli-Q water. A 73 μM recombinant tau 244-372LM protein was mixed with freshly prepared 1 mM dithiothreitol and 18 μM heparin, and then transferred to a small polycarbonate container. A 10 mM magnetic Teflon® stirring bar was added to the container. The solution was cultured at 37°C for 4 days with constant stirring at 750 rpm. The fibrils were recovered by ultracentrifugation at 150,000 rcf for 30 minutes in a Beckman T-55 bio-contained rotor. Following the initial rotation, excess heparin and soluble proteins were removed from the fibril pellet by performing three washing steps. Briefly, the supernatant was discarded, and the fibril pellet was resuspended in several milliliters of 50 mM HEPES, 25 mM NaCl buffer, and then centrifuged again. Before division, the fibril pellet was resuspended in the same buffer as above and then sonicated in a Qsonica cup horn at 50% amplitude for 30 seconds. This step helped to uniformly disperse the fibril in the solution and break up any visible clumps.

[0206] Characterization of tau aggregates by thioflavin T fluorescence. Aggregates of purified recombinant tau-244-372LM monomer were monitored by measuring the fluorescence of thioflavin T (ThT), an amyloid-specific dye, at 485 nm (excitation 443 nm). Samples of tau-244-372LM monomer (50 μM, 25 μM, and 10 μM) were prepared from a 73 μM stock by dilution in buffer (50 mM HEPES, pH 7.4, 30 mM NaCl). Each concentration of tau was transferred three times to a black 96-well microplate with newly prepared ThT in a 2-fold molar excess relative to tau-244-372LM. Heparin was added to three wells at each concentration (12.5 μM, 6.25 μM, and 2.5 μM heparin) in a heparin-to-tau molar ratio of 1:4. Fluorescence was collected over 120 hours using a BMG ClarioStar plate reader. The plate was maintained at a constant 37°C and subjected to constant dual-orbit shaking at 700 rpm. Measurements were recorded every 15 minutes through the bottom of the plate.

[0207] Transmission electron microscopy of recombinant tau-244-372LM fibrils. Tau-244-372LM fibrils were diluted 1:5 with buffer (50 mM HEPES, pH 7.4, 25 mM NaCl). Approximately 8-10 μL of the sample was applied to the center of a 300-mesh formvar-coated copper grid and incubated for 2 minutes. The remaining liquid was then absorbed using filter paper, and simultaneously, 10 μL of 1% uranyl acetate staining solution was applied. Next, excess staining was absorbed using filter paper again. Images were acquired at 80 kV using a JEOL 1200 EX electron microscope.

[0208] Development of stable tau aggregate cell clones as a source of tau seeding material. HEK293T tau-YFP cells were seeded in DMEM medium at a density of 500,000 cells / well in a 6-well dish. The following day, the cells were treated with purified tau 244-372LM fibrils. Tau fibrils (1 μg) were placed in LIPOFECTAMINE® 2000 (Invitrogen, catalog number 11668-019) in Opti-MEM medium, cultured at room temperature for 20 minutes, and then added to the wells. The following day, the cells were passaged in a 96-well plate using serial dilutions, so that each row of the plate received a 2-fold dilution compared to the previous row. The plates were magnified and visually inspected to identify wells containing single colonies. Single-cell derived clones were further examined by fluorescence microscopy to identify clones that contained tau-YFP aggregates, Agg+, in all cells and maintained these aggregates over several passages. Three clones were validated to evaluate their ability to produce tau seeding activity. Briefly, aggregate-containing cells were grown in a T175 flask until dense, the medium was replaced with fresh DMEM medium, and the medium was collected after a 4-day incubation. This conditioned medium was then centrifuged at 800 rpm for 5 minutes to remove necrotic tissue fragments, and divided into aliquots and stored at -80°C. To test tau seeding activity, HEK293T tau-CFP / tau-YFP (tau) biosensor cells were cultured for 3 days in 75% conditioned medium:25% DMEM medium, and then collected for analytical flow cytometry using CytoFLEX LX (Beckman Coulter) to measure the FRET signal in seeded cells. Based on the retention of its aggregates in all cells across many passages, and the ability of the clone 18 conditioned medium to consistently induce approximately 0.1% FRET signaling in approximately 0.1% tau biosensor cells, which we defined as a minimal tau seeding treatment, we selected tau-YFP Agg+ clone 18 for further proliferation.

[0209] As an alternative source of tau seeding activity, whole cell lysates were collected from tau-YFP Agg+ clone 18. Cells were grown in six T175 flasks. When dense, cells were collected by scraping into cold PBS. The whole cell pellet was resuspended in 4 ml of fresh PBS with 40 μL of EDTA (ThermoFisher, catalog no. 1861283), 40 μL of HALT protease, and a phosphatase inhibitor cocktail (ThermoFisher, catalog no. 78446), and sonicated for 3 minutes at 51 Amp using a Qsonica Q500 sonicator. After final centrifugation, the supernatant containing whole cell lysates was collected, protein concentration was determined, then equalized and stored at -80°C.

[0210] Selection of Cas9 expressing tau biosensor clones. HEK293T tau-CFP / tau-YFP (tau) biosensor cells were grown in DMEM medium and transduced in 24-well dishes at high MOI (multiple doses of infection) in the presence of 8 μg / mL Polybrene (Millipore, catalog no. TR-1003-G) using a lentivirus-packaged pLentiCas9-Blast vector (GenScript). After 24 hours, the medium was replaced with DMEM medium containing 10 μg / mL blastosidine (Invivogen, catalog no. ant-bl-1), and the cells were grown selectively. On day 3 after transduction, the cells were passaged in serial dilutions. Eight grown single-cell clones were evaluated for both their Cas9 expression levels and their cleavage activity. Cas9 mRNA expression levels were measured using the Quick RNA96 kit (Zymo Research, catalog number R1053), followed by TaqMan qRT-PCR using the Quantinova One-Step RT-PCR kit (Qiagen, catalog number 208352), and then by the VG_Cas9U2 TaqMan assay and reference assay β2M. Samples were run on a QuantStudio real-time PCR system (ThermoFisher). Cas9 cleavage activity was determined as the percentage of indel alleles after transduction of PERK gRNA6 at the PERK locus. This gRNA was cloned into a pLentiGuide-Puro vector (GenScript), packaged into a lentivirus, and transduced into a Cas9 clone with three replication wells. After 24 hours, the medium was replaced with DMEM medium containing 1.5 μg / mL of puromycin (Invivogen, catalog number ant-pr-1). Cells were grown under puromycin selectivity. Transduced cells were collected on days 3 and 7, and genomic DNA was extracted for digital PCR analysis (dPCR) using the Blood & Cell Culture DNA Mini Kit (Qiagen, catalog number 13323).dPCR was performed using QuantStudio 3D Digital PCR Master Mix V2 (ThermoFisher, catalog number A26358) with VIC-labeled copy number reference assays, human TERT (ThermoFisher, catalog number 4403315), and FAM-labeled assays targeting PERK_gRNA6 cleavage sites. The dPCR reaction products were loaded onto QuantStudio 3D Digital PCR 20K Chip v2 (ThermoFisher, catalog number A26316) and executed using the ProFlex 2X Flat PCR System (ThermoFisher, catalog number 4484078). The luminescence of FAM and VIC dyes was analyzed using QuantStudio 3D Analysis Suite software. PERK gRNA6 cleavage efficiency was determined as a percentage of the FAM / VIC ratio. Clones E were enlarged and genome-wide CRISPRn screening was performed. See Tables 4-9.

[0211] Selection of dCas9-SAMs expressing tau biosensor clones. Tau biosensor cells were transduced with pLentidCas9-P64-Blast and pLentiMS2-P65-HSF1-Hyg vectors (GenScript) packaged in lentivirus (as described above). Cells were grown under selective conditions of 5 μg / mL blastosidine and 100 μg / mL hygromycin (Invivogen, catalog no. ant-hg-1). On day 3 post-transduction, cells were passaged in serial dilutions. Nine single-cell expansion clones were evaluated for both their transgene expression levels and gene activation activity. The levels of dCas9, VP64, MS2, and P65 mRNA expression were evaluated by TaqMan qRT-PCR using the following assays: Cas9D_VG_SAM, VP64_VG_SAM, MS2_VG_SAM, and p65_VG_SAM with reference β2M. Based on the high expression of both components of the dCas9-SAM system, clone DC11 was expanded using the ΔCt method for further validation. See Tables 8 and 9.

[0212] Eleven gRNAs targeting genes (l1B, LIN28A, UBA52, RANBP1, EEF1A1, ZFP42, PIN1, ATG7, RBM17, DDX42, and STUB1) were transduced into the tau biosensor dCas9-SAM clone DC11 (Figure 6E). These genes were selected based on their basal transcription levels in tau biosensor cells, as determined by RNA-seq transcriptional profiling analysis and as previously reported, indicating that SAM-mediated activation ratios are inversely correlated with basal transcript levels. See Duval et al., (2015), Nature, 517:583-588, which is incorporated herein by reference in its entirety. The gRNAs were cloned into the pLenti_sgRNA(MS2)_zeo vector (GenScript) and packaged in lentiviruses. gRNAs 1-3 for each gene target were pooled and transduced into DC11 cells in three replication wells per target. After 24 hours, the culture medium was replaced with DMEM medium containing 600 μg / mL zeosin (Invivogen, catalog no. ant-Zn-1). On day 7, transduced cells were collected and mRNA expression analysis was performed by TaqMan qRT-PCR using TaqMan Gene Expression Assays (ThermoFisher) in conjunction with reference assay β2M. Relative expression was calculated based on the ΔΔCt method and normalized for each assay to the mean of samples transduced with gRNAs targeting other genes as a non-targeting control. See Tables 4-9.

