Neuronal promoters and uses thereof

Abstract

The present disclosure provides a heterologous gene of interest linked to a regulatory element, wherein the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located 3' to the translation start site of the endogenous GPR88 gene. The present disclosure provides a heterologous gene of interest linked to a regulatory element that results in increased gene expression. The present disclosure provides a promoter sequence for use in a method for treating Parkinson's disease. The present disclosure provides a designer receptor exclusively activated by designer drugs (DREADD) for use in a method for treating Parkinson's disease.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 437,220, filed January 5, 2023, and U.S. Provisional Patent Application No. 63 / 589,864, filed October 12, 2023, which applications are incorporated herein by reference.

[0002] Incorporation by reference to sequence listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy, created on December 20, 2023, is named 062692-502001WO_SL.xml and is 69,865 bytes in size. [Background technology]

[0003] background Adeno-associated virus (AAV) is a small (25 nm) virus belonging to the Parvovirus family that infects humans and other primate species. AAV is used as a delivery vector for gene therapy because it can establish a latent infection, allowing the AAV genome to integrate into host chromosomes without triggering a destructive T-cell immune response. Approximately 13 serotypes of AAV have been isolated from the wild. Summary of the Invention [Means for solving the problem]

[0004] overview Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but these effects lack specificity due to the widespread distribution of dopamine receptors in the central nervous system and periphery. This disclosure involves the development of a gene therapy strategy to selectively manipulate the circuitry affected by PD. We designed a therapeutic strategy that targets striatal D1 medium spiny neurons (MSNs), whose activity may be chronically suppressed in PD, and includes a highly efficient novel retrograde AAV, a promoter element with strong D1-MSN activity, and a chemogenetic effector that enables precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach can rescue locomotion, tremor, and motor skill defects in PD, thus supporting the utility of targeted circuit modulation tools for the treatment of PD in humans.

[0005] The present disclosure provides heterologous genes of interest linked to regulatory elements that increase gene expression. The present disclosure provides promoter sequences for use in methods for treating Parkinson's disease. The present disclosure provides designer receptors exclusively activated by designer drugs (DREADDs) for use in methods for treating Parkinson's disease.

[0006] The present disclosure provides promoters and promoter sequences that increase gene expression, particularly in neuronal cells. The promoters allow for enhanced expression of gene therapy, therapeutic proteins, and / or designer receptors in neuronal tissue (e.g., dopaminergic medium spiny neurons). Such enhanced expression can be used to treat Parkinson's disease.

[0007] In some embodiments, the disclosure provides a nucleic acid comprising a heterologous gene of interest operably linked to a regulatory element, wherein the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located 3' to the translation start site of the endogenous GPR88 gene.

[0008] In some embodiments, the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located, in part, within an intron. In some embodiments, the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located, in part, within an intron.

[0009] In some embodiments, the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located less than about 1,000 nucleotides 3' to the translation start site of the endogenous GPR88 gene. In some embodiments, the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO: 39. In some embodiments, the regulatory element comprises a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 39.

[0010] In some embodiments, the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located less than about 900 nucleotides 3' to the translation start site of the endogenous GPR88 gene. In some embodiments, the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in SEQ ID NO: 40. In some embodiments, the regulatory element comprises a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 40.

[0011] In some embodiments, the regulatory element comprises a nucleotide sequence that corresponds to a genomic sequence located 5' to the translation start site of the endogenous GPR88 gene.

[0012] In some embodiments, the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 100 nucleotides 5' to the translation start site of the endogenous GPR88 gene. In some embodiments, the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in SEQ ID NO: 41. In some embodiments, the regulatory element comprises a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 41.

[0013] In some embodiments, the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 600 nucleotides 5' to the translation start site of the endogenous GPR88 gene. In some embodiments, the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in SEQ ID NO: 42. In some embodiments, the regulatory element comprises a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 42.

[0014] In some embodiments, the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 1,500 nucleotides 5' to the translation start site of the endogenous GPR88 gene. In some embodiments, the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO: 43. In some embodiments, the regulatory element comprises a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 43.

[0015] In some embodiments, the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 45, or 46. In some embodiments, the regulatory element comprises a nucleotide sequence that is identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 45, or 46.

[0016] In some embodiments, the disclosure provides a nucleic acid comprising a heterologous gene of interest operably linked to a regulatory element, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in SEQ ID NO:47.

[0017] In some embodiments, the heterologous gene of interest is 3' to the regulatory element. In some embodiments, the heterologous gene of interest has a therapeutic utility.

[0018] In some embodiments, the gene of interest comprises a neurotrophic factor, an RNA-guided nuclease, an enzyme, or a DREADD. In some embodiments, the heterologous gene of interest exhibits increased expression in striatal neurons compared to the promoter of the hSYN1 gene. In some embodiments, the gene of interest comprises a DREADD. In some embodiments, the DREADD is selected from the list consisting of one or more of rM3Ds, hM3Ds, or hM3Ds(A147S-F349Y). In some embodiments, the DREADD is rM3Ds. In some embodiments, the DREADD comprises an amino acid sequence that exhibits at least about 90%, 95%, 97%, 98%, 99% identity to, or is identical to, SEQ ID NO: 38. In some embodiments, the heterologous gene of interest comprises one or more of hM3Dq, hM1Dq, hMD5q, hM4Di, hM2Di, or BDNF. In some embodiments, the DREADD is hM3Ds. In some embodiments, the DREADD comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to, or identical to, SEQ ID NO: 49. In some embodiments, the DREADD is hM3Ds(A147S-F349Y). In some embodiments, the DREADD comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99% identical to, or identical to, SEQ ID NO: 50.

[0019] In some embodiments, the nucleic acid is contained in a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a retroAAV (AAV retro) virion.

[0020] In some embodiments, the gene of interest exhibits at least twice the expression of the heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene. In some embodiments, the gene of interest exhibits at least twice the expression of the heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene. In some embodiments, the gene of interest exhibits at least twice the expression of the heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

[0021] In some embodiments, the present disclosure provides pharmaceutical compositions, comprising pharmaceutically acceptable carriers, additives or diluents and viral vectors.In some embodiments, viral vectors or pharmaceutical compositions are used in the method for expressing polypeptide in striatal neurons.In some embodiments, striatal neurons are D1 dopaminergic medium spiny neurons.

[0022] In some embodiments, the viral vector or pharmaceutical composition is used in a method for genetically manipulating striatal neurons. In some embodiments, the striatal neurons are D1 dopaminergic medium spiny neurons.

[0023] In some embodiments, the viral vector or pharmaceutical composition is used in a method of treating a neurodegenerative disease in an individual. In some embodiments, the neurodegenerative disease comprises Parkinson's disease.

[0024] In some embodiments, the present disclosure provides a method of expressing a polypeptide in a neuron of the striatum of an individual, the method comprising administering a nucleic acid or pharmaceutical composition to the individual to express the polypeptide in the neuron of the striatum. In some embodiments, the neuron of the striatum is a D1 dopaminergic medium spiny neuron.

[0025] In some embodiments, the present disclosure provides a method for genetically manipulating striatal neurons in an individual, comprising administering a nucleic acid or pharmaceutical composition to the individual to genetically manipulate the striatal neurons. In some embodiments, the striatal neurons are D1 dopaminergic medium spiny neurons.

[0026] In some embodiments, the present disclosure provides a method of treating an individual suffering from a neurodegenerative disease, comprising administering a nucleic acid or pharmaceutical composition to the individual suffering from the neurodegenerative disease, thereby treating the neurodegenerative disease. In some embodiments, the neurodegenerative disease comprises Parkinson's disease. In some embodiments, the individual is a mammal. In some embodiments, the individual is human.

[0027] In some embodiments, the present disclosure provides a method for expressing and activating a DREADD in the central nervous system of an individual, the method comprising activating the DREADD in the central nervous system of an individual by administering to the individual a retro-AAV or a pharmaceutical composition and a ligand that activates the DREADD. In some embodiments, the DREADD is expressed and activated in neurons of the striatum. In some embodiments, the neurons of the striatum are D1 dopaminergic medium spiny neurons. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. In some embodiments, activating a DREADD in the central nervous system of an individual treats a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder includes Parkinson's disease. In some embodiments, the ligand that activates the DREADD includes quetiapine or clozapine. In some embodiments, the ligand that activates the DREADD includes quetiapine. In some embodiments, the ligand that activates the DREADD includes clozapine. In some embodiments, the retro-AAV and the ligand that activates the DREADD are administered separately.

[0028] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages thereof will be obtained by reference to the following detailed description that sets forth illustrative examples in which the principles of the features described herein are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0029] [Figure 1A-B] FIG. 1A shows the amino acid sequence from rAAV2 retrovirus at position 588 and N385D, which were introduced into the AAV8 capsid protein at three sites to generate AAV8R. [ka] (where the bolded portion represents an additional 10-aa insertion) and TS720-721IN. Two additional mutations, V183E and N411S, were incorporated into the AAV8R12 capsid protein. The figure discloses SEQ ID NOS: 33, 61, and 61, respectively, in order of appearance. FIG. 1B shows the mutations made to generate AAV1R, AAV5R, and AAV6R. To generate AAV1R, the amino acid sequence of SSSTDP starting at position 586 of AAV1 (SEQ ID NO: 53) was replaced with RGNLADQDYTKTARQA (SEQ ID NO: 54), and two point mutations, N383D and A709I, were made. To generate AAV5R, the amino acid sequence NQSSTTAP (SEQ ID NO: 55) starting at position 575 of AAV5 was replaced with LQRGNLADQDYTKTARQA (SEQ ID NO: 56), and DPQF at positions 695-698 was mutated to KSIN (SEQ ID NO: 57). To generate AAV6R, the amino acid sequence SSSTDP (SEQ ID NO: 53) starting at position 586 of AAV6 was replaced with RGNLADQDYTKTARQA (SEQ ID NO: 54), and two point mutations, N383D and A709I, were made. The figure discloses SEQ ID NOs: 62-66, 57, 62, and 66, respectively, in order of appearance. [Figure 1C]FIG. 1C shows that AAV1R, AAV5R, and AAV6R are insufficient to label striating projection neurons, as almost no neurons are labeled. [Figure 1D] Figure 1D shows images and percentages of retrogradely labeled neurons in the SNr and upstream brain regions by substantia nigra injection of AAV8R12, indicating brain regions with EYFP+ cells. SNr: substantia nigra pars reticulata. STN: subthalamic nucleus. SC: superior colliculus. OFC: orbitofrontal cortex. ACAv: anterior cingulate cortex, ventral. ILA: inferolimbic cortex. PrL: prelimbic cortex. FrA: frontal association cortex. Scale bar, 1 mm. [Figure 1E] Figure 1E shows images and percentages of retrogradely labeled neurons in the SNr and upstream brain regions by substantia nigra injection of AAV8R12, demonstrating quantification of EYFP+ cells. n = 3 mice per group, data are presented as mean ± SEM.

[0030] [Figure 2A] FIG. 2A shows how the virus was injected into the SNr of wild-type mice. [Figure 2B] Figures 2B-2C show the extent to which AAV8R and AAV8R12 exhibit a marked increase in efficiency of labeling striatonigral projection neurons compared to rAAV2-retro. Scale bar (2B), 100 µm. n = 3 mice per group (2C). Error bars indicate SEM. One-way ANOVA with post-hoc Tukey's test (F(2,6) = 48.66; rAAV2-retro vs. AAV8R: 2078 ± 262.4 vs. 10,092 ± 463.9, P = 0.0032; rAAV2-retro vs. AAV8R12: 2078 ± 262.4 vs. 16,026 ± 1654, P = 0.0002; AAV8R vs. AAV8R12: 10,092 ± 463.9 vs. 16,026 ± 1654, P = 0.0137). [Figure 2C]Figures 2B-2C show the extent to which AAV8R and AAV8R12 exhibit a marked increase in efficiency of labeling striatonigral projection neurons compared to rAAV2-retro. Scale bar (2B), 100 µm. n = 3 mice per group (2C). Error bars indicate SEM. One-way ANOVA with post-hoc Tukey's test (F(2,6) = 48.66; rAAV2-retro vs. AAV8R: 2078 ± 262.4 vs. 10,092 ± 463.9, P = 0.0032; rAAV2-retro vs. AAV8R12: 2078 ± 262.4 vs. 16,026 ± 1654, P = 0.0002; AAV8R vs. AAV8R12: 10,092 ± 463.9 vs. 16,026 ± 1654, P = 0.0137). [Figure 2D-E] Figures 2D-2E demonstrate robust improvement in retrograde labeling efficiency of nucleus accumbens MSNs compared to rAAV2-retro. Figure 2D shows representative images of mouse nucleus accumbens-projecting neurons labeled with rAAV2-retro, AAV8R, and AAV8R12 after delivery to the dorsolateral ventral pallidum (scale bar, 100 µm). Figure 2E shows representative images of mouse nucleus accumbens-projecting neurons labeled with rAAV2-retro, AAV8R, and AAV8R12 after delivery to the lateral hypothalamus (LH) (scale bar, 100 µm).