[0213] Design and cloning of dCas9-KRAB expression vector. The Streptococcus pyogenes Cas9 nuclease sequence was obtained from the National Center for Biotechnology Information (NCBI, accession number NP_269215) in the United States. The nucleic acid sequence was modified to include the Kozak signal, the N-terminus nuclear localization signal (NLS), and a C-terminal NLS linker fused to the KRAB domain of human zinc finger protein 10 (accession number CAA36558). The nucleic acid sequence was codon-optimized using MacVector 18.1.5, and the nuclease cleavage domain was inactivated by incorporating D10A and N863A amino acid substitutions. The A2A peptide was incorporated to support the co-expression of dCas9-KRAB and blastosidine-S deaminase from the EF1α promoter. The complete sequence was synthesized using GenScript and cloned into a lentiviral skeleton for packaging.

[0214] Selection of dCas9-KRAB expressing tau biosensor clones. Tau biosensor cells were transduced with lentivirus-packaged pLentidCas9-KRAB-Blast. Cells were grown using 5 μg / mL blastosidine selection. Two days after transduction, cells were passaged in serial dilutions. Ten single-cell expansion clones were evaluated for transgene expression. The level of dCas9-KRAB mRNA expression was assessed by TaqMan qRT-PCR using VG_Cas9D and 2629_KRAB.P assays and reference assay β2M. Transcriptional repression activity was determined in three clones showing the highest levels of dCas9-KRAB expression. Activity was determined by measuring the mRNA levels of four target genes (EGFR, HGF, HSPA8, and NEDD4) after transduction of gRNA targeting a specific sequence located within 200 bp of the transcription start site of these genes. The gRNA was cloned into a pLentiGuide-Puro vector (GenScript), packaged into a lentivirus, and transduced into a dCas9-KRAB clone. Cells were grown under selective 1.5 μg / mL puromycin conditions, and cells were collected 7 days post-transduction for target mRNA expression analysis by TaqMan qRT-PCR using TaqMan Gene Expression Assays (ThermoFisher) and reference assay β2M. Clone TK-B4 was determined to have the highest level of dCas9-KRAB expression, inducing the strongest transcriptional repression, and was selected for subsequent validation experiments. See Tables 4-9.

[0215] Genome-wide CRISPRn screening using the hGeCKO library combined with minimal tau seeding. The combined hGeCKO-A and B half libraries contain 111,985 unique single gRNAs (6 gRNAs per gene) targeting 19,050 genes in the human genome, and 1,000 untargeted gRNAs as negative controls. The inventors performed transduction of the library at a low infection multiplicity (MOI of 0.3) to ensure that most cells accepted at most one gRNA construct within the cell coverage range of 300 cells per gRNA, and selected for viral vector integration events in cells transduced by growth in the presence of puromycin. The inventors sampled transduced cell cultures 3 and 6 days after transduction of the GeCKO library and then induced tau aggregates by transferring the cells to a fresh medium consisting of a 3:1 mixture of Agg+ conditioned medium:fresh growth medium. After further growth for 4 days, the culture was harvested and FRET+ cells were purified by FACS (Figure 1B). Cas9-expressing clone E tau biosensor cells were grown under blastosidine selection and 15 × 10¹⁶ cells were placed in four T175 flasks. 6 Cells were seeded at the density of the flasks in DMEM medium. On day 0, cells were transduced in DMEM medium containing 8 μg / m2 of polyblen using an hGeCKO-A or hGeCKO-B lentiviral packaging gRNA library at a MOI of 0.3, with an applicability of 300 cells per intrinsic gRNA in the library. After 24 hours, the medium was replaced with DMEM medium containing 1.5 μg / mL of puromycin and 10 μg / mL of blastosidine, and the cells were grown under both blastosidine and puromycin selectivity. On day 3 posttransduction, the flasks were washed with PBS using 0.05% trypsin-EDTA (GIBCO, catalog no. 25300-054) neutralized in DMEM medium, and the cells were detached. The cell suspensions from the four T175 flasks were combined and transferred to three T175 flasks at a dilution ratio of 1:4 in DMEM medium. The remaining cells were counted and approximately 20 x 10⁶ cells were selected for genomic DNA isolation and NGS analysis. 6The cells were pelleted. Six days after transduction, the cells were transferred to two T175 flasks at a 1:4 dilution ratio (no selection). In each T175 flask, 30 mL of tau-YFP Agg+ conditioned medium was added to 10 mL of cell suspension. Approximately 20 × 10 6 Individual cells were also collected for genomic DNA isolation and NGS analysis. Ten days after transduction, cells were collected for flow cytometry using a MoFlo Astios Cell Sorter (Beckman Coulter). The cells were pelleted into 10 × 10⁶ cells. 6 Cells were resuspended via a cell-strainer cap tube (Falcon, catalog no. 352235) in Hanks' Balanced Salt Solution (HBSS, GIBCO, catalog no. 14175-079) containing 2% FBS at a concentration of cells / mL. The collection tube was coated with 80% FBS and 20% DMEM medium. FRET-positive cells, FRET+, were pelleted for genomic DNA (gDNA) isolation and NGS analysis. CRISPRn screening was repeated 5 times using both hGeCKO sublibraries, for a total of 10 genome-wide CRISPRn screenings.

[0216] Next-generation sequencing was performed. The gRNA library was multiplexed and sequenced on NextSeq 500 (Illumina) to generate 1×80 base pair (bp) single-end reads. After demultiplexing using bcl2fastq (Illumina), the reads were screened for the 16 bp rival vector sequence leading to the gRNA, and the downstream 20 bp gRNA reads were extracted for gRNA count. DESeq2 analysis was performed on OmicSoft Studio software version 10.0.1.118 (Qiagen).

[0217] Gene-centered enrichment analysis. The plicativity change for each gene was obtained by calculating the arithmetic mean of the log2-converted FRET+10 day to 3 or 6 day ratios generated for the corresponding gRNA by DESEq2. See Love et al., (2014), "Genome Biol.", 15:550, which is incorporated herein by reference in its entirety. The enrichment p-values ​​for each gene were calculated as follows: Firstly, in each FRET+10 day sample, gRNA was considered present if its DESeq2-normalized read count was 30 or greater, and the inventors considered read counts less than 30 as background noise. The enrichment of the presence of gRNA corresponding to a gene in the FRET+10 day sample was calculated using the hypergeometric distribution-based function phyper(x, m, n, k, lower.tail=FALSE) in R (https: / / www.R-project.org / ). The number of gRNAs corresponding to gene minus 1 is x, the total number of gRNAs corresponding to the gene in the library is m, the total number of gRNAs in the library minus m is n, and the total number of gRNAs present in the FRET+ sample is k. Next, the p-values ​​were converted to log10 and averaged over all 10 FRET+ day 10 samples.

[0218] Genome-wide CRISPrA screening using an hSAM library combined with maximal tau seeding. dCas9-SAM clone DC11 tau biosensor cells were grown under selective blastosidine and hygromycin conditions, resulting in 23 × 10⁶ cells per flask. 6Cells were seeded at a density of 100 cells into four T175 flasks. On day 0, cells were transduced in DMEM medium with an hSAM lentiviral packaging gRNA library at a MOI of 0.3, with an application range of 300 cells per intrinsic gRNA in the library, in the presence of 8 μg / mL polyblen. After 24 hours, the medium was replaced with DMEM medium containing 600 μg / mL zeosin, and cells were grown under triple zeosin, blastosidine, and hygromycin selective conditions. On days 3 and 7 post-transduction, cells were detached, and the cell suspensions from the four T175 flasks were combined and transferred to three T175 flasks in DMEM medium at a dilution ratio of 1:4. On day 10, cells were detached and transferred to two T175 flasks in DMEM medium (unselective) at a dilution ratio of 1:4. A maximum seeding treatment consisting of a mixture of LIPOFECTAMINE® 2000 (4 μL / mL medium) combined with tau-YFP Agg+ whole cell lysate (5 μg / mL medium) was directly added to the cells. Cell samples were also collected on days 7 and 10 for genomic DNA isolation and NGS analysis. On day 13, cells were collected for flow cytometry using a MoFlo Astios Cell Sorter to isolate FRET+ and FRET- cells, which were then pelleted for DNA isolation and NGS analysis. Genome-wide CRISPrA screening was repeated five times.

[0219] Next-generation sequencing was performed on NextSeq (Illumina) using an 80-cycle multiplexed single-read run. The generated data was demultiplexed using unique index reads. The gRNA count was determined based on perfectly matched sequencing reads of both the reader sequence and the gRNA sequence.