[0031] [Figure 3A-B] Figures 3A-3C show the strategy for identifying highly active MSN promoters. A list of striatum-enriched genes was identified based on in situ hybridization data (3A). Putative promoter sequences were determined by H3K4me1 and H3K27ac epigenetic marks (3B) and cloned into an AAV vector expressing EYFP (3C). WPRE is the woodchuck hepatitis posttranscriptional regulatory element, pA is the polyadenylation signal, and ITR is the inverted terminal repeat. [Figure 3C]Figures 3A-3C show the strategy for identifying highly active MSN promoters. A list of striatum-enriched genes was identified based on in situ hybridization data (3A). Putative promoter sequences were determined by H3K4me1 and H3K27ac epigenetic marks (3B) and cloned into an AAV vector expressing EYFP (3C). WPRE is the woodchuck hepatitis posttranscriptional regulatory element, pA is the polyadenylation signal, and ITR is the inverted terminal repeat.

[0032] [Figure 4A] Figures 4A-4B show the extent to which the G88P2, G88P3, and G88P7 promoters exhibit increased efficiency in driving reporter expression in MSNs, as evidenced by the outgrowth of labeled neurons, compared to several commonly used promoters, including human synapsin-1 (hSyn), CMV early enhancer / chicken β-actin (CAG), and elongation factor 1 alpha (EF1α). Scale bar (4A), 100 µm. n = 3 mice per group (4B). Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test, (F(5,12) = 13.49; hSyn vs. EF1α: 16052 ± 1630 vs. 6954 ± 698.7 P = 0.4749; hSyn vs. CAG: 16052 ± 1630 vs. 4610 ± 707.7, P = 0.2824; hSyn vs. G88P2: 16052 ± 1630 vs. 36290 ± 3705, P = 0.0258; hSyn vs. G88P3: 16052 ± 1630 vs. 37026 ± 6646, P = 0.0209; hSyn vs. G88P7: 16052 ± 1630 vs. 39460 ± 7281, P = 0.0104). [Figure 4B]Figures 4A-4B show the extent to which the G88P2, G88P3, and G88P7 promoters exhibit increased efficiency in driving reporter expression in MSNs, as evidenced by the outgrowth of labeled neurons, compared to several commonly used promoters, including human synapsin-1 (hSyn), CMV early enhancer / chicken β-actin (CAG), and elongation factor 1 alpha (EF1α). Scale bar (4A), 100 µm. n = 3 mice per group (4B). Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test, (F(5,12) = 13.49; hSyn vs. EF1α: 16052 ± 1630 vs. 6954 ± 698.7 P = 0.4749; hSyn vs. CAG: 16052 ± 1630 vs. 4610 ± 707.7, P = 0.2824; hSyn vs. G88P2: 16052 ± 1630 vs. 36290 ± 3705, P = 0.0258; hSyn vs. G88P3: 16052 ± 1630 vs. 37026 ± 6646, P = 0.0209; hSyn vs. G88P7: 16052 ± 1630 vs. 39460 ± 7281, P = 0.0104).

[0033] [Figure 5A-B] Figure 5A shows striatal neurons (arrowheads) colabeled with AAV8R12-G88P3-EYFP for Drd1, but not Drd2, after viral injection into the SNr. Scale bar, 5 μm. Figure 5B is a plot showing quantification of Drd1 and Drd2 cells among EYFP cells; n = 3 mice per group, and data are presented as mean ± SEM.

[0034] [Figure 6A]Figure 6A shows that after delivery of AAV8R12-G88P3-HA-hM3Dq to the substantia nigra, intraperitoneal (ip) delivery of CNO induced isotropic rotation, whereas intracranial (i.c.) injection of CNO into the dorsomedial striatum induced retrograde rotation. (n = 8 mice per group. Error bars indicate mean ± SEM. Unpaired t-test.) (Saline i.c. vs. CNO i.c.: 45.25 ± 4.128 vs. 87.84 ± 4.666, t = 6.836, P < 0.0001; saline i.c. vs. CNO i.c.: 49.63 ± 5.879 vs. 15.08 ± 3.435, t = 5.074, P = 0.0002). [Figure 6B] Figure 6B shows AAV8R12-G88P3-HA-hM3Dq and c-Fos colabeled substantia nigra neurons after intraperitoneal, but not intracranial, injection of CNO. Scale bar, 20 μm. [Figure 6C] Figure 6C shows that after delivery of AAV8R12-G88P3-EYFP to the substantia nigra, both intraperitoneal (ip) delivery of CNO and intracranial (ic) injection of CNO into the dorsomedial striatum had no effect on rotational behavior in mice. (n = 5 mice per group. Error bars indicate mean ± SEM. Unpaired t-test.) (Saline ip vs. CNO ip: 47.08 ± 10.5 vs. 44.72 ± 10.89, t = 0.1561, df = 8, P = 0.8798; saline ic vs. CNO ic: 54.22 ± 8.766 vs. 61.39 ± 5.774, t = 0.6838, df = 8, P = 0.5134). [Figure 6D] FIG. 6D includes representative images of neurons retrogradely labeled by substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the SNr and upstream brain regions, where the scale bar represents 1 mm. [Figure 6E-F]Figure 6E shows retrograde labeling of mouse striatal neurons with Drd1 ISH (left panel, arrowhead) and Drd2 ISH (right panel, arrowhead) after AAV8R12-G88P3-HA-hM3Dq injection into the SNr, where the scale bar represents 10 μm. Figure 6F is a plot showing quantification of Drd1 and Drd2 cells among all HA cells, where n = 3 mice per group, and data are presented as mean ± SEM. [Figure 6G] Figure 6G shows the electrophysiological response to CNO in retrogradely labeled D1-MSNs after AAV8R12-G88P3-HA-hM3Dq-2A-EYFP injection into the SNr. Whole-cell patch-clamp recordings were performed from EYFP+ cells in ex vivo slices. Figure 6G includes representative traces (left panel) and quantification (right panel) of action potentials induced by somatic current injection at baseline and after CNO administration. n = 7 cells from 4 mice. Data are presented as mean ± SEM, paired two-tailed t-test; and ***p < 0.001. [Figure 6H-I] Figures 6H-6I depict chemogenetic BG direct tract manipulation in mice 3 weeks after substantia nigra delivery of AAV8R12-G88P3-HA-hM3Dq and CNO delivery via either intraperitoneal (i.p.) injection (Figure 6H) or intracranial (i.c.) injection targeting the dorsomedial striatum (Figure 6I). The percentage of retrograde rotation was quantified (left panel); n = 8 mice per group. Data are presented as mean ± SEM. Unpaired two-tailed t-test, ***p < 0.001. HA and c-Fos antibody staining in the mouse SNr after CNO delivery (right panel). Scale bar, 20 µm.

[0035] [Figure 7A]Figure 7A shows that after substantia nigra delivery of AAV8R12-G88P7-rM3Ds-2A-EYFP, both intraperitoneal and intracranial delivery of CNO induced retrograde rotation. (n = 8 mice per group. Error bars indicate mean ± SEM.) (Unpaired t-test) (Saline i.p. vs. CNO i.p.: 45.95 ± 4.972 vs. 80.05 ± 5.168, t = 4.754, P = 0.0003; Saline i.c. vs. CNO i.c.: 58.31 ± 4.742 vs. 80.77 ± 4.187, t = 3.551, P = 0.0032). [Figure 7B] Figure 7B shows that no nigral neurons were co-labeled with AAV8R12-G88P7-rM3Ds-2A-EYFP and c-Fos after intraperitoneal or intracranial injection of CNO. Scale bar, 20 μm. [Figure 7C] Figure 7C shows that after substantia nigra delivery of AAV8R12-G88P7-EYFP, both intraperitoneal and intracranial delivery of CNO had no effect on mouse rotational behavior. (n = 5 mice per group; error bars indicate mean ± SEM; unpaired t-test) (saline i.p. vs. CNO i.p.: 48.68 ± 7.203 vs. 46.88 ± 7.497, t = 0.1733, df = 8, P = 0.8667; saline i.c. vs. CNO i.c.: 41.11 ± 12.73 vs. 34.55 ± 7.536, t = 0.4435, df = 8, P = 0.6691). [Figure 7D] FIG. 7D includes representative images of neurons retrogradely labeled by substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP in the SNr and upstream brain regions, where the scale bar represents 1 mm. [Figure 7E-F] Figure 7E includes images of retrograde labeling of mouse striatal neurons with Drd1 ISH (left, arrowhead) and Drd2 ISH (right, arrowhead) after AAV8R12-G88P7-rM3Ds-2A-EYFP injection into the SNr, where the scale bar represents 10 μm. Figure 7F is a plot showing quantification of Drd1 and Drd2 cells among EYFP cells, where n=3 mice per group and data are presented as mean ± SEM. [Figure 7G] Figure 7G shows the electrophysiological response to CNO in retrogradely labeled D1-MSNs after AAV8R12-G88P7-rM3Ds-2A-EYFP injection into the SNr. Whole-cell patch-clamp recordings were performed from EYFP+ cells in ex vivo slices. Figure 7G contains representative traces (left panel) and quantification (right panel) of action potentials induced by somatic current injection at baseline and after CNO administration. n = 10 cells from 6 mice. Data are presented as mean ± SEM. Paired two-tailed t-test, ****p < 0.0001. [Figure 7H-I] Figures 7H-7I depict chemogenetic manipulation of the BG direct pathway in mice 3 weeks after delivery of AAV8R12-G88P7-rM3Ds-2A-EYFP to the substantia nigra and CNO delivery via either i.p. injection (Figure 7H) or i.c. injection targeting the dorsomedial striatum (Figure 7I). The percentage of retrograde rotation was quantified (left panel); n = 8 mice per group. Data are presented as mean ± SEM. Unpaired two-tailed t-test, **p < 0.01, ***p < 0.001). EYFP and c-Fos antibody staining in the mouse SNr after CNO delivery (right panel). Scale bar, 20 µm.

[0036] [Figure 8A] Figures 8A-8B show representative heat maps of macaques receiving substantia nigra injections of AAV8R12-G88P7-HA-hM3Dq (Figure 8A) or AAV8R12-G88P7-rM3Ds-2A-EYFP (Figure 8B) after saline or CNO infusion. Animals administered CNO spent less time in the higher part of the observation cage. [Figure 8B] Figures 8A-8B show representative heat maps of macaques receiving substantia nigra injections of AAV8R12-G88P7-HA-hM3Dq (Figure 8A) or AAV8R12-G88P7-rM3Ds-2A-EYFP (Figure 8B) after saline or CNO infusion. Animals administered CNO spent less time in the higher part of the observation cage. [Figure 8C-H]Figures 8C-8H show quantification of the speed of isotropic rotation (Figures 8C, 8F), total distance traveled (Figures 8D, 8G), and immobility time (Figures 8E, 8H) for macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (Figures 8C-8E) or AAV8R12-G88P7-rM3Ds-2A-EYFP (Figures 8F-8H) after saline or CNO infusion. Monkeys per group (n = 3) (Figures 8C-8E) and monkeys per group (n = 6) (Figures 8F-8H). Data are presented as mean ± SEM; paired two-tailed t-test; ns, not significant.

[0037] [Figure 9A] Figure 9A shows AAV8R12-G88P3-mCherry injected into the SNr of a cynomolgus macaque; labeled neurons were found throughout the caudate and putamen nuclei. The location of coronal sections along the anterior-posterior axis is shown as the distance from EBZ (ear bar zero). Scale bar, 5 mm. [Figure 9B] Figure 9B shows labeled neurons in the caudate and putamen nuclei. Scale bar, 100 μm. [Figure 9C] Figure 9C shows retrograde labeling of striatal neurons with DRD1 ISH (upper panel, arrowheads) and DRD2 ISH (lower panel, arrowheads) after AAV8R12-G88P3-mCherry injection into the macaque SNr. Only co-labeling was observed for DRD1. Scale bar, 20 µm. [Figure 9D-E] Figures 9D-9E show activation of the BG direct pathway (9D) and the percentage of rotational behavior (i.e., i.e., i.v. and i.p. rotations) in mice 12 months after substantia nigra delivery of AAV8R12-G88P7-rM3Ds-2A-EYFP and CNO delivery by i.p. injection were quantified (Figure 9E). n=6 mice per group. Data error bars indicate mean ± SEM. Unpaired two-tailed t-test. **P<0.01. [Figure 9F-G]Figure 9F shows robust labeling of D1-MSNs after substantia nigra injection of AAV8R12 in a cynomolgus macaque, with neuronal cell bodies retrogradely labeled throughout the caudate nucleus and putamen, extracted from the fluorescence image (9A). The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 2 mm. Figure 9G is a plot showing quantification of DRD1+ and DRD2+ cells among mCherry+ cells, where n=6 sections from one macaque and data are presented as mean ± SEM.