[0220] Differential gRNA read count analysis. To compare gRNA read counts between two groups of samples, the inventors used a DESeq2 Generalized Linear Model (GLM) and tested for differences in abundance using Wald's test based on a negative binomial distribution in ArrayStudio (OmicSoft). Using the terms "experiment" and "FRET status," a FRET+ vs. FRET- comparison model was constructed between samples at 13 days across five experiments. gRNAs with significant differences in read count between the comparison groups were identified using the criteria of i) multiplicative change ≥ 1.5 in either direction, and ii) p-value < 0.05. The gRNAs enriched in the FRET samples were further defined using the following three criteria: i) FRET- significantly increased in the 13-day sample compared to FRET+, ii) FRET- significantly increased in the 13-day sample compared to the 10-day sample, and iii) FRET+ did not significantly increase in the 13-day sample compared to the 10-day sample (either no significant difference or a significant decrease). The inventors paid particular attention to gene targets having multiple gRNAs enriched in the FRET samples.

[0221] Preparation of cell samples transduced with -hGeCKO or -hSAM libraries for NGS. Genomic DNA was extracted from cell pellets using the Blood & Cell Culture DNA Midi & Cell Culture DNA Midi kit (Qiagen, catalog no. 13343) or the QIAamp DNA mini-kit (Qiagen, catalog no. 51304). For the hGeCKO library, genomes from samples on day 3 and day 6 were prepared using a two-step nested PCR strategy. In the first PCR, 130 μg of genomic DNA per sample was amplified to achieve a 300-fold coverage of the total intrinsic gRNA in the library. For each sample, 13 PCR reactions of 100 μL each, containing 10 μg of genomic DNA in each reaction, were performed using NEB Next High-Fidelity PCR Master Mix (New England BioLabs, catalog no. M0541S) and then mixed. For the second nested PCR, to bind the Illumina adapter, and for multiplexing the NGS run, ten PCR reactions were performed on the barcoded sample. FRET+ samples on day 10 (approximately 50,000 cells per sample) were amplified from the whole cell population based on 10 μg of amplified DNA per 100 μL of the first PCR reaction. 5 μL of the first PCR reaction, along with equimolar amounts of nine different forward primers and reverse primers with unique barcodes, were used for the second PCR (Table 3). All primers were used at a final concentration of 0.5 μM. PCR cycles began with initial denaturation at 98°C for 30 seconds; followed by 18 cycles for PCR1 and 15 cycles for PCR2, with denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, and final extension at 72°C for 5 minutes. The PCR products were combined, concentrated using the DNA Clean & Concentrator kit (Zymo Research, catalog number D4034), purified using the Pippin Prep instrument (DNA size selection system, Sage Science), and then subjected to NGS.For the hSAM library, 140 μg of genomic DNA from cell samples on day 7 and day 10 was amplified over a 300-fold coverage range of the SAM gRNA library. Both FRET+ and FRET- samples on day 13 were amplified from the entire cell population. Only single-step PCR was performed using 2.5 μg of DNA per 100 μL reaction, 10 different forward primers, and a reverse primer with a unique barcode. The PCR cycle consisted of 26 cycles starting with initial denaturation at 98°C for 30 seconds; followed by denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds; and final extension at 72°C for 5 minutes. After purification of the combined PCR products, they were subjected to NGS. See Table 3.

[0222] Validation of individual tau modifying factor targets by minimal tau seeding treatment. Based on the analysis of CRISPRn screening using the hGeCKO library, 30 gRNAs targeting 14 different candidate genes, as well as a control gRNA, were purchased (GenScript) and packaged in lentiviruses. Individual gRNAs were transduced into Cas9 clone E tau biosensor cells and grown for 6 days under puromycin selectivity. Each gRNA was transduced into three replication wells. On day 7, transduced cells were supplemented with either tau-YFP Agg+ cell-derived conditioned medium or control fresh DMEM medium in a 75% conditioned medium vs. 25% DMEM medium ratio. On day 10, cells were collected for analytical flow cytometry using CytoFLEX LX to assess the percentage of cells positive for the FRET signal. Briefly, cells were washed with PBS, trypsin-treated to form single-cell suspensions, centrifuged at 800 rpm for 5 minutes, and resuspended in 200 μL of HBSS + 2% FBS. Cells were filtered through a cell strainer tube, transferred to a 96-well round-bottom plate, and stained with 0.5 μL of 7-aminoactinomycin D (7-AAD, ThermoFisher, catalog no. A1310) to assess cell viability. The FRET+ cell percentage was assessed as the percentage of live single CFP+ / YFP+ cells that were FRET+. Integrated FRET Density (IFD) was calculated as the product of the percentage of FRET+ cells and the median fluorescence intensity (MFI) of FRET+ cells. See Tables 4-9.

[0223] Determination of gene editing at gRNA cleavage sites of BANF1, ANKLE2, and PPP2CA. Cas9 clone E tau biosensor cells were transduced with BANF1 gRNA1, PPP2CA gRNA5, and a control gRNA, and grown under puromycin selectivity. Three days after transduction, the cells were passaged and grown using serial dilutions. Nine single-cell-derived clones (seven for BANF1 and two for PPP2CA) were evaluated for FRET signaling in response to seeding in conditioned medium from tau-YFP Agg+ and tau-YFP Agg- cells, as well as for the level of gene editing at BANF1 and PPP2CA gRNA cleavage sites. This was evaluated by amplifying the region surrounding the cleavage site and performing NGS to characterize the sequence and prevalence of indel alleles. The percentage of NGS reads from each clone characterized by indels, as a percentage of total reads, was determined to represent the percentage of gene editing.

[0224] NGS unit replication sequence library preparation. Target-specific oligonucleotides (21–27 base pairs, bp) were designed, and a maximum unit replication sequence size of 350 bp was generated at a primer melting temperature (Tm) of 60–65°C. Barcode adapter sequences were added to the target-specific oligonucleotides (Tables 8 and 9), and the complete sequences were ordered from Integrated DNA Technologies (IDT). PCR was completed for each DNA sample. Briefly, in each reaction, 4 ng of DNA was mixed with IDT oligonucleotides, Q5 polymerase (New England Biolabs, catalog number M0491), 10 μM dNTPs, buffer, and water, according to the manufacturer's specifications. The amplified product was then diluted 1:100 and used in PCR barcoding reactions to prepare the final sequencing library. Each barcoding reaction contained a single amplified target with forward and reverse primers containing unique barcodes and indices. Each PCR plate was pooled by volume and then purified in a single tube using AMPure XP reagent (Beckmann-Coulter, catalog number A63881) according to the manufacturer's instructions. The final library concentration was measured using a Qubit fluorometer (Invitrogen, catalog number Q32866). The 4 nanomolar prepared library was loaded into an Illumina MiSeq using a 2 × 300 read kit (Illumina, catalog number MS-102-3003) according to the manufacturer's instructions.

[0225] Sequence mapping and characterization. Barcoded samples were demultiplexed into individual reads (FASTQ format). Next, the forward and reverse reads of each FASTQ file were merged using PEAR (ncbi.nlm.nih.gov / pmc / articles / PMC3933873 / ). The merged reads were mapped to the Mus musculus genome version 9 (mm9) using Bowtie2 (ncbi.nlm.nih.gov / pmc / articles / PMC3322381 / ). Each sample was sequenced with a minimum of 20,000 merged reads across the expected cleavage site. Finally, barcoded samples were characterized using a custom Perl script. Briefly, all insertions, deletions, or base changes (INDELs) within a 20-base window upstream and downstream of the expected cleavage site were considered to be CRISPR / Cas9-induced modifications. The number of reads containing INDEL was compared to the number of reads with wild-type sequences to determine the edit percentage per animal and tissue.

[0226] STRING database search. BANF1 was used as input for the String database search (https: / / string-db.org). The settings were a minimum required interaction score of 0.4 and a maximum number of interactions for the first shell <= 10.

[0227] Gene expression analysis by qRT-PCR. For all other gene expression analyses in tau cells, RNA was extracted using the Zymo Quick RNA Cell 96 Kit, and the samples were diluted to 10 ng / μL. Gene expression analysis was performed using the QuantiNova Pathogen+IC Kit (Qiagen, catalog number 208654) on a QuantStudio thermocycler (ThermoFisher) in a 384-well PCR plate. TaqMan gene expression assays (ThermoFisher or custom-designed) were labeled with FAM, and the reference assay GAPDH was labeled with VIC, allowing them to function as internal controls in each well. See Tables 8 and 9.