[0038] [Figure 10A]Figure 10A shows a representative top-view movement tracing plot of an observation cage housing a macaque that received a substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the observation cage after an icCNO injection into the dorsomedial caudate nucleus of the macaque. Figure 10B shows a representative top-view tracing plot of a macaque that received a substantia nigra injection of AAV8R12-NP3-rM3Ds-2A-EYFP in the observation cage after an intramuscular (i.m.) CNO injection into the macaque. Quantification of time in the top compartment of the observation cage after intracranial or systemic CNO injection is shown. Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed a significant increase in retrograde rotation (10C). n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired two-tailed t-test (t = 3.276, P = 0.0469; saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP spent less time in the top part of the observation cage (10D). (Figure 1 shows quantification of the speed of retrograde rotation after CNO injection in macaques, n = 4 monkeys per group. Error bars indicate mean ± SEM, paired two-tailed t-test, *p <0.05 (t = 3.605, P = 0.0366, saline vs. CNO: 68.42 ± 10.7 vs. 33.74 ± 2.641)), and showed increased velocity during retrograde rotation (10 G) after intracranial or systemic CNO injection, respectively (paired t test, t = 4.06, P = 0.0269, saline vs. CNO: 38.42 ± 5.465 vs. 46.98 ± 6.669).No significant differences were found for immobility time (10E) (paired t-test, t = 2.211, P = 0.1140; saline vs. CNO: 107.1 ± 2.322 vs. 89.13 ± 8.379), total distance (10F) (paired t-test, t = 2.272, P = 0.1077; saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), velocity of retrograde rotation (10G), or velocity of isograde rotation (10H) (paired t-test, t = 1.001, P = 0.3905; saline vs. CNO: 37.43 ± 3.264 vs. 41.37 ± 6.566). n = 4. Error bars represent mean ± SEM (paired t-test). Figure 10I includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10J includes higher-magnification images of labeled hM3Dq neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10K includes retrograde labeling of hM3Dq striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10L shows quantification of DRD1 and DRD2 cells among all HA cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM. Figure 10M includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10N includes high-magnification images of labeled rM3Ds+ neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10O shows retrograde labeling of rM3Ds+ striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10P shows quantification of DRD1+ and DRD2+ cells among all EYFP+ cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM.Figure 10Q depicts a representative top-view behavioral tracking plot of an observation cage housing a macaque that received an icCNO injection into the dorsomedial caudate nucleus 8 weeks after the substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10Q. n = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figure 10S is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10B. n = 6 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figures 10T-10U depict quantification of time spent in the top compartment of the observation cage (Figure 10T) and speed of retrograde rotation (Figure 10U) after icCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq. n = 3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p < 0.05. Figures 10V-10W depict quantification of time spent in the top compartment of the observation cage (Figure 10V) and speed of retrograde rotation (Figure 10W) ​​after imCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 6 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01, ***p < 0.001. [Figure 10B]Figure 10A shows a representative top-view movement tracing plot of an observation cage housing a macaque that received a substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the observation cage after an icCNO injection into the dorsomedial caudate nucleus of the macaque. Figure 10B shows a representative top-view tracing plot of a macaque that received a substantia nigra injection of AAV8R12-NP3-rM3Ds-2A-EYFP in the observation cage after an intramuscular (i.m.) CNO injection into the macaque. Quantification of time in the top compartment of the observation cage after intracranial or systemic CNO injection is shown. Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed a significant increase in retrograde rotation (10C). n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired two-tailed t-test (t = 3.276, P = 0.0469; saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP spent less time in the top part of the observation cage (10D). (Figure 1 shows quantification of the speed of retrograde rotation after CNO injection in macaques, n = 4 monkeys per group. Error bars indicate mean ± SEM, paired two-tailed t-test, *p <0.05 (t = 3.605, P = 0.0366, saline vs. CNO: 68.42 ± 10.7 vs. 33.74 ± 2.641)), and showed increased velocity during retrograde rotation (10 G) after intracranial or systemic CNO injection, respectively (paired t test, t = 4.06, P = 0.0269, saline vs. CNO: 38.42 ± 5.465 vs. 46.98 ± 6.669).No significant differences were found for immobility time (10E) (paired t-test, t = 2.211, P = 0.1140; saline vs. CNO: 107.1 ± 2.322 vs. 89.13 ± 8.379), total distance (10F) (paired t-test, t = 2.272, P = 0.1077; saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), velocity of retrograde rotation (10G), or velocity of isograde rotation (10H) (paired t-test, t = 1.001, P = 0.3905; saline vs. CNO: 37.43 ± 3.264 vs. 41.37 ± 6.566). n = 4. Error bars represent mean ± SEM (paired t-test). Figure 10I includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10J includes higher-magnification images of labeled hM3Dq neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10K includes retrograde labeling of hM3Dq striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10L shows quantification of DRD1 and DRD2 cells among all HA cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM. Figure 10M includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10N includes high-magnification images of labeled rM3Ds+ neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10O shows retrograde labeling of rM3Ds+ striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10P shows quantification of DRD1+ and DRD2+ cells among all EYFP+ cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM.Figure 10Q depicts a representative top-view behavioral tracking plot of an observation cage housing a macaque that received an icCNO injection into the dorsomedial caudate nucleus 8 weeks after the substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10Q. n = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figure 10S is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10B. n = 6 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figures 10T-10U depict quantification of time spent in the top compartment of the observation cage (Figure 10T) and speed of retrograde rotation (Figure 10U) after icCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq. n = 3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p < 0.05. Figures 10V-10W depict quantification of time spent in the top compartment of the observation cage (Figure 10V) and speed of retrograde rotation (Figure 10W) ​​after imCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 6 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01, ***p < 0.001. [Figure 10C-H]Figure 10A shows a representative top-view movement tracing plot of an observation cage housing a macaque that received a substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the observation cage after an icCNO injection into the dorsomedial caudate nucleus of the macaque. Figure 10B shows a representative top-view tracing plot of a macaque that received a substantia nigra injection of AAV8R12-NP3-rM3Ds-2A-EYFP in the observation cage after an intramuscular (i.m.) CNO injection into the macaque. Quantification of time in the top compartment of the observation cage after intracranial or systemic CNO injection is shown. Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed a significant increase in retrograde rotation (10C). n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired two-tailed t-test (t = 3.276, P = 0.0469; saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP spent less time in the top part of the observation cage (10D). (Figure 1 shows quantification of the speed of retrograde rotation after CNO injection in macaques, n = 4 monkeys per group. Error bars indicate mean ± SEM, paired two-tailed t-test, *p <0.05 (t = 3.605, P = 0.0366, saline vs. CNO: 68.42 ± 10.7 vs. 33.74 ± 2.641)), and showed increased velocity during retrograde rotation (10 G) after intracranial or systemic CNO injection, respectively (paired t test, t = 4.06, P = 0.0269, saline vs. CNO: 38.42 ± 5.465 vs. 46.98 ± 6.669).No significant differences were found for immobility time (10E) (paired t-test, t = 2.211, P = 0.1140; saline vs. CNO: 107.1 ± 2.322 vs. 89.13 ± 8.379), total distance (10F) (paired t-test, t = 2.272, P = 0.1077; saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), velocity of retrograde rotation (10G), or velocity of isograde rotation (10H) (paired t-test, t = 1.001, P = 0.3905; saline vs. CNO: 37.43 ± 3.264 vs. 41.37 ± 6.566). n = 4. Error bars represent mean ± SEM (paired t-test). Figure 10I includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10J includes higher-magnification images of labeled hM3Dq neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10K includes retrograde labeling of hM3Dq striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10L shows quantification of DRD1 and DRD2 cells among all HA cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM. Figure 10M includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10N includes high-magnification images of labeled rM3Ds+ neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10O shows retrograde labeling of rM3Ds+ striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10P shows quantification of DRD1+ and DRD2+ cells among all EYFP+ cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM.Figure 10Q depicts a representative top-view behavioral tracking plot of an observation cage housing a macaque that received an icCNO injection into the dorsomedial caudate nucleus 8 weeks after the substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10Q. n = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figure 10S is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10B. n = 6 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figures 10T-10U depict quantification of time spent in the top compartment of the observation cage (Figure 10T) and speed of retrograde rotation (Figure 10U) after icCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq. n = 3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p < 0.05. Figures 10V-10W depict quantification of time spent in the top compartment of the observation cage (Figure 10V) and speed of retrograde rotation (Figure 10W) ​​after imCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 6 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01, ***p < 0.001. [Figure 10I-L]Figure 10A shows a representative top-view movement tracing plot of an observation cage housing a macaque that received a substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the observation cage after an icCNO injection into the dorsomedial caudate nucleus of the macaque. Figure 10B shows a representative top-view tracing plot of a macaque that received a substantia nigra injection of AAV8R12-NP3-rM3Ds-2A-EYFP in the observation cage after an intramuscular (i.m.) CNO injection into the macaque. Quantification of time in the top compartment of the observation cage after intracranial or systemic CNO injection is shown. Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed a significant increase in retrograde rotation (10C). n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired two-tailed t-test (t = 3.276, P = 0.0469; saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP spent less time in the top part of the observation cage (10D). (Figure 1 shows quantification of the speed of retrograde rotation after CNO injection in macaques, n = 4 monkeys per group. Error bars indicate mean ± SEM, paired two-tailed t-test, *p <0.05 (t = 3.605, P = 0.0366, saline vs. CNO: 68.42 ± 10.7 vs. 33.74 ± 2.641)), and showed increased velocity during retrograde rotation (10 G) after intracranial or systemic CNO injection, respectively (paired t test, t = 4.06, P = 0.0269, saline vs. CNO: 38.42 ± 5.465 vs. 46.98 ± 6.669).No significant differences were found for immobility time (10E) (paired t-test, t = 2.211, P = 0.1140; saline vs. CNO: 107.1 ± 2.322 vs. 89.13 ± 8.379), total distance (10F) (paired t-test, t = 2.272, P = 0.1077; saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), velocity of retrograde rotation (10G), or velocity of isograde rotation (10H) (paired t-test, t = 1.001, P = 0.3905; saline vs. CNO: 37.43 ± 3.264 vs. 41.37 ± 6.566). n = 4. Error bars represent mean ± SEM (paired t-test). Figure 10I includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10J includes higher-magnification images of labeled hM3Dq neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10K includes retrograde labeling of hM3Dq striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10L shows quantification of DRD1 and DRD2 cells among all HA cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM. Figure 10M includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10N includes high-magnification images of labeled rM3Ds+ neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10O shows retrograde labeling of rM3Ds+ striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10P shows quantification of DRD1+ and DRD2+ cells among all EYFP+ cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM.Figure 10Q depicts a representative top-view behavioral tracking plot of an observation cage housing a macaque that received an icCNO injection into the dorsomedial caudate nucleus 8 weeks after the substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10Q. n = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figure 10S is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10B. n = 6 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figures 10T-10U depict quantification of time spent in the top compartment of the observation cage (Figure 10T) and speed of retrograde rotation (Figure 10U) after icCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq. n = 3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p < 0.05. Figures 10V-10W depict quantification of time spent in the top compartment of the observation cage (Figure 10V) and speed of retrograde rotation (Figure 10W) ​​after imCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 6 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01, ***p < 0.001. [Figure 10M-P]Figure 10A shows a representative top-view movement tracing plot of an observation cage housing a macaque that received a substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the observation cage after an icCNO injection into the dorsomedial caudate nucleus of the macaque. Figure 10B shows a representative top-view tracing plot of a macaque that received a substantia nigra injection of AAV8R12-NP3-rM3Ds-2A-EYFP in the observation cage after an intramuscular (i.m.) CNO injection into the macaque. Quantification of time in the top compartment of the observation cage after intracranial or systemic CNO injection is shown. Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed a significant increase in retrograde rotation (10C). n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired two-tailed t-test (t = 3.276, P = 0.0469; saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP spent less time in the top part of the observation cage (10D). (Figure 1 shows quantification of the speed of retrograde rotation after CNO injection in macaques, n = 4 monkeys per group. Error bars indicate mean ± SEM, paired two-tailed t-test, *p <0.05 (t = 3.605, P = 0.0366, saline vs. CNO: 68.42 ± 10.7 vs. 33.74 ± 2.641)), and showed increased velocity during retrograde rotation (10 G) after intracranial or systemic CNO injection, respectively (paired t test, t = 4.06, P = 0.0269, saline vs. CNO: 38.42 ± 5.465 vs. 46.98 ± 6.669).No significant differences were found for immobility time (10E) (paired t-test, t = 2.211, P = 0.1140; saline vs. CNO: 107.1 ± 2.322 vs. 89.13 ± 8.379), total distance (10F) (paired t-test, t = 2.272, P = 0.1077; saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), velocity of retrograde rotation (10G), or velocity of isograde rotation (10H) (paired t-test, t = 1.001, P = 0.3905; saline vs. CNO: 37.43 ± 3.264 vs. 41.37 ± 6.566). n = 4. Error bars represent mean ± SEM (paired t-test). Figure 10I includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10J includes higher-magnification images of labeled hM3Dq neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10K includes retrograde labeling of hM3Dq striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10L shows quantification of DRD1 and DRD2 cells among all HA cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM. Figure 10M includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10N includes high-magnification images of labeled rM3Ds+ neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10O shows retrograde labeling of rM3Ds+ striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10P shows quantification of DRD1+ and DRD2+ cells among all EYFP+ cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM.Figure 10Q depicts a representative top-view behavioral tracking plot of an observation cage housing a macaque that received an icCNO injection into the dorsomedial caudate nucleus 8 weeks after the substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10Q. n = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figure 10S is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10B. n = 6 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figures 10T-10U depict quantification of time spent in the top compartment of the observation cage (Figure 10T) and speed of retrograde rotation (Figure 10U) after icCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq. n = 3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p < 0.05. Figures 10V-10W depict quantification of time spent in the top compartment of the observation cage (Figure 10V) and speed of retrograde rotation (Figure 10W) ​​after imCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 6 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01, ***p < 0.001. [Figure 10Q-S]Figure 10A shows a representative top-view movement tracing plot of an observation cage housing a macaque that received a substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the observation cage after an icCNO injection into the dorsomedial caudate nucleus of the macaque. Figure 10B shows a representative top-view tracing plot of a macaque that received a substantia nigra injection of AAV8R12-NP3-rM3Ds-2A-EYFP in the observation cage after an intramuscular (i.m.) CNO injection into the macaque. Quantification of time in the top compartment of the observation cage after intracranial or systemic CNO injection is shown. Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed a significant increase in retrograde rotation (10C). n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired two-tailed t-test (t = 3.276, P = 0.0469; saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP spent less time in the top part of the observation cage (10D). (Figure 1 shows quantification of the speed of retrograde rotation after CNO injection in macaques, n = 4 monkeys per group. Error bars indicate mean ± SEM, paired two-tailed t-test, *p <0.05 (t = 3.605, P = 0.0366, saline vs. CNO: 68.42 ± 10.7 vs. 33.74 ± 2.641)), and showed increased velocity during retrograde rotation (10 G) after intracranial or systemic CNO injection, respectively (paired t test, t = 4.06, P = 0.0269, saline vs. CNO: 38.42 ± 5.465 vs. 46.98 ± 6.669).No significant differences were found for immobility time (10E) (paired t-test, t = 2.211, P = 0.1140; saline vs. CNO: 107.1 ± 2.322 vs. 89.13 ± 8.379), total distance (10F) (paired t-test, t = 2.272, P = 0.1077; saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), velocity of retrograde rotation (10G), or velocity of isograde rotation (10H) (paired t-test, t = 1.001, P = 0.3905; saline vs. CNO: 37.43 ± 3.264 vs. 41.37 ± 6.566). n = 4. Error bars represent mean ± SEM (paired t-test). Figure 10I includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10J includes higher-magnification images of labeled hM3Dq neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10K includes retrograde labeling of hM3Dq striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10L shows quantification of DRD1 and DRD2 cells among all HA cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM. Figure 10M includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10N includes high-magnification images of labeled rM3Ds+ neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10O shows retrograde labeling of rM3Ds+ striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10P shows quantification of DRD1+ and DRD2+ cells among all EYFP+ cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM.Figure 10Q depicts a representative top-view behavioral tracking plot of an observation cage housing a macaque that received an icCNO injection into the dorsomedial caudate nucleus 8 weeks after the substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10Q. n = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figure 10S is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10B. n = 6 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figures 10T-10U depict quantification of time spent in the top compartment of the observation cage (Figure 10T) and speed of retrograde rotation (Figure 10U) after icCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq. n = 3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p < 0.05. Figures 10V-10W depict quantification of time spent in the top compartment of the observation cage (Figure 10V) and speed of retrograde rotation (Figure 10W) ​​after imCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 6 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01, ***p < 0.001. [Figure 10T-W]Figure 10A shows a representative top-view movement tracing plot of an observation cage housing a macaque that received a substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq in the observation cage after an icCNO injection into the dorsomedial caudate nucleus of the macaque. Figure 10B shows a representative top-view tracing plot of a macaque that received a substantia nigra injection of AAV8R12-NP3-rM3Ds-2A-EYFP in the observation cage after an intramuscular (i.m.) CNO injection into the macaque. Quantification of time in the top compartment of the observation cage after intracranial or systemic CNO injection is shown. Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed a significant increase in retrograde rotation (10C). n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired two-tailed t-test (t = 3.276, P = 0.0469; saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP spent less time in the top part of the observation cage (10D). (Figure 1 shows quantification of the speed of retrograde rotation after CNO injection in macaques, n = 4 monkeys per group. Error bars indicate mean ± SEM, paired two-tailed t-test, *p <0.05 (t = 3.605, P = 0.0366, saline vs. CNO: 68.42 ± 10.7 vs. 33.74 ± 2.641)), and showed increased velocity during retrograde rotation (10 G) after intracranial or systemic CNO injection, respectively (paired t test, t = 4.06, P = 0.0269, saline vs. CNO: 38.42 ± 5.465 vs. 46.98 ± 6.669).No significant differences were found for immobility time (10E) (paired t-test, t = 2.211, P = 0.1140; saline vs. CNO: 107.1 ± 2.322 vs. 89.13 ± 8.379), total distance (10F) (paired t-test, t = 2.272, P = 0.1077; saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), velocity of retrograde rotation (10G), or velocity of isograde rotation (10H) (paired t-test, t = 1.001, P = 0.3905; saline vs. CNO: 37.43 ± 3.264 vs. 41.37 ± 6.566). n = 4. Error bars represent mean ± SEM (paired t-test). Figure 10I includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10J includes higher-magnification images of labeled hM3Dq neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10K includes retrograde labeling of hM3Dq striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10L shows quantification of DRD1 and DRD2 cells among all HA cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM. Figure 10M includes representative images of retrograde labeling throughout the basal ganglion after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. The location of coronal sections along the anterior-posterior axis is shown as the distance from the EBZ. Scale bar, 5 mm. Figure 10N includes high-magnification images of labeled rM3Ds+ neurons in the caudate and putamen nuclei of macaques. Scale bar, 20 μm. Figure 10O shows retrograde labeling of rM3Ds+ striatal neurons (green) with DRD1 ISH (left panel, magenta, arrowheads) and DRD2 ISH (right panel, magenta, arrowheads). Scale bar, 20 μm. Figure 10P shows quantification of DRD1+ and DRD2+ cells among all EYFP+ cells. n = 6 sections from one macaque per group; data are presented as mean ± SEM.Figure 10Q depicts a representative top-view behavioral tracking plot of an observation cage housing a macaque that received an icCNO injection into the dorsomedial caudate nucleus 8 weeks after the substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10Q. n = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figure 10S is a plot showing quantification of the ratio of retrograde to ipsidirectional rotations after CNO injection for Figure 10B. n = 6 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p < 0.05. Figures 10T-10U depict quantification of time spent in the top compartment of the observation cage (Figure 10T) and speed of retrograde rotation (Figure 10U) after icCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq. n = 3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p < 0.05. Figures 10V-10W depict quantification of time spent in the top compartment of the observation cage (Figure 10V) and speed of retrograde rotation (Figure 10W) ​​after imCNO injection in macaques receiving substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 6 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01, ***p < 0.001.

[0039] [Figure 11A]Figure 11 shows representative images of naive macaques after saline or CNO injection from the top (Figure 11A) and side (Figure 11B) perspectives of the observation cage. The animals showed no differences in rotational behavior or time spent in the higher part of the observation cage. After intracranial or systemic CNO injection, there was no significant difference in the percentage of retrograde and ipsilateral rotations (Figure 11C) (paired t-test, t = 1.709, df = 2, P = 0.2296, saline vs. CNO: 0.767 ± 0.4296 vs. 0.8384 ± 0.4152), time spent in the higher part of the observation cage (Figure 11D) (paired t-test, t = 1.141, df = 2, P = 0.3720, saline vs. CNO: 55.73 ± 2.64 vs. 60.43 ± 4.542), and time spent immobile (Figure 11E) (paired t-test, t = 0.02596, df = 3, P = 0.9816, saline vs. CNO: 149.9 ± 21.61 vs. 149.8 ± 21.61). No significant differences were found in the total distance (Fig. 6F) (paired t-test, t = 1.505, df = 2, P = 0.2713, saline vs. CNO: 53.12 ± 15.3 vs. 59.48 ± 18.65), the speed of retrograde rotation (Fig. 6G) (paired t-test, t = 0.134, df = 2, P = 0.9057, saline vs. CNO: 39.07 ± 5.889 vs. 39.63 ± 7.092), or the speed of isotropic rotation (Fig. 6H) (paired t-test, t = 1.129, df = 2, P = 0.3762, saline vs. CNO: 41.49 ± 3.96 vs. 42.79 ± 4.705). n = 3 monkeys per group, error bars indicate mean ± SEM, paired t test. [Figure 11B]Figure 11 shows representative images of naive macaques after saline or CNO injection from the top (Figure 11A) and side (Figure 11B) perspectives of the observation cage. The animals showed no differences in rotational behavior or time spent in the higher part of the observation cage. After intracranial or systemic CNO injection, there was no significant difference in the percentage of retrograde and ipsilateral rotations (Figure 11C) (paired t-test, t = 1.709, df = 2, P = 0.2296, saline vs. CNO: 0.767 ± 0.4296 vs. 0.8384 ± 0.4152), time spent in the higher part of the observation cage (Figure 11D) (paired t-test, t = 1.141, df = 2, P = 0.3720, saline vs. CNO: 55.73 ± 2.64 vs. 60.43 ± 4.542), and time spent immobile (Figure 11E) (paired t-test, t = 0.02596, df = 3, P = 0.9816, saline vs. CNO: 149.9 ± 21.61 vs. 149.8 ± 21.61). No significant differences were found in the total distance (Fig. 6F) (paired t-test, t = 1.505, df = 2, P = 0.2713, saline vs. CNO: 53.12 ± 15.3 vs. 59.48 ± 18.65), the speed of retrograde rotation (Fig. 6G) (paired t-test, t = 0.134, df = 2, P = 0.9057, saline vs. CNO: 39.07 ± 5.889 vs. 39.63 ± 7.092), or the speed of isotropic rotation (Fig. 6H) (paired t-test, t = 1.129, df = 2, P = 0.3762, saline vs. CNO: 41.49 ± 3.96 vs. 42.79 ± 4.705). n = 3 monkeys per group, error bars indicate mean ± SEM, paired t test. [Figure 11C-H]Figure 11 shows representative images of naive macaques after saline or CNO injection from the top (Figure 11A) and side (Figure 11B) perspectives of the observation cage. The animals showed no differences in rotational behavior or time spent in the higher part of the observation cage. After intracranial or systemic CNO injection, there was no significant difference in the percentage of retrograde and ipsilateral rotations (Figure 11C) (paired t-test, t = 1.709, df = 2, P = 0.2296, saline vs. CNO: 0.767 ± 0.4296 vs. 0.8384 ± 0.4152), time spent in the higher part of the observation cage (Figure 11D) (paired t-test, t = 1.141, df = 2, P = 0.3720, saline vs. CNO: 55.73 ± 2.64 vs. 60.43 ± 4.542), and time spent immobile (Figure 11E) (paired t-test, t = 0.02596, df = 3, P = 0.9816, saline vs. CNO: 149.9 ± 21.61 vs. 149.8 ± 21.61). No significant differences were found in the total distance (Fig. 6F) (paired t-test, t = 1.505, df = 2, P = 0.2713, saline vs. CNO: 53.12 ± 15.3 vs. 59.48 ± 18.65), the speed of retrograde rotation (Fig. 6G) (paired t-test, t = 0.134, df = 2, P = 0.9057, saline vs. CNO: 39.07 ± 5.889 vs. 39.63 ± 7.092), or the speed of isotropic rotation (Fig. 6H) (paired t-test, t = 1.129, df = 2, P = 0.3762, saline vs. CNO: 41.49 ± 3.96 vs. 42.79 ± 4.705). n = 3 monkeys per group, error bars indicate mean ± SEM, paired t test.