[0228] Cloning of modifying factor cDNAs into the lentiviral scaffold. Based on the analysis of CRISPrA screening using the hSAM library, cDNA sequences encoding full-length LEMD2, short isoform LEMD2 (LEMDi2), LEMD3, CHMP7, or luciferase (Luc) were cloned into expression vectors as control cDNAs and packaged into lentiviral LV-cDNA. The cDNA and protein sequences were obtained from Ensembl and confirmed with Uniprot. Expression plasmids for cDNA sequence expression were prepared by synthesizing (GenScript), and by subcloning human and control luciferase cDNAs into the pLVX-pEF1α-IRES-Hyg expression vector, and mouse and control luciferase cDNAs into the pLVX-phSynapsin1-IRES-Hyg expression vector (an expression vector developed by Regeneron). The cDNA fragments were inserted downstream of the EF1α promoter using Spe-I restriction sites and Not-I restriction sites (GenScript). Nucleic acid sequences were obtained by reverse translation of protein sequences (using the MacVector 18.1.5 reverse translator tool) and codon-optimized for expression in human and mouse cells. Human protein catalog numbers: BANF1 (O75531), LEMD2 (Q8NC56), LEMD2-isoform 2 (Q8NC56-2), LEMD3 (Q9Y2U8), CHMP7 (Q8WUX9). Mouse protein catalog numbers: Lemd2 (Q6DVA0), Lemd3 (Q9WU40), Chmp7 (Q8R1T1). As a control cDNA, the coding sequence of the reporter gene luciferase was cloned into the same expression vector. For live-cell imaging studies, NLS:mCherry cDNA was cloned into the EF1α promoter upstream of the same expression vector. Plasmids were sequenced by Sanger sequencing before lentiviral packaging.

[0229] Expression of tau modifier genes by transduced cDNAs. To test the effect of overexpression of LEMD2, LEMDi2, LEMD3, CHMP7, or luciferase cDNAs on tau aggregates, packaged lentiviruses were transduced into tau biosensor cells. The sequences of the lentiviral constructs are set forth in SEQ ID NO: 32, SEQ ID NO: 34 to SEQ ID NO: 40. After 24 hours, the medium was replaced with DMEM medium containing 50 μg / mL hygromycin, and cells were grown under hygromycin selection. On day 3 post-transduction, the transduced cells were seeded with maximum tau seeding treatment as LIPOFECTAMINE™ 2000 (4 μL / mL) and tau-YFP Agg+ cell lysate (5 μg / mL). On day 4, cells were harvested for analytical flow cytometry using a CytoFLEX LX, and FRET signals were measured in the seeded cells. Transduced cells were also harvested for cDNA expression analysis by TaqMan qRT-PCR using a TaqMan assay designed to specifically amplify codon-optimized cDNA and the MAPT-4RD transgene. See Figures 6F, 6G, Table 8, and Table 9.

[0230] To test the effects of overexpression of these modifying factor targets on biosensor cells with increased tau aggregate tendency, cDNA encoding LEMD2, LEMDi2, LEMD3, CHMP7, or luciferase was transduced into tau biosensor cells with reduced BANF1 or ANKLE2 expression. Briefly, dCas9-KRAB cloned TK-B4 tau biosensor cells were seeded in 6-well dishes, transduced with gRNA-targeted BANF1 (kBANF1_gRNA6), ANKLE2 (kANKLE2_gRNA2), or control gRNA, untargeted 303, and control gNT303, cloned into pLentiGuide-Puro expression vectors, and packaged in lentivirus (GenScript). Cells were grown under both puromycin and blastosidine selection and passaged into 12-well plates on day 3 post-transduction, with each sample replicated in two wells. On day 4, cells were transduced with LEMD2, LEMDi2, LEMD3, CHMP7, or luciferase LV-cDNA as described above. After 24 hours, the medium was changed to DMEM medium + 1.5 μg / mL puromycin + 50 μg / mL hygromycin. On day 7, cells were passaged into three sets of 24-well plates and one set of 6-well plates for the protein cell fraction (Luc and LEMD2 cDNA-expressing cells). On day 9, the DMEM medium was changed and minimal tau seeding was added to the wells as follows: the first set of plates received 1 μg / mL myelolysis derived from 9-month-old P301S Het transgenic mice in each well, and the second set of plates received 1 μg / mL tau-YFP Agg + whole cell lysate in each well. Densely packed cells in a 6-well format were treated with 10 μg / mL cell lysates from tau-YFP Agg+ cells or tau-YFP Agg- cells.On day 11, cells from the first two sets of plates were collected for analytical flow cytometry using CytoFLEX LX to measure FRET signaling in seeded cells, and cells from the third set of plates were collected for RNA extraction to evaluate the expression of transduced cDNA constructs and the knockdown of KRAB gRNA target genes by TaqMan qRT-PCR. Cells in a 6-well format were collected for protein cell fractionation.

[0231] Intracellular protein fractionation combined with Western blot analysis. Whole cell lysates were prepared from densely packed cells on a 6-well plate scraped into 1 mL of ice-cold PBS and centrifuged at 3000 g for 5 minutes. The pellet was resuspended in 150 μL of Novex Tris-glycine SDS sample buffer (ThermoFisher, catalog no. LC2676) and 150 μL of PBS, homogenized with a 23G needle, and heated at 95°C for 5 minutes. Proteins were quantified using the RC DC Protein Assay Kit II (Bio-Rad, catalog no. 5000122). Protein fractionation was performed from densely packed cells in 6 wells using the Intracellular Cell Fractionation Kit for Cultured Cells (ThermoFisher, catalog no. 78840), which contains four extraction buffers. According to the manufacturer's protocol, the first buffer added to the cell pellet induced selective membrane permeabilization, releasing soluble cytoplasmic proteins. The second buffer dissolved the plasma, mitochondria, and ER-Golgi membrane, but did not solubilize the nuclear membrane. After recovering intact nuclei by centrifugation, the third buffer extracted soluble nucleoproteins. Further nuclear extraction was performed using micrococcal nucleases to release chromatin-bound nucleoproteins. The recovered insoluble pellet was resuspended in the final buffer. The fractions were quantified using the Qubit Protein Assay Kit (ThermoFisher, catalog number Q33212). For Western blot analysis, Novex Tris-glycine SDS sample buffer (2X) was added to each fraction and heated at 95°C for 5 minutes. 10 μL of protein extract was transferred onto a 4-20% Novex Tris-glycine wedgewell protein gel and dried onto a nitrocellulose membrane using the Invitrogen iBlot2 system (ThermoFisher). The membrane was blocked for 1 hour at room temperature with a 5% emulsion in Tris-buffered saline with 0.05% Tween® 20 (TBST). Primary antibodies were cultured overnight at 4°C in a locker.Primary antibodies used: tau (Dako, A0024, 1:250,000), phosphorylated tau serine 356 (Abcam, ab92682 or ab75603, 1:10,000), BANF1 (Abcam, ab129184, 1:1000), PPP2CA (Proteintech, 13482-1-AP, 1:1000). HRP-conjugated rabbit secondary antibody was added, imaging was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher, catalog number 34580), and recording was performed with a FluorChem M imaging system (ProteinSimple).

[0232] Antisense oligonucleotides. 25 ASOs targeting mouse Banf1 and 96 ASOs targeting mouse Ankle2 mRNA transcripts (Banf1 transcript ID NM_011793.3, Ankle2 transcript ID NM_001253814.1) were designed in silico by scanning across the entire mature mRNA transcript. All ASOs were designed using a 5-10-5 "gapmer" format, wherein the 5-nucleotide "wing" has 2'-methoxyethyl (Methoxy Ethyl, MOE) modification and the 10-nucleotide core has DNA bases to promote RNaseH-mediated knockdown. Furthermore, the ASOs have phosphorothioate (phosphorothioate, PS) linkages throughout. To assay for mRNA knockdown, ASOs were individually transfected at 100 nM into NSC34 cells using LIPOFECTAMINE RNAiMaX according to the manufacturer's recommendations. mRNA knockdown was measured 72 hours later using a transcript-specific TAQMAN qRT-PCR assay.

[0233] Six potent ASO target mice, Banf1 and Ankle2, identified from these screenings, were secondary screened in primary mouse cortical neurons to determine the best hit. Gymnotic delivery was obtained by diluting a 1 mM ASO stock solution (synthesized by IDT) in Neurobasal culture medium to a final ASO concentration of 2.5 μM.

[0234] For the NLS:mCherry experiment, neurons were seeded (gymnotic delivery) in a 96-well format and treated with individual ASOs three days later. Half of the neurobasal medium volume was replaced and replenished with ASOs after four days. Eight days after the initial ASO treatment, neurons were transduced with the "EF1α-NLS:mCherry" LV-cDNA construct. Neurons were maintained in culture for an additional two days before sample collection for live-cell imaging and expression analysis by TaqMan qRT-PCR.

[0235] For cDNA rescue experiments, neurons were treated with individual ASOs (gymnotic delivery) at a final concentration of 2.5 μM three days after seeding. Four days later, neurons were transduced with Lemd2, Lemd3, Chmp7, or the LVhSyn 1-cDNA construct encoding luciferase as a control cDNA. Six hours later, the Neurobasal medium was replaced and replenished with ASOs. Half of the Neurobasal medium volume was replaced every four days. On day 10, neurons were fixed for immunofluorescence studies. Samples were similarly collected for expression analysis by TaqMan qRT-PCR.