[0040] [Figure 12A-C]Figures 12A-12F show electrophysiological analysis of macaques after chemogenetic activation of the basal ganglion direct pathway. Figure 12A shows a schematic diagram of electrophysiological recordings in the caudate nucleus (monkey ID: CM045) after AAV8R12-G88P3-HA-hM3Dq injection into the SNr and CNO injection into the dorsomedial caudate nucleus. Figure 12B shows the time course of a typical neuronal response in the caudate nucleus after CNO injection into the dorsomedial caudate nucleus. The x-axis indicates the spike counting period (in minutes), and the y-axis indicates the normalized spike count value. Each inset shows the raw spike trace (upper part of the inset) and waveform (lower part of the inset) at the specific time point indicated by the arrow. Figure 12C shows that a total of 34 cells in the caudate nucleus were recorded from monkey CM045, of which 70.6% (n = 24 cells) were activated, 8.8% (n = 3 cells) were inactivated, and 20.6% (n = 7 cells) remained unchanged. Figure 12D shows a schematic diagram of electrophysiological recordings in the caudate nucleus (monkey ID: CM048) after AAV8R12-G88P7-rM3Ds-2A-EYFP injection into the SNr and intramuscular CNO injection. Figure 12E shows the time course of a typical neuronal response in the caudate nucleus after intramuscular CNO injection. The x-axis indicates the spike counting period (in minutes), and the y-axis indicates the normalized spike count value. Each inset shows the raw spike trace (upper part of the inset) and waveform (lower part of the inset) at the specific time point indicated by the arrow. Figure 12F shows that a total of 38 cells in the caudate nucleus were recorded from monkey CM048, of which 65.8% (n = 25 cells) were activated, 7.9% (n = 3 cells) were inactivated, and 26.3% (n = 10 cells) remained unchanged. [Fig. 12D-F]Figures 12A-12F show electrophysiological analysis of macaques after chemogenetic activation of the basal ganglion direct pathway. Figure 12A shows a schematic diagram of electrophysiological recordings in the caudate nucleus (monkey ID: CM045) after AAV8R12-G88P3-HA-hM3Dq injection into the SNr and CNO injection into the dorsomedial caudate nucleus. Figure 12B shows the time course of a typical neuronal response in the caudate nucleus after CNO injection into the dorsomedial caudate nucleus. The x-axis indicates the spike counting period (in minutes), and the y-axis indicates the normalized spike count value. Each inset shows the raw spike trace (upper part of the inset) and waveform (lower part of the inset) at the specific time point indicated by the arrow. Figure 12C shows that a total of 34 cells in the caudate nucleus were recorded from monkey CM045, of which 70.6% (n = 24 cells) were activated, 8.8% (n = 3 cells) were inactivated, and 20.6% (n = 7 cells) remained unchanged. Figure 12D shows a schematic diagram of electrophysiological recordings in the caudate nucleus (monkey ID: CM048) after AAV8R12-G88P7-rM3Ds-2A-EYFP injection into the SNr and intramuscular CNO injection. Figure 12E shows the time course of a typical neuronal response in the caudate nucleus after intramuscular CNO injection. The x-axis indicates the spike counting period (in minutes), and the y-axis indicates the normalized spike count value. Each inset shows the raw spike trace (upper part of the inset) and waveform (lower part of the inset) at the specific time point indicated by the arrow. Figure 12F shows that a total of 38 cells in the caudate nucleus were recorded from monkey CM048, of which 65.8% (n = 25 cells) were activated, 7.9% (n = 3 cells) were inactivated, and 26.3% (n = 10 cells) remained unchanged. [Figure 12G-J]Figure 12G includes a schematic (left panel) of electrophysiological recordings combined with intramuscular CNO injection in an anesthetized macaque, AAV8R12-G88P7-HA-rM3Ds-2A-Cre injection into the SNr, AAV9-EF1α-DIO-ChR2-EYFP injection into the caudate / putamen, and optical stimulation in the caudate / putamen. Raw spike traces and waveforms (right panel) of a typical neuron in the caudate nucleus upon blue light (473 nm) illumination. Figure 12H shows spike counts in response to repeated optogenetic stimulation of the typical neuron shown in Figure 12G. Figure 12I shows raw spike traces and waveforms at baseline and after intramuscular CNO administration for the typical neuron shown in Figure 12G. Figure 12J plots the time course of the response of retrogradely labeled caudate / putamen neurons (n ​​= 5) identified by optotagging after intramuscular CNO injection. The x-axis indicates the period (in minutes) over which spikes were counted, and the y-axis indicates the normalized population response. [Figure 12K-N]Figure 12K includes a schematic diagram (left panel) of electrophysiological recordings in the caudate nucleus after AAV8R12-G88P3-HA-hM3Dq injection into the SNr and CNO injection into the dorsomedial caudate nucleus in an anesthetized macaque. The right panel shows the time course of the responses of activated caudate nucleus neurons (n ​​= 23) after CNO injection into the dorsomedial caudate nucleus. The x-axis indicates the period (in minutes) over which spikes were counted, and the y-axis indicates the normalized population response. The inset shows the raw spike traces and waveforms of a typical neuron in the caudate nucleus at baseline and 50–60 min after CNO injection. Figure 12L shows that for CNO injection, a total of 34 cells were recorded in the caudate nucleus, of which 67.6% (n = 23 cells) were activated, 2.9% (n = 1 cell) were inactivated, and 29.4% (n = 10 cells) remained unchanged; for saline injection, a total of 32 cells were recorded in the caudate nucleus, of which 18.8% (n = 6 cells) were activated, 40.6% (n = 13 cells) were inactivated, and 40.6% (n = 13 cells) remained unchanged. Figure 12M includes a schematic diagram (left panel) of electrophysiological recordings in the caudate / putamen after AAV8R12-G88P7-rM3Ds-2A-EYFP injection into the SNr and intramuscular CNO injection in an anesthetized macaque. The right panel shows the time course of the responses of activated caudate / putamen neurons (n ​​= 19) after CNO injection. The x-axis indicates the period (in minutes) over which spikes were counted, and the y-axis indicates the normalized population response. The inset shows the raw spike traces and waveforms of a typical neuron in the caudate nucleus at baseline and 50–60 min after CNO injection. Figure 12N shows that for CNO administration, a total of 38 cells were recorded in the caudate nucleus / putamen, of which 50% (n = 19 cells) were activated, 13.2% (n = 5 cells) were inactivated, and 36.8% (n = 14 cells) remained unchanged. For saline administration, a total of 30 cells were recorded in the caudate nucleus / putamen, of which 20% (n = 6 cells) were activated, 53.3% (n = 16 cells) were inactivated, and 26.7% (n = 8 cells) remained unchanged.

[0041] [Figure 13A] Figures 13A-13F show that chemogenetic activation of D1 MSNs reversed PD symptoms in mice. Figure 13A shows a scheme of stereotaxic injection and behavioral analysis in PD mice. Figure 13B shows representative images of tyrosine hydroxylase (TH) staining in control and PD animals. Dopaminergic neurons in the SNc (bottom) and their terminals in the Cpu (top) robustly degenerated. Scale bars: 1000 μm (top), 500 μm (bottom). Figure 13C shows representative tracking images of mice in the open field test. Figures 13D-13E show that motor behavior was significantly reduced after 6-OHDA lesions, as quantified by total distance traveled (Figure 13D) and immobility time (Figure 13E). Chemogenetic activation of D1 MSNs significantly rescued the motor deficits in the PD model (n = 8 mice). Error bars indicate mean ± SEM. Paired t-test, **p < 0.01. Figure 13F shows that motor skills were significantly impaired after 6-OHDA lesioning. Chemogenetic activation of D1 MSNs partially rescued motor deficits in the PD model (n=8 mice). Error bars indicate mean±SEM. Paired t-test, *p<0.05, **p<0.01). [Figure 13B]Figures 13A-13F show that chemogenetic activation of D1 MSNs reversed PD symptoms in mice. Figure 13A shows a scheme of stereotaxic injection and behavioral analysis in PD mice. Figure 13B shows representative images of tyrosine hydroxylase (TH) staining in control and PD animals. Dopaminergic neurons in the SNc (bottom) and their terminals in the Cpu (top) robustly degenerated. Scale bars: 1000 μm (top), 500 μm (bottom). Figure 13C shows representative tracking images of mice in the open field test. Figures 13D-13E show that motor behavior was significantly reduced after 6-OHDA lesions, as quantified by total distance traveled (Figure 13D) and immobility time (Figure 13E). Chemogenetic activation of D1 MSNs significantly rescued the motor deficits in the PD model (n = 8 mice). Error bars indicate mean ± SEM. Paired t-test, **p < 0.01. Figure 13F shows that motor skills were significantly impaired after 6-OHDA lesioning. Chemogenetic activation of D1 MSNs partially rescued motor deficits in the PD model (n=8 mice). Error bars indicate mean±SEM. Paired t-test, *p<0.05, **p<0.01). [Figure 13C]Figures 13A-13F show that chemogenetic activation of D1 MSNs reversed PD symptoms in mice. Figure 13A shows a scheme of stereotaxic injection and behavioral analysis in PD mice. Figure 13B shows representative images of tyrosine hydroxylase (TH) staining in control and PD animals. Dopaminergic neurons in the SNc (bottom) and their terminals in the Cpu (top) robustly degenerated. Scale bars: 1000 μm (top), 500 μm (bottom). Figure 13C shows representative tracking images of mice in the open field test. Figures 13D-13E show that motor behavior was significantly reduced after 6-OHDA lesions, as quantified by total distance traveled (Figure 13D) and immobility time (Figure 13E). Chemogenetic activation of D1 MSNs significantly rescued the motor deficits in the PD model (n = 8 mice). Error bars indicate mean ± SEM. Paired t-test, **p < 0.01. Figure 13F shows that motor skills were significantly impaired after 6-OHDA lesioning. Chemogenetic activation of D1 MSNs partially rescued motor deficits in the PD model (n=8 mice). Error bars indicate mean±SEM. Paired t-test, *p<0.05, **p<0.01). [Figure 13D-F]Figures 13A-13F show that chemogenetic activation of D1 MSNs reversed PD symptoms in mice. Figure 13A shows a scheme of stereotaxic injection and behavioral analysis in PD mice. Figure 13B shows representative images of tyrosine hydroxylase (TH) staining in control and PD animals. Dopaminergic neurons in the SNc (bottom) and their terminals in the Cpu (top) robustly degenerated. Scale bars: 1000 μm (top), 500 μm (bottom). Figure 13C shows representative tracking images of mice in the open field test. Figures 13D-13E show that motor behavior was significantly reduced after 6-OHDA lesions, as quantified by total distance traveled (Figure 13D) and immobility time (Figure 13E). Chemogenetic activation of D1 MSNs significantly rescued the motor deficits in the PD model (n = 8 mice). Error bars indicate mean ± SEM. Paired t-test, **p < 0.01. Figure 13F shows that motor skills were significantly impaired after 6-OHDA lesioning. Chemogenetic activation of D1 MSNs partially rescued motor deficits in the PD model (n=8 mice). Error bars indicate mean±SEM. Paired t-test, *p<0.05, **p<0.01). [Figure 13G-H] Figure 13G contains plots showing quantification of the number of SNc TH+ cells in control and 6-OHDA-treated mice (n=4 mice per group, data expressed as mean±SEM, unpaired two-tailed t-test, ****p<0.0001). Figure 13H contains additional representative traces for mice in the open field test before and after 6-OHDA lesion, and after saline or CNO treatment in lesioned animals. [Figure 13I-K] [Figure 13L-O]Figure 13L shows stereotactic injection of AAV8R12-G88P7-EYFP into the SNr followed by CNO administration in a mouse model of Parkinson's disease via 6-OHDA-mediated dopaminergic cell death. Figure 13M includes plots quantifying total distance traveled in the open field test in mice receiving substantia nigra AAV8R12-G88P7-EYFP injections. n = 8 mice per group; data are presented as mean ± SEM; one-way ANOVA with post-hoc Tukey's test; ***p < 0.001, ****p < 0.0001; ns, not significant. Figure 13N includes plots quantifying immobility time in the open field test in mice receiving substantia nigra AAV8R12-G88P7-EYFP injections. n=8 mice per group, data are presented as mean ± SEM, one-way ANOVA with post-hoc Tukey's test, ***p<0.001, ****p<0.0001, ns, not significant. Figure 13O is a plot quantifying the latency to fall in the rotarod test in mice receiving substantia nigra AAV8R12-G88P7-EYFP injections. n=8 mice per group, data are presented as mean ± SEM, one-way ANOVA with post-hoc Dunnett's test, **p<0.01, ***p<0.001, ns, not significant. [Figure 13P-Q]Figure 13P shows the electrophysiological response to CNO in retrogradely labeled D1-MSNs after injection of AAV8R12-G88P7-rM3Ds-2A-EYFP into the SNr. Whole-cell patch clamp recordings were performed from EYFP+ cells in ex vivo slices. Representative traces (left panel) and quantification (right panel) of action potentials induced by somatic current injection at baseline and after CNO administration. n = 11 cells from 7 mice (L). Data are presented as mean ± SEM. Paired two-tailed t-test. **p < 0.01; ns, not significant. Figure 13P shows the electrophysiological response to CNO in retrogradely labeled D1-MSNs after injection of AAV8R12-G88P7-EYFP into the SNr. Whole-cell patch clamp recordings were performed from EYFP+ cells in ex vivo slices. Representative traces (left panel) and quantification (right panel) of action potentials induced by somatic current injection at baseline and after CNO administration. n=8 cells from 3 mice (M), data are presented as mean±SEM, paired two-tailed t-test, **p<0.01, ns, not significant.

[0042] [Figure 14] Figure 14 shows a scheme of stereotaxic injection and behavioral analysis in PD monkeys.

[0043] [Figure 15A-B] Figure 15A shows representative images of tyrosine hydroxylase (TH) staining in control and MPP+-injected animals. Dopaminergic neurons show robust degradation in the SNc and at their terminals in the Cpu. Scale bars, 5000 μm (Cd and Put), 50 μm (SNc). Figure 15B is a plot showing quantification of the number of SNc TH+ cells in control and MPP+-treated macaques. n=6 sections from one macaque per group; data are presented as mean ± SEM; unpaired two-tailed t-test; ***p<0.001. [Figure 15C-F]Figures 15C-15F show the raw spike traces and waveforms of a typical neuron in the caudate nucleus at baseline and 50-60 min after CNO (Figure 15C) or DCZ (Figure 15E) administration. Time course of the response of activated caudate / putamen neurons after CNO (Figure 15D, cells n = 19) or DCZ (Figure 15F, cells n = 13) administration in anesthetized macaques. The x-axis shows the period (in minutes) over which spikes were counted, and the y-axis shows the normalized population response. [Figure 15G-H] Figure 15G shows that after CNO administration, a total of 36 cells were recorded in the caudate / putamen, of which 52.8% (n = 19 cells) were activated, 13.9% (n = 5 cells) were inactivated, and 33.3% (n = 12 cells) remained unchanged. After DCZ administration, a total of 31 cells were recorded in the caudate / putamen, of which 41.9% (n = 13 cells) were activated, 9.7% (n = 3 cells) were inactivated, and 48.4% (n = 15 cells) remained unchanged. After saline administration, a total of 39 cells were recorded in the caudate / putamen, of which 17.9% (n = 7 cells) were activated, 33.4% (n = 13 cells) were inactivated, and 48.7% (n = 19 cells) remained unchanged. Figure 15H contains plots showing quantification of total PD scores in macaques before and after DCZ treatment. n=4 monkeys per group, data are presented as mean±SEM, one-way ANOVA with post-hoc Dunnett's test, *p<0.05, **p<0.01, ns, not significant. [Figure 15I] Figure 15I plots the total activity of macaques in the observation cage, divided into low, medium, and high activity. Quantification of the percentage of high activity fraction showed changes in macaque activity after MPP + injury and DCZ treatment compared to the pre-injury state. n = 4 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test. *p < 0.05, **p < 0.01.

[0044] [Figure 16A]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16B]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16C]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16D]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16E]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16F]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16G]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16H]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16I-J]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16K]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L). [Figure 16L]Figures 16A-16J show that chemogenetic activation of D1-MSNs reverses Parkinson's disease symptoms in macaques. Representative traces of distance traveled (Figures 16A and 16C) and activity plots of time traveled (Figures 16B and 16D) are shown. Macaques receiving MPP+ showed significant reductions in total activity (Figures 16E), distance traveled (Figures 16G), and immobility time (Figures 16H). DCZ treatment successfully rescued the motor deficits. n = 4 monkeys per group. Error bars indicate mean ± SEM. One-way ANOVA with post-hoc Dunnett's test (Figure 16C), paired two-tailed t-test (Figure 16D). *p<0.05, **p<0.01, ns, not significant. Macaques' total activity was classified as low, moderate, or high mobility. Macaques with MPP+ lesions exhibited impaired motor balance and rarely stayed in the top section of the observation cage. DCZ treatment significantly reversed this phenotype. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16F). Total and individual PD scores of PD macaques before and after DCZ treatment. n = 4 monkeys per group. Error bars indicate mean ± SEM. Paired t-test, *p < 0.05, **p < 0.01. (16I-16J). Representative EMG plots of the biceps muscle in macaques. The typical PD-related 4-6 Hz tremor signal (16K) was eliminated after DCZ treatment. Macaques receiving DCZ treatment significantly reversed their dyskinesia phenotype, as shown by representative quantification of dyskinesia scores at 2 weeks and 1 month after treatment with DCZ or L-dopa. n = 3 monkeys per group, data are expressed as mean ± SEM, unpaired two-tailed t test, *p < 0.05, ****p < 0.0001 (L).

[0045] [Figure 17A-B]Figure 17A shows quantification of the percentage of tremor episodes per 10 minutes. EMG was recorded continuously for 120 minutes after intramuscular delivery of DCZ. n=3 monkeys per group, error bars indicate mean±SEM, one-way ANOVA with post-hoc Dunnett's test, **p<0.01, ns, not significant. Figure 17B shows quantification of the success rate of hand-to-mouth movements. DCZ treatment partially restored this motor skill. n=3 monkeys per group, error bars indicate mean±SEM, one-way ANOVA with post-hoc Dunnett's test, **p<0.01. [Figures 17C-E] Figure 17C is a plot showing quantification of distance traveled after MPP + lesion and DCZ treatment compared to the pre-lesion state. n=4 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test. *p<0.05, ns, not significant. Figure 17D is a plot showing quantification of immobility time after MPP + lesion and DCZ treatment compared to the pre-lesion state. n=4 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test. *p<0.05, ns, not significant. Figure 17D is a plot showing quantification of time in the top compartment of the observation cage after MPP + lesion and DCZ treatment compared to the pre-lesion state. n=4 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test. *p<0.05, ns, not significant.

[0046] [Figure 18A] Figures 18A-18C show total PD scores in Parkinson's macaques before and after L-dopa treatment. n = 3 monkeys per group; error bars indicate mean ± SEM; paired t-test; *p < 0.05, **p < 0.01 (18A). Comparison of the efficacy of DCZ and L-dopa. DCZ achieved efficacy comparable to L-dopa (18B). DCZ reached stable efficacy faster than L-dopa, and its effects persisted over 24 hours (18C). n = 3 monkeys per group; error bars indicate mean ± SEM; paired t-test; *p < 0.05 (18C). [Figure 18B]Figures 18A-18C show total PD scores in Parkinson's macaques before and after L-dopa treatment. n = 3 monkeys per group; error bars indicate mean ± SEM; paired t-test; *p < 0.05, **p < 0.01 (18A). Comparison of the efficacy of DCZ and L-dopa. DCZ achieved efficacy comparable to L-dopa (18B). DCZ reached stable efficacy faster than L-dopa, and its effects persisted over 24 hours (18C). n = 3 monkeys per group; error bars indicate mean ± SEM; paired t-test; *p < 0.05 (18C). [Figure 18C] Figures 18A-18C show total PD scores in Parkinson's macaques before and after L-dopa treatment. n = 3 monkeys per group; error bars indicate mean ± SEM; paired t-test; *p < 0.05, **p < 0.01 (18A). Comparison of the efficacy of DCZ and L-dopa. DCZ achieved efficacy comparable to L-dopa (18B). DCZ reached stable efficacy faster than L-dopa, and its effects persisted over 24 hours (18C). n = 3 monkeys per group; error bars indicate mean ± SEM; paired t-test; *p < 0.05 (18C).

[0047] [Figure 19A] Figure 19A depicts the alignment of AAV2 (SEQ ID NO:20), rAAV2-retro (SEQ ID NO:21), rAAV8-retro (SEQ ID NO:1), and AAV8 (SEQ ID NO:30). The alignment was performed using ClustalOmega Multiple Sequence Alignment (www.ebi.ac.uk / Tools / msa / clustalo / ). The "*" symbol indicates a perfect alignment, the ":" symbol indicates sites that belong to groups showing strong similarity, and the "." symbol indicates sites that belong to groups showing weak similarity. [Figure 19B]Figure 19B depicts the alignment of Cap protein sequences of AAV2, rAAV2-retro, AAV8, AAV8R, and AAV8R12 aligned with Clustal Omega, with the results shown displayed by MViewer 1.63. The figure discloses, in order of appearance, SEQ ID NOS: 20-21 and 67-69, respectively.

[0048] [Figure 20] FIG. 20 shows that Seroquel (quetiapine; QTP) stimulates behavior in mice with SNr expression of DREADD rM3Ds.

[0049] [Figure 21] FIG. 21 shows that Seroquel (quetiapine; QTP) does not stimulate behavior in mice with SNr expression of DREADD hM3Ds.

[0050] [Figure 22] 22 shows the alignment of rM3Ds and hM3Ds. The figure discloses, in column order, SEQ ID NOs: 70 to 73, respectively.

[0051] [Figure 23] FIG. 23 shows that Seroquel increased luciferase levels in hM3Ds-A147S-F349Y to the same level as observed for rM3Ds, but did not increase luciferase levels for wild-type hM3Ds.

[0052] [Figure 24A-B]Figure 24A plots quantification of macaque total PD scores before, 3 days, 1 week, and 2 weeks after L-dopa treatment. N=3 monkeys per group, data are presented as mean±SEM, one-way ANOVA with post-hoc Dunnett's test, *p<0.05, ns, not significant. Figure 24B plots quantification of change in PD scores after 3 days, 1 week, and 2 weeks of initial, continuous administration of DCZ or L-dopa. N=3 monkeys per group, data are presented as mean±SEM, paired two-tailed t-test, *p<0.05, ns, not significant. [Figure 24C-F] Figure 24C plots the quantification of total PD scores following a single dose of DCZ or L-dopa after the drugs had reached steady-state efficacy. N = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed paired t-test. *p<0.05; ns, not significant. Figure 24D plots the corticospinal fluid (CSF) concentrations of DCZ measured by LC-MS 6, 12, and 24 hours after im delivery (0.3 mg / kg). N = 3 monkeys per group. Data are presented as mean ± SEM. Figure 24E plots the quantification of dyskinesia scores after 2 weeks, 1 month, and 4 months of treatment with DCZ or L-dopa. N = 3 monkeys per group. Data are presented as mean ± SEM. Two-tailed unpaired t-test. *p<0.05; ****p<0.0001. Figure 24F shows that for extended L-dopa treatment, animals received L-dopa once daily for 4 months, with a 1-month washout period before DCZ administration. [Figure 24G-L]Figure 24G plots quantification of total PD scores in macaques before, 1, 2, and 4 months after L-dopa treatment. N=3 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test, *p<0.05. Figure 24H plots quantification of total PD scores in macaques before, 1, and 2 months after DCZ treatment following extended L-dopa administration and washout (shown in Figure 24F). N=3 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test, *p<0.05. Figure 24I plots quantification of distance traveled before, 1, and 2 months after DCZ treatment following extended L-dopa administration and washout (shown in Figure 24F). N=3 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test, *p<0.05. Figure 24J plots the quantification of immobility time before, 1, and 2 months after DCZ treatment after extended L-dopa administration and washout (shown in Figure 24F). n=3 monkeys per group; data are presented as mean ± SEM; one-way ANOVA with post-hoc Dunnett's test, *p<0.05. Figure 24K plots the quantification of time in the top compartment of the observation cage before, 1, and 2 months after DCZ treatment after extended L-dopa administration and washout (shown in Figure 24F). n=3 monkeys per group; data are presented as mean ± SEM; one-way ANOVA with post-hoc Dunnett's test, *p<0.05. Figure 24L plots the quantification of dyskinesia scores after 1 and 2 months of DCZ treatment after extended L-dopa administration and washout (shown in Figure 24F). n=3 monkeys per group; data are presented as mean ± SEM.

[0053] [Figure 25]Figure 25 is a schematic diagram showing the locations of mutations introduced at two or three sites within the AAV1 / 5 / 6 capsid protein to create AAV1R, AAV5R, and AAV6R. This schematic is related to the retrograde AAV tracer for D1-MSN and Figure 1C. This figure discloses, in order of appearance, SEQ ID NOS: 74, 63, 55, 65-66, 57, 75, and 66, respectively.

[0054] [Figure 26A-C] Figures 26A-26E depict characterization of labeling specificity after intravenous delivery of AAV-PHP.eB-G88P7-EYFP. Figure 26A includes co-staining of transduced neurons (EYFP) with parvalbumin (PV) after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Arrowheads indicate double+ cells. Scale bar, 50 μm. Figure 26B includes co-staining of transduced neurons (EYFP) with somatostatin (SST) after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Arrowheads indicate double+ cells. Scale bar, 50 μm. Figure 26C shows co-staining of transduced neurons (EYFP) with ChAT after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Figure 26D shows quantification of PV+, SST+, and ChAT+ cells among EYFP+ cells in the striatum after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAV-PHP.eB-hSyn-EYFP. n = 6 mice per group. Data are presented as mean ± SEM. Figure 26E shows co-staining of transduced neurons (EYFP) with Drd1 and Drd2 after intravenous delivery of AAV-PHP.eB-G88P7-EYFP. Arrowheads indicate double+ cells. Scale bar, 20 μm. [Figure 26D-E]Figures 26A-26E depict characterization of labeling specificity after intravenous delivery of AAV-PHP.eB-G88P7-EYFP. Figure 26A includes co-staining of transduced neurons (EYFP) with parvalbumin (PV) after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Arrowheads indicate double+ cells. Scale bar, 50 μm. Figure 26B includes co-staining of transduced neurons (EYFP) with somatostatin (SST) after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Arrowheads indicate double+ cells. Scale bar, 50 μm. Figure 26C shows co-staining of transduced neurons (EYFP) with ChAT after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Figure 26D shows quantification of PV+, SST+, and ChAT+ cells among EYFP+ cells in the striatum after intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAV-PHP.eB-hSyn-EYFP. n = 6 mice per group. Data are presented as mean ± SEM. Figure 26E shows co-staining of transduced neurons (EYFP) with Drd1 and Drd2 after intravenous delivery of AAV-PHP.eB-G88P7-EYFP. Arrowheads indicate double+ cells. Scale bar, 20 μm.