[0236] Culture of mouse cortical neurons. Primary mouse cortical neurons were purchased from ThermoFisher (GIBCO, catalog number A15586), and according to the manufacturer's user guide, the cells were seeded in a poly-D-lysine-treated 96-well plate (Greiner Bio-One, catalog number 655946) with Neurobasal Plus Medium (GIBCO, catalog number A35829-01) + 1% B-27 Plus Supplement (GIBCO, catalog number A35828-01) + 1X GlutaMAX Supplement (GIBCO, catalog number 35050-061) at a density of approximately 20,000 neurons per well, with a volume of 100 μL. Three days after seeding, neurons were transduced with a (Cas9+gRNA) construct as either a mouse-targeted gRNA (Banf1_gRNA3, Ankle2_gRNA3, Ppp2ca_gRNA2) or a control non-targeted gRNA (NT303). These constructs were cloned into a pLentiCRISPR-v2 Cas9 expression vector and packaged in a lentivirus (GenScript). After six hours, half of the neurobasal medium volume was replaced and replenished every 3-4 days. Neurons were maintained in culture for two weeks before fixation for immunofluorescence analysis.

[0237] Immunofluorescence staining. Primary mouse cortical neurons seeded in a 96-well plate were pre-fixed in 2% paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog no. 15714) in PBS solution on ice for 5 minutes. After gentle aspiration, the fixation solution was added as 100 μL of cold 4% PFA in PBS on ice for 15 minutes. The fixation solution was washed three times with 150 μL of cold PBS. Finally, 100 μL of cell permeabilization and blocking solution in 0.2% TBS-Triton® solution (TBST, Millipore, catalog no. 807423) as 10% donkey normal serum (DNS) (Sigma-Aldrich, catalog no. S30-100ML) was added at room temperature for 1 hour. Immunostaining was performed overnight at 4°C in blocking solution using the following antibodies: rabbit antiphosphorylated tau Ser356 1:1,000 (Abcam, catalog number ab92682), rabbit anti-tau 1:10,000 (Dako, A0024), and chicken anti-MAP 2 1:20,000 (Abcam, catalog number ab5392). The following day, each well was washed three times with 150 μL of TBST and incubated with 100 μL of the following secondary antibody solution in TBST at room temperature for 1 hour: Alexa Fluor568 donkey anti-rabbit IgG 1:1000 (Invitrogen, catalog number A10042), Alexa Fluor647 goat anti-chicken IgG. 1:1,000 (Invitrogen, catalog number A21449) and DAPI (ThermoFisher, catalog number 62248). Finally, the plates were washed three times with 150 μL of TBST, and 100 μL of PBS was added to each well. The plates were maintained at 4°C until imaging.

[0238] Image analysis. Unbiased image analysis and calculations were performed using the analysis software Harmony 4.9 (PerkinElmer). Immunofluorescence imaging was performed using the Opera Phenix High-Content Screening System (PerkinElmer) with a 40x water objective lens to capture approximately 40 fields of view per 96 wells. Harmony was used to quantify the percentage of SRRM2 in the cell nuclei. The nuclei were labeled using DAPI staining, and the cytoplasm was labeled using YFP immunostaining. For HEK293T cells, 30 μm 2 The nucleus was identified using Method C, which has a hyper-region. The cytoplasm was defined based on the nucleus (Harmony Method A). The percentage of total SRRM2 intensity within the nucleus (Abcam, catalog number ab11826) was calculated by dividing the nuclear SRRM2 intensity by the sum of the nuclear intensity and cytosolic intensity for each image. Significance was determined using an unpaired two-sided t-test.

[0239] To quantify phosphorylated tau Ser356 in cultured mouse cortical neurons, nuclei were labeled using DAPI staining and cell bodies were labeled using MAP2 immunostaining. Nuclear detection method C was selected for its robustness regarding the contrast variation of nuclear size and fluorescence signal. Cytoplasm was defined based on the nucleus (Harmony method A). Perinuclear and cell body regions were labeled using DAPI. + It was defined as a ring surrounding the nucleus cell. For quantification, a minimum of 1,000 cells were analyzed for each condition.

[0240] For imaging and recording of living cells, the inventors used a Zeiss Axio Observer 7 microscope equipped with culture components, a Colibir 7 LED light source, and an Axiocam 703 Mono sCMOS camera. In Zen Blue (Zeiss), mCherry fluorescence intensity measurements were performed by calculating the average intensity of three subject regions of the same area within the cell body in each individual cell.

[0241] Statistical analysis of NGS data from CRISPR screening. Gene-centered enrichment analysis. Similar to DESeq2, our alternative algorithm has two components: enrichment coefficient (referred to as multiplicative change in DESeq2) and enrichment p-value. The enrichment coefficient is the same as that used in DESeq2, except that it is summarized at the gene level by averaging the enrichment of all gRNAs targeting the same gene. Unlike existing CRISPR data analysis methods, our method uses a hypergeometric distribution-based p-value calculation that applies only to the enrichment of different gRNAs targeting the gene in each FRET+10 day sample. The final enrichment p-value for a gene is the average of the p-values ​​obtained from each of the 10 FRET+10 day samples. The multiplicative change for each gene was obtained by calculating the arithmetic mean of the log2-converted FRET+10 day to day 3 or day 6 ratios generated by DESeq2 for the corresponding gRNA (Love et al., 2014). The enrichment p-value for each gene was calculated as follows: Firstly, in each FRET+10 day sample, gRNA was considered to be present if its DESeq2 normalized read count was 30 or greater, and the inventors considered read counts less than 30 as background noise. The enrichment of gRNAs corresponding to genes in FRET+10 day samples was calculated using the hypergeometric distribution-based function phyper(x, m, n, k, lower.tail=FALSE) in R (R-project.org / ). x is the number of present gRNAs corresponding to gene minus 1, m is the total number of gRNAs corresponding to genes in the library, n is the total number of gRNAs in the library minus m, and k is the total number of gRNAs present in the FRET+ sample. Next, the p-values ​​were log10 transformed and averaged over all 10 FRET+10 day samples.

[0242] Differential gRNA read count analysis. To compare gRNA read counts between two groups of samples, the inventors used a DESeq2 Generalized Linear Model (GLM) and tested for differences in abundance using Wald's test based on a negative binomial distribution in ArrayStudio (OmicSoft). Using the terms "experiment" and "FRET state," a FRET+ vs. FRET- comparison model was constructed between samples at 13 days across five experiments. gRNAs with significant differences in read count between the comparison groups were identified using the criteria of i) multiplicative change ≥ 1.5 in either direction, and ii) p-value < 0.05. The gRNAs enriched in the FRET samples were further defined using the following three criteria: i) FRET- significantly increased in the 13-day sample compared to FRET+, ii) FRET- significantly increased in the 13-day sample compared to the 10-day sample, and iii) FRET+ did not significantly increase in the 13-day sample compared to the 10-day sample (either no significant difference or a significant decrease). The inventors paid particular attention to gene targets having multiple gRNAs enriched in the FRET samples.

[0243] Statistical analysis of immunofluorescence. Statistical analysis was performed using GraphPad Prism8. Specifically, the significance of immunofluorescence staining and NLS:mCherry fluorescence intensity for primary mouse cortical neurons was determined using the Mann-Whitney U (two-tailed test). Quantitative results are reported as mean ± SEM (error bars).

[0244] [Table 3]

[0245] [Table 4-1] [Table 4-2]

[0246]

Table 5

[0247]

Table 6

[0248]

Table 7

[0249]

Table 8

[0250]

Table 9

[0251] Example 2. Overexpression of LEMD2, LEMD3, or CHMP7 as a treatment modality for tauopathy The present inventors identified LEMD2, LEMD3 and CHMP7 as modifiers of tau aggregates (see Example 1). The inventors further validated these modifiers using cDNAs designed to enable overexpression in tau biosensor cells. These targets are further validated ex vivo in mouse cortical neurons, and in vivo using a mouse model of tauopathy.

[0252] Mouse cortical neurons Experiments are performed to assess whether overexpression of mouse LEMD2, LEMD3, or CHMP7 can reduce tau hyperphosphorylation at Ser356, as detected by immunofluorescence in the nucleus and perinuclear region of mouse cortical neurons, after treatment with either ASO targeting Banf1 or Ankle2, which induces greater tau hyperphosphorylation, or a non-targeting scrambled ASO control.

[0253] Mouse cortical neurons were treated with ASO-targeted Banf1 or Ankle2 or a non-targeted scrambled ASO control, and then transduced with AAV (5000VG / neuron) or lentivirus (LV) (10000VG / neuron) encoding codon-optimized mouse LEMD2, LEMD3, or CHMP7. The sequences of the AAV constructs are described in SEQ ID NOs. 41 to 43. The sequences of the LV constructs are described in SEQ ID NOs. 35, 38, and 40. The expression of mouse LEMD2, LEMD3, or CHMP7 was confirmed by a codon-optimized TAQMAN assay specific to codon-optimized cDNA, compared to the endogenous gene. Phosphorylated tau was analyzed by immunofluorescence and Western blotting in combination with cell fractions. Neuronal cell lysates are combined with LIPOFECTAMINE® 2000, and treated tau biosensor cells are added (as a seeding agent) for qualitative FRET analysis to evaluate the neurons for evidence of misfolded tau.