[0055] [Figure 27A-D]Figures 27A-27D depict characterization of striatonigral projection neurons after nigral delivery of AAV8R12-G88P7-EYFP. Figure 27A shows retrograde labeling by stereotaxic injection of AAV8R12-G88P7-EYFP into the SNr and AAV9-G88P7-DIO-tdTomato into the striatum in Drd1-Cre or Drd2-Cre mice. Figure 27B shows retrograde labeling of striatal neurons (EYFP, green, arrowheads) and Cre-driven tdTomato expression (tdT, magenta) in Drd1-Cre (upper panel) or Drd2-Cre (lower panel) mice. Scale bars, 50 μm (low magnification, left), 10 μm (high magnification, right). Figure 27C shows quantification of tdT+ and tdT- cells among all EYFP+ cells from the densely labeled striatal region after substantia nigra injection of AAV8R12-G88P7-EYFP and striatal injection of AAV9-G88P7-DIO-tdTomato in Drd1-Cre mice. n=3 mice per group. Figure 27D shows quantification of tdT+ and tdT- cells among all EYFP+ cells from the densely labeled striatal region after substantia nigra injection of AAV8R12-G88P7-EYFP and striatal injection of AAV9-G88P7-DIO-tdTomato in Drd2-Cre mice. n=3 mice per group.

[0056] [Figure 28A-B] Figures 28A-28B depict the percentage of neurons retrogradely labeled by substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq and AAV8R12-G88P7-rM3Ds-2A-EYFP in the SNr and upstream brain regions. Figure 28A shows quantification of EYFP+ cells after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq (i.e., 1 μg / mL). n=3 mice per group; data are presented as mean ± SEM. Figure 28B shows quantification of EYFP+ cells after substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP (i.e., 3 mice per group; data are presented as mean ± SEM).

[0057] [Figure 29A-F] Figures 29A-29F depict striatal slice electrophysiological recordings after substantia nigra injection of AAV8R12 expressing DREADDs. Figure 29A shows the latency of the first AP after current injection before and after CNO incubation. n=5 cells from 3 mice, data are presented as mean ± SEM, paired two-tailed t-test, ***p<0.001. Figure 29B shows basal activity without current injection recorded in slices prepared from mice that received substantia nigra AAV8R12-G88P3-HA-hM3Dq-2A-EYFP injections before and after CNO incubation. n=6 cells from 4 mice, data are presented as mean ± SEM, paired two-tailed t-test, ns, not significant. Figure 29C shows resting membrane potentials recorded in slices prepared from mice receiving substantia nigra AAV8R12-G88P3-HA-hM3Dq-2A-EYFP injections before and after CNO incubation. n=6 cells from 4 mice; data are presented as mean ± SEM; paired two-tailed t-test; ns, not significant. Figure 29D shows the latency of the first AP after current injection before and after CNO incubation. n=7 cells from 5 mice; data are presented as mean ± SEM; paired two-tailed t-test; ***p<0.001. Figure 29E shows basal activity without current injection recorded in slices prepared from mice receiving substantia nigra AAV8R12-G88P7-rM3Ds-2A-EYFP injections before and after CNO incubation. n=8 cells from 7 mice; data are presented as mean ± SEM; paired two-tailed t-test; ns, not significant. Figure 29F shows resting membrane potentials recorded in slices prepared from mice receiving substantia nigra AAV8R12-G88P7-rM3Ds-2A-EYFP injections before and after CNO incubation. n=8 cells from 7 mice, data are presented as mean ± SEM, paired two-tailed t-test, ns, not significant.

[0058] [Figure 30A-D]Figures 30A-30D depict chemogenetic manipulation of mice injected with AAV8R12-G88P3 / G88P7-EYFP. Figure 30A shows the effect of CNO on rotational behavior in mice after substantia nigra injection of AAV8R12-G88P3-EYFP followed by intraperitoneal (i.p.) delivery. n=5 mice per group. Data are presented as mean ± SEM; unpaired two-tailed t-test; ns, not significant. Figure 30B shows the effect of CNO on rotational behavior in mice after substantia nigra injection of AAV8R12-G88P3-EYFP followed by intracranial (i.c.) injection into the dorsomedial striatum. n=5 mice per group. Data are presented as mean ± SEM; unpaired two-tailed t-test; ns, not significant. Figure 30C shows the effect of CNO on rotational behavior in mice after substantia nigra injection of AAV8R12-G88P7-EYFP followed by i.p. delivery. n=5 mice per group. Data are presented as mean ± SEM; unpaired two-tailed t-test; ns, not significant. Figure 30D shows the effect of CNO on rotational behavior in mice after substantia nigra injection of AAV8R12-G88P7-EYFP followed by injection into the dorsomedial striatum. n=5 mice per group. Data are presented as mean ± SEM; unpaired two-tailed t-test; ns, not significant.

[0059] [Figure 31A-E]Figures 31A-31E depict a study of optotagging in mouse striatonigral projection neurons. Figure 31A shows retrograde labeling by stereotaxic injection of AAV8R12-G88P7-HA-rM3Ds-2A-Cre into the SNr and AAV9-EF1a-DIO-ChR2-EYFP into the striatum in C57BL / 6J mice. Figure 31B shows HA and EYFP staining in striatal sections. Arrowheads indicate cells that were positive for both HA and EYFP. Scale bar, 10 μm. Figure 31C shows whole-cell patch-clamp recordings from EYFP+ cells in ex vivo slices. Raw spike traces of a typical neuron in the striatum upon blue light (473 nm) illumination. Figure 31D shows electrophysiological responses to CNO in optically identified striatonigral projection neurons. Representative traces (left panel) and quantification (right panel) of action potentials induced by somatic current injection at baseline and after CNO administration. n=6 cells from 3 mice, data are presented as mean±SEM, paired two-tailed t-test, *p<0.05. Figure 31E is a plot of induced action potentials comparing baseline and CNO.

[0060] [Figure 32A-F]Figures 32A-32F depict striatal slice electrophysiological recordings after substantia nigra injection of AAV8R12 in Parkinson's disease mice. Figure 32A shows the latency of the first AP after current injection before and after CNO incubation. n=8 cells from 7 mice. Data are presented as mean ± SEM. Paired two-tailed t-test. ****p<0.0001. Figures 32B-32C show basal activity without current injection (Figure 32B) and resting membrane potential (Figure 32C) recorded in slices prepared from Parkinson's disease mice receiving substantia nigra AAV8R12-G88P7-rM3Ds-2A-EYFP injection before and after CNO incubation. n=12 cells from 8 mice. Data are presented as mean ± SEM. Paired two-tailed t-test. ns, not significant. Figure 32D shows the latency of the first AP after current injection before and after CNO incubation. Cells n = 6 from 3 mice. Data are presented as mean ± SEM. Two-tailed paired t-test. ns, not significant. Figures 32E-32F show that basal activity without current injection (Figure 32E) and resting membrane potential (Figure 32F) were recorded in slices prepared from Parkinson's disease mice receiving substantia nigra AAV8R12-G88P7-EYFP injections before and after CNO incubation. Cells n = 7 from 3 mice. Data are presented as mean ± SEM. Two-tailed paired t-test. ns, not significant.

[0061] [Figure 33A-B] Figure 33A shows BG direct pathway manipulation in a macaque model of Parkinson's disease by stereotactic injection of AAV8R12-G88P7-rM3Ds-2A-EYFP into the SNr, followed by MPP+-mediated depletion of SNc dopaminergic neurons and DCZ-mediated activation of rM3Ds. Figure 33B is a schematic diagram of the injection site in the SNr of a macaque. A guiding grid attached above the SN was used to obtain coordinates from MRI images and to guide targeting during injection. Nine discrete sites (green) were selected for injection to cover as much of the SNr as possible.

[0062] [Fig. 34A-D] Figures 34A-34D depict the effects of saline administration on Parkinson's disease symptoms in macaques. Figure 34A shows quantification of macaque total PD scores before, 3 days, 1 week, and 2 weeks after saline administration. n=4 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test; ns, not significant. Figures 34B-34D show the distance traveled (Figure 34B), immobility time (Figure 34C), and time spent in the top compartment of the observation cage (Figure 34D) in macaques after MPP+lesion and saline administration compared to the pre-lesion condition. n=4 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test; *p<0.05; ns, not significant.

[0063] [Fig. 35A-G]Figures 35A-35G illustrate that DCZ alone does not alter motor-related behavior in naive macaques. Figure 35A includes representative top-view motion tracking plots of naive macaques housed in an observation cage after intramuscular (i.m.) saline or DCZ (0.3 mg / kg) injection. Figure 35B includes representative side-view heat maps of macaques after i.m. saline or DCZ (0.3 mg / kg) injection. Figure 35C shows quantification of the ratio of retrograde to i.v. rotations in macaques after saline or DCZ (0.3 mg / kg) injection. n = 4 monkeys per group. Data are presented as mean ± SEM; paired two-tailed t-test; ns, not significant. Figure 35D shows quantification of time spent in the top compartment of the observation cage in macaques after saline or DCZ (0.3 mg / kg) injection. n = 4 monkeys per group. Data are presented as mean ± SEM; paired two-tailed t-test; ns, not significant. Figures 35E-35F show quantification of total distance traveled (Figure 35E) and immobility time (Figure 35F) in macaques after saline or DCZ (0.3 mg / kg) injection. n = 4 monkeys per group. Data are presented as mean ± SEM, paired two-tailed t-test, ns, not significant. Figure 35G shows that total activity of macaques in the observation cage after saline or DCZ (0.3 mg / kg) injection was divided into low, medium, and high mobility. n = 4 monkeys per group. Data are presented as mean ± SEM, paired two-tailed t-test, ns, not significant.

[0064] [Figure 36A-F]Figures 36A-36F depict the effects of D1-MSN activation on dyskinesia-like behavior and health-related blood factors in macaques with Parkinson's disease. Figure 36A shows quantification of dyskinesia scores after 2, 4, and 8 months of treatment with DCZ. n = 3 monkeys per group. Figures 36B-36F show blood test results for alanine aminotransferase (ALT, Figure 36B), aspartate aminotransferase (AST, Figure 36C), gamma-glutamyltransferase (GGT, Figure 36D), creatinine (CREA, Figure 36E), and blood urea nitrogen (BUN, Figure 36F) in macaques that received substantia nigra injections of AAV8R12-G88P7-rM3Ds-2A-EYFP before and after DCZ treatment. n = 3 monkeys per group. Data are presented as mean ± SEM.

[0065] [Figure 37A-E] Figures 37A-37F depict the effects of L-dopa treatment on Parkinson's disease symptoms in macaques. Figure 37A shows individual PD scores of MPP+-treated macaques before and after L-dopa treatment. n=3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p<0.05, **p<0.01. ns, not significant. Figure 37B contains representative top-view movement traces of macaques in the observation cage. Figures 37C-37E show the distance traveled (Figure 37C), time spent in the top compartment of the observation cage (Figure 37D), and immobility time (Figure 37E) in macaques after MPP+-lesion and treatment with L-dopa compared to the pre-lesion condition. n=3 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test. *p<0.05. ns, not significant. Figure 37F shows that the total activity of macaques in the observation cage was divided into low, medium, and high mobility. Quantification of the percentage of high mobility fraction showed changes in macaque activity after MPP + injury and L-dopa treatment compared to the pre-lesion state. n=3 monkeys per group, data are presented as mean ± SEM, one-way ANOVA with post-hoc Dunnett's test, *p<0.05, ns, not significant. [Figure 37F]Figures 37A-37F depict the effects of L-dopa treatment on Parkinson's disease symptoms in macaques. Figure 37A shows individual PD scores of MPP+-treated macaques before and after L-dopa treatment. n=3 monkeys per group. Data are presented as mean ± SEM. Paired two-tailed t-test. *p<0.05, **p<0.01. ns, not significant. Figure 37B contains representative top-view movement traces of macaques in the observation cage. Figures 37C-37E show the distance traveled (Figure 37C), time spent in the top compartment of the observation cage (Figure 37D), and immobility time (Figure 37E) in macaques after MPP+-lesion and treatment with L-dopa compared to the pre-lesion condition. n=3 monkeys per group. Data are presented as mean ± SEM. One-way ANOVA with post-hoc Dunnett's test. *p<0.05. ns, not significant. Figure 37F shows that the total activity of macaques in the observation cage was divided into low, medium, and high mobility. Quantification of the percentage of high mobility fraction showed changes in macaque activity after MPP + injury and L-dopa treatment compared to the pre-lesion state. n=3 monkeys per group, data are presented as mean ± SEM, one-way ANOVA with post-hoc Dunnett's test, *p<0.05, ns, not significant. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description Gene therapy using viral vectors works by using the vector to introduce genetic material (e.g., a transgene or nuclease) into the nucleus of a cell. Viral vectors, which are vectors designed to resemble viruses but do not cause viral infection, are used to deliver gene therapy to cells (e.g., mammalian cells) because they can cross cell membranes and deliver their cargo genetic material into the nucleus of host cells. As a result, the host cell can utilize the newly introduced genetic material to produce the desired therapeutic effect. Described herein are promoters that can be used with viral and non-viral vectors to achieve neuron-specific expression.