[0254] Tauopathy mouse model AAV encoding mouse LEMD2 or CHMP7 or the mCherry control was injected into neonatal P301S transgenic mice (a P19 mouse model of tauopathy) via intracerebral (intracerebroventricular, ICV) injection to determine whether overexpression of LEMD2 or CHMP7 could prevent tauopathy-related phenotypes. The sequences of the AAV constructs are listed in SEQ ID NOs: 41 to 43.

[0255] Multiple AAV titers (5E10, 2.5E10, and 1.25E10) will be tested. Following cDNA expression, qRT-PCR will be performed. Once the optimal AAV titer is determined, it will be injected into P301S heterozygous neonates, and long-term studies will be conducted for 6–9 months.

[0256] AAVs encoding mouse LEMD2, CHMP7, or mCherry control are also injected into 3-month-old P301S animals at different AAV titers (1E11 or 5E10 VG / animal). cDNA expression is evaluated by qRT-PCR. Once the AAV titer is optimized, it is injected into 3-month-old P301S heterozygous mice, and long-term studies are conducted over 6–9 months.

[0257] PS19 mouse model. PS19 (Tau P301S (System PS19); PS19Tg; B6; C3-Tg (Prnp-MAPT) *The P301S)PS19Vle / J) mouse lineage is a tauopathy model. The genetic background of this lineage is C57BL / 6xC3H. PS19 transgenic mice express a mutant human microtubule-associated protein tau MAPT driven by a mouse prion protein (Prnp) promoter. The transgene encodes the disease-associated P301S mutation and contains four microtubule-binding domains and one N-terminal insert (4R / 1N). The transgene was inserted into Chr3:140354280-140603283 (build GRCm38 / mm10), resulting in a 249Kb deletion that does not affect the known gene. See Goodwin et al., (2019), "Genome Res.", 29(3):494-505, which is incorporated herein by reference in its entirety. Expression of mutant human tau is five times higher than expression of endogenous mouse protein. See Yoshiyama et al., (2007), "Neuron", 53(3):337-351, which is incorporated herein by reference in its entirety. PS19 mice develop neuronal loss and brain atrophy by 8 months of age. They also develop extensive tau aggregates, known as neurofibrillary condensate-like inclusions, in the neocortex, amygdala, hippocampus, brainstem, and spinal cord. See Yoshiyama et al., (2007). Prior to the appearance of overt tau pathology by histological methods, the brains of these mice have been shown to exhibit tau dissemination activity. That is, tau aggregates present in brain homogenates may induce further tau aggregates, possibly via a prion-like mechanism. See Holmes et al., (2014), "Proc.Natl.Acad.Sci.USA" 111(41):E4376–E4385, which is incorporated in its entirety herein by reference.

[0258] Cloning of cDNA into lentiviral expression plasmids. cDNA and protein sequences were obtained from Ensembl (ensembl.org) and verified with UniProt. Nucleic acid fragments were synthesized and subcloned into the pLVX-Ef1a IRES-Hyg expression plasmid vector to generate expression plasmids for overexpression of the cDNA sequence. The cDNA fragments were inserted downstream of the Ef1a promoter using Spe-I and Not-I restriction sites. The nucleic acid sequences were obtained by backtranslation of protein sequences and codons optimized for expression in humans (Homo sapiens) and mice (Mus Musculus), respectively. Protein sequences were obtained from UniProt with the following catalog numbers: for humans (Homo sapiens), BANF1 (O75531), ANKLE2 (Q86XL3), Lemd2 (Q8NC56), Lemd2-isoform 2 (Q8NC56-2), LEMD3 (Q9Y2U8), and CHMP7 (Q8WUX9); for mice (Mus Musculus), Lemd2 (Q6DVA0), LEMD3 (Q9WU40), and CHMP7-isoform 1 (Q8R1T1). As a control cDNA, the coding sequence of the reporter gene luciferase was also cloned into the expression vector. Plasmids were sequenced by Sanger sequencing before lentiviral packaging.

[0259] Cloning of cDNA to an AAV expression plasmid. The cDNA and protein sequences were obtained from Ensembl and confirmed with UniProt. The nucleic acid sequences were codon-optimized and synthesized into a lentiviral backbone. Next, mouse Lemd2 and mouse Chmp7 cDNA were amplified using PCR, and restriction sites for subcloning were added to our AAV backbone using the human synapsin 1 promoter. Specifically, 44 mL of Invitrogen AccuPrime Pfx DNA polymerase (catalog number 12344024) was combined with 0.2 mL each of appropriate forward and reverse oligos at a 100 mM concentration, and 1 mL of template. The PCR reaction was performed for 29 cycles on an Eppendorf MasterCycler ProS thermal cycler using the following parameters: 96°C for 2 minutes; (96°C for 20 seconds + 50°C for 30 seconds + 72°C for 45 seconds) 29 times; 4°C.

[0260] PCR products were purified using the QIAquick PCR Purification Kit (catalog number 28106), and then New England BioLabs enzymes EcoRI-HF (catalog number R3101L) and NotI-HF (catalog number R3189L) were pulverized in CutSmart buffer (catalog number B7204S) according to the manufacturer's protocol. The pulverized products were visualized on an e-gel with 1.2% Invitrogen (catalog number 5018-01) and then purified using QIAquick Spin Columns (catalog number 28115). The ssAAV skeleton was pulverized as described above, and then cultured on a 1.2% agarose gel (catalog number 2120-100GM) for 4 hours before the ssAAV skeleton was extracted. DNA fragments were extracted from agar plates using QIAquick Spin Columns (catalog number 28115). Each cDNA fragment was ligated to a DNA backbone using New England Biolabs T4 DNA ligase (catalog number M0202L 400U / mL) according to the manufacturer's protocol. The ligated cells were converted to MAX Efficiency Stbl2 competent cells (catalog number 10268019) and then recovered in Invitrogen SOC recovery medium (catalog number 15544-34) at 30°C for 1 hour, according to the manufacturer's protocol. One-third of the recovered cells were seeded on Teknova LB Agar Plate with carbenicillin-100 (catalog number L1010) and grown overnight at 30°C. The resulting colonies were seeded in 5 mL of Sigma-Aldrich Terrific Broth (catalog number T5574-500 mL) and grown overnight at 30°C. Plasmid DNA was collected using the QiaPrep Spin Miniprep Kit (catalog number 27106). The extracted DNA was sequenced by Sanger sequencing before AAV packaging using a pHP.eB capsid to cross the blood-brain barrier.

[0261] Example 3. Overexpression of LEMD2, LEMD3, or CHMP7 in PS19 mice. To further study Lemd2, Lemd3, and Chmp7 as genetic modifiers of tau aggregates, and particularly as therapeutic mechanisms for tauopathy, the inventors designed an experiment to test whether overexpression of these mouse genes by cDNA introduction can delay the onset of tauopathy-related disease phenotypes in a mouse model of tauopathy.

[0262] The inventors utilized the AAV capsid pHP.eB, which enables the delivery of cargo such as modifying factors cDNA to the CNS across the blood-brain barrier. The inventors also developed a transgenic mouse model of tauopathy (PS19 (Tau P301S (PS19 strain); PS19Tg; B6; C3-Tg (Prnp-MAPT)). * In the P301S)PS19Vle / J) mouse strain, two different AAV injection routes, intraperitoneal (IP) and intraventricular (ICV), were tested.

[0263] cDNA and protein sequences were obtained from Ensembl and verified using UniProt. Plasmids for cDNA sequence expression were generated by synthesizing nucleic acids and subcloning them into expression vectors for mouse and control cDNAs. Nucleic acid sequences were obtained by reverse translation of protein sequences (Reverse translator tool, MacVector 18.1.5) and codons optimized for expression in mouse cells (mouse protein catalog numbers: Lemd2 (Q6DVA0), Lemd3 (Q9WU40), and Chmp7 (Q8R1T1)). The coding sequences of the reporter gene mCherry or GFP were cloned into the same expression vector to function as non-specific control cDNAs. Plasmid sequences were verified by Sanger sequencing before AAV packaging using plasmids encoding the CNS-specific AAV_pHP.eB capsid. The sequences for the AAV-mLemd2, AAV-mLemd3, and AAV-mChmp7 vectors are described in SEQ ID NOs. 249 to 251, respectively.

[0264] The inventors first optimized the titer necessary for robust cDNA expression. They injected neonatal mice (p0 or p1) with different doses of AAV_cDNA (1.25E10 VG / mouse, 2.5E10 VG / mouse, 5E10 VG / mouse) via ICV, and sacrificed the animals after 4 months. They also injected 2-month-old mice with different doses of AAV_cDNA (5E10 VG / mouse, 7.5E10 VG / mouse, 1E11 VG / mouse) via IP, and sacrificed the animals after 4 months. They collected tissue from the liver, spinal cord, and brain, isolated RNA therefrom, and performed qRT-PCR expression analysis. The inventors demonstrated that cDNA-driven expression was robust and reproducible across animals after ICV injection. The human synapsin-1 promoter enabled greater CNS specificity in cDNA expression.

[0265] Three different modes of treatment for tauopathy will be evaluated, including prevention, delay, and / or cure of the disease. Lemd2, Lemd3, and Chmp7 cDNAs will be introduced at birth, before the onset of the disease, at 3 months of age, or after the onset of the disease, at 6 months of age.

[0266] In the second experiment, PS19 newborns were injected with 2.5E10 VG / mouse via ICV for all cDNAs. PS19 heterozygous animals were also injected with 1.5E11 VG / mouse via IP.

[0267] We will conduct a long-term study on serum neuronal filament light chains (sNfLs), a powerful indicator of neurodegenerative processes and a biomarker of neuronal damage. To assess sNfL levels, animals will have blood drawn (submandibular method) at 4 and 6 months, considered baseline in this mouse model, and then every 5 weeks thereafter to observe sNfL levels over time. The present invention provides, for example, the following items: (Item 1) A method for inhibiting tau aggregates in cells, comprising LEM domain-containing protein 2 (LEMD2), charged multivesicular body protein 7 (CHMP7), or inner nuclear membrane protein Man 1 (inner nuclear membrane protein Man1, LEMD3), or the aforementioned LEMD2, A method comprising administering the nucleic acid encoding CHMP7 or LEMD3 to the cells. (Item 2) A method for inhibiting or reducing taur phosphorylation in cells, comprising administering to the cells LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or a nucleic acid encoding said LEMD2, said CHMP7, or said LEMD3. (Item 3) The method according to item 2, for inhibiting or reducing the phosphorylation of tau on serine 356. (Item 4) The method according to item 2 or 3, wherein tau phosphorylation is reduced in the cell body, perinuclear region, and / or nucleocytoplasm of the cell. (Item 5) A method for inhibiting or reducing the accumulation of insoluble tau in cells, comprising administering to the cells LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or a nucleic acid encoding LEMD2, CHMP7, or LEMD3. (Item 6) The method according to any one of items 1 to 5, comprising administering the LEMD2 or the nucleic acid encoding the LEMD2 to the cells. (Item 7) The method according to item 6, wherein the LEMD2 is human LEMD2, optionally the LEMD2 includes SEQ ID NO: 1 or SEQ ID NO: 5, and optionally the nucleic acid encoding the LEMD2 includes SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7. (Item 8) The method according to item 6, wherein the LEMD2 is mouse LEMD2, optionally the LEMD2 includes SEQ ID NO: 10, and optionally the nucleic acid encoding the LEMD2 includes SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 255. (Item 9) The method according to any one of items 1 to 8, comprising administering the CHMP7 or the nucleic acid encoding the CHMP7 to the cells. (Item 10) The method according to item 9, wherein the CHMP7 is human CHMP7, optionally the CHMP7 includes SEQ ID NO: 15, and optionally the nucleic acid encoding the CHMP7 includes SEQ ID NO: 16 or SEQ ID NO: 17. (Item 11) The method according to item 9, wherein the CHMP7 is mouse CHMP7, optionally the CHMP7 includes SEQ ID NO: 19, and optionally the nucleic acid encoding the CHMP7 includes SEQ ID NO: 20 or SEQ ID NO: 21. (Item 12) The method according to any one of items 1 to 11, comprising administering the LEMD3 or the nucleic acid encoding the LEMD3 to the cells. (Item 13) The method according to item 12, wherein the LEMD3 is human LEMD3, optionally the LEMD3 includes SEQ ID NO: 23, and optionally the nucleic acid encoding the LEMD3 includes SEQ ID NO: 24 or SEQ ID NO: 25. (Item 14) The method according to item 12, wherein the LEMD3 is mouse LEMD3, optionally the LEMD3 includes SEQ ID NO: 27 or SEQ ID NO: 29, and optionally the nucleic acid encoding the LEMD3 includes SEQ ID NO: 29 or SEQ ID NO: 30. (Item 15) The method according to any one of items 1 to 14, wherein the nucleic acid encoding LEMD2, CHMP7, or LEMD3 is administered to the cells, and the nucleic acid is optionally codon-optimized for expression in human or mouse cells. (Item 16) The method according to item 15, wherein the nucleic acid comprises complementary DNA encoding LEMD2, CHMP7, or LEMD3. (Item 17) The method according to item 15, wherein the nucleic acid comprises a messenger RNA encoding LEMD2, CHMP7, or LEMD3. (Item 18) The method according to item 15 or 16, comprising administering an expression construct comprising the nucleic acid encoding LEMD2, CHMP7, or LEMD3, which is operably coupled to a promoter. (Item 19) The method according to item 18, wherein the promoter is a different type of promoter. (Item 20) The method according to item 18 or 19, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inductive promoter. (Item 21) The method according to item 20, wherein the promoter is a neuron-specific promoter. (Item 22) The method according to item 21, wherein the promoter is the synapsin-1 promoter. (Item 23) The method according to item 22, wherein the promoter is the human synapsin-1 promoter. (Item 24) The method according to any one of items 15 to 23, wherein the nucleic acid is present in the vector. (Item 25) The method according to item 24, wherein the vector is a viral vector. (Item 26) The method according to item 25, wherein the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector. (Item 27) The method according to item 26, wherein the vector is the AAV vector, and optionally the AAV vector is the AAV-PHP.eB vector. (Item 28) The method according to any one of items 1 to 27, wherein the cells are mammalian cells. (Item 29) The method according to item 28, wherein the mammalian cells are human cells, rodent cells, mouse cells, or rat cells. (Item 30) The method according to item 29, wherein the cells are human cells. (Item 31) The method according to any one of items 1 to 30, wherein the cell is a nerve cell. (Item 32) The method according to any one of items 1 to 31, wherein the aforementioned cells are present in the subject in vivo. (Item 33) The method according to item 32, wherein the cells are nerve cells in the brain of the subject. (Item 34) The method according to item 32 or 33, wherein the nucleic acid encoding LEMD2, CHMP7, or LEMD3, or LEMD2, CHMP7, or LEMD3, is administered to the subject by intraventricular injection, intracranial injection, or intrathecal injection. (Item 35) The method according to any one of items 1 to 34, further comprising evaluating one or more signs or symptoms of tauopathy or tau aggregation in the cells. (Item 36) The method according to item 35, further comprising evaluating the phosphorylated tau level in the aforementioned cells. (Item 37) The method according to any one of items 1 to 36, for reducing the amount of new tau aggregate formation in the aforementioned cells. (Item 38) The method according to any one of items 1 to 37, for reducing the amount of existing tau aggregate formation in the aforementioned cells. (Item 39) A method for treating tauopathy in a subject, comprising administering to the subject LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or a nucleic acid encoding LEMD2, CHMP7, or LEMD3, wherein LEMD2, CHMP7, or LEMD3 inhibits tau aggregates in the subject's cells. (Item 40) A method for preventing tauopathy in a subject, comprising administering to the subject LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), or a nucleic acid encoding LEMD2, CHMP7, or LEMD3, wherein LEMD2, CHMP7, or LEMD3 inhibits tau aggregates in the subject's cells. (Item 41) The method according to item 39 or 40, comprising administering the LEMD2 or the nucleic acid encoding the LEMD2 to the subject. (Item 42) The method according to item 41, wherein the LEMD2 is human LEMD2, optionally the LEMD2 includes SEQ ID NO: 1 or SEQ ID NO: 5, and optionally the nucleic acid encoding the LEMD2 includes SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7. (Item 43) The method according to item 41, wherein the LEMD2 is mouse LEMD2, optionally the LEMD2 includes SEQ ID NO: 10, and optionally the nucleic acid encoding the LEMD2 includes SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 255. (Item 44) The method according to any one of items 39 to 43, comprising administering the CHMP7 or the nucleic acid encoding the CHMP7 to the subject. (Item 45) The method according to item 44, wherein the CHMP7 is human CHMP7, optionally the CHMP7 includes SEQ ID NO: 15, and optionally the nucleic acid encoding the CHMP7 includes SEQ ID NO: 16 or SEQ ID NO: 17. (Item 46) The method according to item 44, wherein the CHMP7 is mouse CHMP7, optionally the CHMP7 includes SEQ ID NO: 19, and optionally the nucleic acid encoding the CHMP7 includes SEQ ID NO: 20 or SEQ ID NO: 21. (Item 47) The method according to any one of items 39 to 46, comprising administering the LEMD3 or the nucleic acid encoding the LEMD3 to the subject. (Item 48) The method according to item 47, wherein the LEMD3 is human LEMD3, optionally the LEMD3 includes SEQ ID NO: 23, and optionally the nucleic acid encoding the LEMD3 includes SEQ ID NO: 24 or SEQ ID NO: 25. (Item 49) The method according to item 47, wherein the LEMD3 is mouse LEMD3, optionally the LEMD3 includes SEQ ID NO: 27 or SEQ ID NO: 29, and optionally the nucleic acid encoding the LEMD3 includes SEQ ID NO: 29 or SEQ ID NO: 30. (Item 50) The method according to any one of items 39 to 49, wherein the nucleic acid encoding LEMD2, CHMP7, or LEMD3 is administered to the subject, and the nucleic acid is optionally codon-optimized for expression in human or mouse cells. (Item 51) The method according to item 50, wherein the nucleic acid comprises complementary DNA encoding LEMD2, CHMP7, or LEMD3. (Item 52) The method according to item 50, wherein the nucleic acid comprises a messenger RNA encoding LEMD2, CHMP7, or LEMD3. (Item 53) The method according to item 50 or 51, comprising administering an expression construct comprising the nucleic acid encoding LEMD2, CHMP7, or LEMD3, which is operably coupled to a promoter. (Item 54) The method according to item 53, wherein the promoter is a different type of promoter. (Item 55) The method according to item 53 or 54, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inductive promoter. (Item 56) The method according to item 55, wherein the promoter is a neuron-specific promoter. (Item 57) The method according to item 56, wherein the promoter is the synapsin-1 promoter. (Item 58) The method according to item 57, wherein the promoter is the human synapsin-1 promoter. (Item 59) The method according to any one of items 50 to 58, wherein the nucleic acid is present in the vector. (Item 60) The method according to item 59, wherein the vector is a viral vector. (Item 61) The method according to item 60, wherein the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector. (Item 62) The method according to item 61, wherein the vector is the AAV vector, and optionally the AAV vector is the AAV-PHP.eB vector. (Item 63) The method according to any one of items 39 to 62, wherein the subject is a mammal. (Item 64) The method according to item 63, wherein the subject is a human, rodent, mouse, or rat. (Item 65) The method according to item 64, wherein the subject is a human being. (Item 66) The method according to any one of items 39 to 65, wherein the cell is a nerve cell. (Item 67) The method according to item 66, wherein the nerve cells are present in the brain of the subject. (Item 68) The method according to any one of items 39 to 67, wherein the nucleic acid encoding LEMD2, CHMP7, or LEMD3, or LEMD2, CHMP7, or LEMD3, is administered to the subject by intraventricular injection, intracranial injection, or intrathecal injection. (Item 69) The method according to any one of items 39 to 68, further comprising evaluating one or more signs or symptoms of tauopathy or tau agglutination in the cells or the subject. (Item 70) The method according to item 69, further comprising evaluating the level of phosphorylated tau in the cells or the subject. (Item 71) The method according to item 69 or 70, further comprising evaluating the serum neurofilament light chain (sNfL) level in the subject. (Item 72) The method according to any one of items 39 to 71, for reducing the amount of new tau aggregate formation in the cells or the subject. (Item 73) The method according to any one of items 39 to 72, for reducing the amount of existing tau aggregate formation in the cells or the subject. (Item 74) An expression construct comprising nucleic acids encoding LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), operably linked to a heterologous promoter. (Item 75) The expression construct according to item 74, comprising the nucleic acid encoding LEMD2. (Item 76) The expression construct according to item 75, wherein the LEMD2 is human LEMD2, optionally comprising SEQ ID NO: 1 or SEQ ID NO: 5, and optionally comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7. (Item 77) The expression construct according to item 75, wherein the LEMD2 is mouse LEMD2, optionally comprising SEQ ID NO: 10, and optionally comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 255. (Item 78) The expression construct according to item 74, comprising the nucleic acid encoding CHMP7. (Item 79) The expression construct according to item 78, wherein the CHMP7 is human CHMP7, optionally comprising SEQ ID NO: 15, and optionally comprising SEQ ID NO: 16 or SEQ ID NO: 17. (Item 80) The expression construct according to item 78, wherein the CHMP7 is mouse CHMP7, optionally comprising SEQ ID NO: 19, and optionally comprising SEQ ID NO: 20 or SEQ ID NO: 21. (Item 81) The expression construct according to item 74, comprising the nucleic acid encoding the LEMD3. (Item 82) The expression construct according to item 81, wherein the LEMD3 is human LEMD3, optionally comprising SEQ ID NO: 23, and optionally comprising SEQ ID NO: 24 or SEQ ID NO: 25. (Item 83) The expression construct according to item 81, wherein the LEMD3 is mouse LEMD3, optionally comprising SEQ ID NO: 27 or SEQ ID NO: 29, and optionally comprising the nucleic acid encoding the LEMD3 comprising SEQ ID NO: 29 or SEQ ID NO: 30. (Item 84) An expression construct according to any one of items 74 to 83, wherein the nucleic acid encoding LEMD2, CHMP7, or LEMD3 is codon-optimized for expression in human or mouse cells. (Item 85) An expression construct according to any one of items 74 to 84, wherein the nucleic acid comprises complementary DNA encoding LEMD2, CHMP7, or LEMD3. (Item 86) The expression construct according to any one of items 74 to 85, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. (Item 87) The expression construct described in item 86, wherein the promoter is a neuron-specific promoter. (Item 88) The expression construct described in item 87, wherein the promoter is the synapsin-1 promoter. (Item 89) The expression construct described in item 88, wherein the promoter is the human synapsin-1 promoter. (Item 90) The nucleic acid is present in the vector in any one of the expression constructs described in items 74 to 89. (Item 91) The expression construct described in item 90, wherein the vector is a viral vector. (Item 92) The expression construct according to item 91, wherein the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector. (Item 93) The expression construct according to item 92, wherein the vector is the AAV vector, and optionally the AAV vector is the AAV-PHP.eB vector.

Claims

1. A composition for use in a method of inhibiting tau aggregates in cells, inhibiting or reducing tau phosphorylation on serine 356, or inhibiting or reducing the accumulation of insoluble tau, comprising a nucleic acid encoding LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), wherein the cells are present in vivo in a subject.

2. The composition for use according to claim 1, wherein tau phosphorylation is reduced in the cell body, perinuclear region, and / or nucleocytoplasm of the cell.

3. The composition for use according to claim 1, wherein the composition comprises the nucleic acid encoding the LEMD2.

4. The composition for use according to claim 3, wherein the LEMD2 is human LEMD2 or mouse LEMD2.

5. The composition for use according to claim 1, wherein the composition comprises the nucleic acid encoding CHMP7.

6. The composition for use according to claim 5, wherein the CHMP7 is human CHMP7 or mouse CHMP7.

7. The composition for use according to claim 1, wherein the composition comprises the nucleic acid encoding the LEMD3.

8. The composition for use according to claim 7, wherein the LEMD3 is human LEMD3 or mouse LEMD3.

9. (I) The nucleic acid contains complementary DNA encoding LEMD2, CHMP7, or LEMD3, (II) The nucleic acid contains messenger RNA encoding LEMD2, CHMP7, or LEMD3, or (III) The composition comprises an expression construct comprising the nucleic acid encoding the LEMD2, CHMP7, or LEMD3, which is operably connected to a promoter. A composition for use according to claim 1.

10. (I) Whether the promoter is a different type of promoter, (II) The composition for use according to claim 9, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inductive promoter.

11. The composition for use according to claim 1, wherein the nucleic acid is present in a viral vector.

12. (I) The cells are human cells or mouse cells, and / or (II) The cell is a nerve cell. A composition for use according to claim 1.

13. The composition for use according to claim 1, wherein the cells are nerve cells in the brain of the subject.

14. The composition for use according to claim 1, further comprising the method for evaluating one or more signs or symptoms of tauopathy or tau aggregation in the cells.

15. The method reduces the amount of new tau aggregate formation in the cells, or The method reduces the amount of existing tau aggregate formation in the cells. A composition for use according to claim 1.

16. A method for inhibiting tau aggregates in cells, inhibiting or reducing tau phosphorylation on serine 356, or inhibiting or reducing the accumulation of insoluble tau, comprising administering to the cells a composition comprising a nucleic acid encoding LEM domain-containing protein 2 (LEMD2), charged multisplenic protein 7 (CHMP7), or inner nuclear membrane protein Man1 (LEMD3), wherein the cells are in vitro.

17. The method according to claim 16, wherein tau phosphorylation is reduced in the cell body, perinuclear region, and / or nucleocytoplasm of the cell.

18. The method according to claim 16, wherein the composition comprises the nucleic acid encoding the LEMD2.

19. The method according to claim 18, wherein the LEMD2 is human LEMD2 or mouse LEMD2.

20. The method according to claim 16, wherein the composition comprises the nucleic acid encoding CHMP7.

21. The method according to claim 20, wherein the CHMP7 is human CHMP7 or mouse CHMP7.

22. The method according to claim 16, wherein the composition comprises the nucleic acid encoding the LEMD3.

23. The method according to claim 22, wherein the LEMD3 is human LEMD3 or mouse LEMD3.

24. (i) The nucleic acid comprises complementary DNA encoding LEMD2, CHMP7, or LEMD3, (II) The nucleic acid contains messenger RNA encoding LEMD2, CHMP7, or LEMD3, or (III) The composition comprises an expression construct comprising the nucleic acid encoding the LEMD2, CHMP7, or LEMD3, which is operably connected to a promoter. The method according to claim 16.

25. (i) The promoter is a different type of promoter, or (II) The method according to claim 24, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inductive promoter.

26. The method according to claim 16, wherein the nucleic acid is present in a viral vector.

27. ​​(i) The cells are human cells or mouse cells, and / or (II) The cell is a nerve cell. The method according to claim 16.

28. The method according to claim 16, further comprising evaluating one or more signs or symptoms of tauopathy or tau aggregation in the cells.

29. The method reduces the amount of new tau aggregate formation in the cells, or The method reduces the amount of existing tau aggregate formation in the cells. The method according to claim 16.

Images