[0067] Adeno-associated viruses (AAVs) can be used as delivery vectors for gene therapy because they can establish latent infections, allowing the AAV genome to integrate into host chromosomes without eliciting a destructive T-cell immune response. Different types of AAVs can target various cells, allowing for more nuanced delivery of gene therapy into the body.

[0068] Gene therapy relies on the ability to express a heterologous gene of interest to provide a desired therapeutic effect. The present disclosure provides a promoter that can enhance the translation of a gene of interest for use in disease treatment methods. The present disclosure also provides a recombinant AAV (rAAV) linked to a regulatory element (e.g., a promoter) that increases gene expression of a gene of interest in medium spiny neurons. In one embodiment, the rAAV linked to a regulatory element (e.g., a promoter) targets medium spiny neurons and is used to treat Parkinson's disease.

[0069] The AAV receptor (AAVR) is a receptor essential for AAV entry into cells. Engineered AAVR can be used to create designer receptors (DREADDs) that are exclusively activated by designer drugs to specifically target neuronal tissue. In one embodiment, a DREADD for medium spiny neurons is used to guide AAV gene therapy treatment for the treatment of Parkinson's disease.

[0070] Parkinson's disease (PD) is a common neurodegenerative disorder affecting more than 6 million people worldwide. The pathophysiological signature of PD may include loss of dopaminergic neurons in the midbrain, but its cause may be unclear. PD symptoms can be treated with the dopamine precursor levodopa (L-dopa) or dopamine receptor agonists to restore activity of the basal ganglion (BG) motor control pathway. However, the effects of these drugs may lack specificity due to the widespread distribution of dopamine receptors in the brain and peripheral organs, which may contribute to non-BG drug consumption or disorders of other central and peripheral dopamine systems. Therefore, there is a need for the development of precise therapeutic solutions for PD that enable selective modulation of specific neuronal populations and circuits affected by PD without interfering with other dopaminergic pathways.

[0071] While an effective and precise method for manipulating specific cell types can involve the use of genetically encoded recombinases specifically expressed in the cell type of interest, this approach is often impractical for clinical intervention. An alternative approach can utilize promoters or enhancers of genes expressed by specific cell types to drive cell-type-specific expression, but only a few identified neuronal promoters can maintain endogenous gene expression specificity across rodent and primate models. Retrograde AAV tracers have been developed, which may differ from traditional AAV vectors in their ability to infect neurons via axon terminals. A recombinase-free system for targeting and modulating specialized projection neuron types, which contains any of the following components, may be useful or may be constructed: (1) a retrograde AAV that can effectively infect the axons of selected projection neurons; (2) a promoter or enhancer that drives high-level gene expression in the target projection neurons; and (3) a chemogenetic effector that can control neuronal excitation of specifically labeled projection neurons. This strategy may not require genetically modified animals and may therefore be more useful for clinical application in humans. In rodents with Parkinson's disease, dopamine reduction induces suppression of direct pathway activity and targeted activation of striatal D1 dopamine receptor-expressing medium spiny neurons (D1-MSNs), effectively relieving core motor symptoms. Because D1-MSNs are the only major cell type in the striatum that, in some cases, projects to the substantia nigra pars reticulata (SNr), they may represent an ideal target for implementing circuit-specific modulatory approaches to PD. Therefore, this disclosure includes the development of a recombinase-free, retrograde AAV-based strategy for precisely isolating and modulating D1-MSNs and investigating their effectiveness in reversing PD symptoms.

[0072] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, it will be understood by those skilled in the art that the provided embodiments may be practiced without these details. Unless the context requires otherwise, throughout the following specification and claims, the word "comprise" and its variants, such as "comprises" and "comprising," should be construed in an open, inclusive sense, i.e., "including but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the term "or" is generally used in its ordinary sense, including "and / or," unless the context clearly dictates otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0073] definition As used herein, the term "about" refers to an amount that is close to or less than 10% of the stated amount.

[0074] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of suffering from, or at risk of developing at least one disease that the described compositions and methods are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0075] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and connecting peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, a polypeptide may contain modifications relative to its native or native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as due to mutations of hosts producing the protein or errors resulting from PCR amplification.

[0076] The percent sequence identity (%) with respect to the reference polypeptide sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity.The alignment for determining percent amino acid sequence identity can be achieved by various known methods, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.The appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequence being compared.However, for the purposes herein, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and the source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or it can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0077] In situations where ALIGN-2 is utilized for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 upon its alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0078] The polypeptides described herein can be encoded by nucleic acids. Nucleic acids are a type of polynucleotide containing two or more nucleotide bases. The terms "nucleic acid" and "nucleic acid molecule" can be used interchangeably. These terms refer to nucleic acids in any compositional form, such as deoxyribonucleic acid (DNA, e.g., complementary DNA (cDNA), genomic DNA (gDNA), etc.), ribonucleic acid (RNA, e.g., messenger RNA (mRNA), small inhibitory RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA, RNA highly expressed by the fetus or placenta, etc.), and / or DNA or RNA analogs (e.g., those containing base analogs, sugar analogs, and / or non-native backbones, etc.), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which can be in single-stranded or double-stranded form. Unless otherwise limited, nucleic acids can contain known analogs of natural nucleotides, some of which can function similarly to naturally occurring nucleotides. Nucleic acids may be in any form useful for carrying out the processes herein (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.). Nucleic acids may be or may be derived from a plasmid, phage, autonomously replicating sequence (ARS), centromere, artificial chromosome, chromosome, or other nucleic acid that can or may be replicated in vitro or in a host cell, a cell, and in certain embodiments, the nucleus or cytoplasm of a cell. In some embodiments, the nucleic acid may be from a single chromosome (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism). Nucleic acids also include derivatives, variants, and analogs of RNA or DNA synthesized, replicated, or amplified from single-stranded ("sense" or "antisense," "plus" or "minus" strand, "forward" or "reverse" reading frame) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine.For RNA, the base cytosine is replaced with uracil, and the sugar 2' position contains a hydroxyl moiety. The nucleic acid can be prepared using nucleic acid obtained from a subject as a template. In some embodiments, the nucleic acid is a component of a vector that can be used to transfer a polynucleotide encoding a polypeptide into cells. A heterologous nucleic acid is a nucleic acid that is foreign to the cell or cell population to be modified. A heterologous nucleic acid can include a gene or nucleotide sequence that is modified from an endogenous gene, or can include a recombinant gene or nucleic acid sequence. A heterologous nucleic acid can include a regulatory sequence and encode a fusion or other modification to an endogenous gene that increases the therapeutic or diagnostic potential of the gene or nucleotide sequence.

[0079] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genome-integrating vector, or "integrating vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of a gene encoded by the vector is referred to herein as an "expression vector." An expression vector can appropriately initiate the expression of a gene of interest operably linked to a promoter, which can be "universal," i.e., active in all or many different cell types (e.g., a CMV promoter), or tissue- or cell-specific, i.e., active in a specific subset of cells or tissues. Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements used to control transcription, such as promoters, enhancers, and polyadenylation signals, can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene for facilitating recognition of transformants may also be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc., can be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors can contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or a limited series of sites within the genome. In addition, vectors can contain sequences derived from transposable elements.

[0080] "Heterologous," as used herein with respect to a nucleic acid, gene, polypeptide, or protein, is a nucleic acid, gene, polypeptide, or protein that is not a native component of the adeno-associated virus (AAV) described herein or that is naturally regulated in cis by any of the promoters described herein. Heterologous nucleic acids can encode genes or RNAs (e.g., antisense or siRNA) that are not normally expressed by the AAVs described herein, including synthetic, mammalian, or human genes or RNAs.

[0081] As used herein, "operably linked" refers to the placement of a promoter or regulatory region on a nucleic acid molecule to an open reading frame (e.g., a gene of interest or target gene) that results in transcription of the open reading frame. Generally, a regulatory region is located 5' to such an open reading frame and may include one or more intervening nucleotides that do not significantly inhibit transcription of the open reading frame.

[0082] Designer receptors that are exclusively activated by designer drugs (DREADD) are a class of artificially engineered protein receptors used in chemical genetics, which are selectively activated by certain ligands.They can be used in biomedical research, such as neuroscience, to manipulate neuronal activity.Non-limiting examples of DREADDs can be found in Urban DJ and Roth BL, 2015, DREADDs (Designer Receptors Exclusively Activated by Designer Drugs): Chemogenetic Tools with Therapeutic Utility, Annu. Rev. Pharmacol. Toxicol. 55:15.1-15.19 and Roth, 2016, DREADDs for Neuroscientists, Neuron. 89:683-694.

[0083] As used herein, the terms "homology," "homology," or "percent homology," when used herein to describe an amino acid sequence or a nucleic acid sequence compared to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, as modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula has been incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent sequence homology can be determined using the most recent version of BLAST as of the filing date of this application.

[0084] As used herein, the term "serotype" refers to a distinguishable strain of a microorganism. A serotype can be defined as a group of organisms that have the same type and number of surface antigens. A serotype may or may not be different from a strain that is an isolate of a single culture. A serotype may or may not be different from a genotype that has a different set of genes.

[0085] In some embodiments, nucleic acid or protein sequences are disclosed herein. In the event of any discrepancy between the sequences in the sequence listing and the description in the specification, the description in the specification will generally control.

[0086] Expression vector The promoter described herein can be a component of different types of expression vectors and can be used to initiate the expression of different heterologous genes.In certain embodiments, the expression vector is a viral vector.In certain embodiments, the expression vector is a naked DNA vector, such as a plasmid, a bacterial artificial chromosome, or a yeast artificial chromosome.In certain embodiments, the viral vector is an adenovirus, a lentivirus, or an adeno-associated virus.

[0087] Adeno-associated virus (AAV) AAV is a virus that has no envelope and is composed of a icosahedral capsid protein shell containing a linear single-stranded DNA genome.The genome of AAV vectors retains their packaging signal (also known as inverted terminal repeats or ITRs), but replaces other viral sequences with selected foreign DNA.The target DNA flanked by AAV ITRs can be called a transgene expression cassette.

[0088] The transgene expression cassette is packaged in an AAV capsid for infection and transduction of target cells. After entering the body, the viral capsid interacts with receptors on the surface of the target cell. The viral capsid is then internalized into the target cell by endocytosis. After intracellular transport through the endocytic and / or proteasomal compartments, endosomal escape, nuclear translocation, virion uncoating, and viral DNA double-strand conversion occur, leading to transgene transcription and expression. AAV vectors can be produced as in Kimura et al., "Production of adeno-associated virus vectors for in vitro and in vivo applications," Sci Rep 9, 12601 (2019).

[0089] There are several AAV serotypes, which may include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, Rh10, PHP.B, PHP.eB, and PHP.S. An AAV vector may contain elements from any one serotype, a mixture of serotypes, a hybrid or chimera of different serotypes, or a combination thereof.

[0090] Recombinant AAV (rAAV) is made of single-stranded DNA (ssDNA). These ssDNA viral vectors have high transduction rates and the ability to stimulate endogenous homologous recombination, a DNA repair mechanism, without causing double-stranded DNA breaks within the genome. In various embodiments, the recombinant AAV vector contains sequences derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, Rh10, PHP.B, PHP.eB, or PHP.S serotypes, or mixtures, hybrids, or chimeras of any of the aforementioned AAV serotypes. In one embodiment, the recombinant AAV vector contains sequences derived from AAV2. In one embodiment, the recombinant AAV vector contains sequences derived from AAV8. In one embodiment, the recombinant AAV vector contains sequences derived from AAV9. In one embodiment, the recombinant AAV vector contains sequences derived from AAV2 and AAV8. In one embodiment, the recombinant AAV vector comprises sequences derived from AAV2 and AAV9. In one embodiment, the recombinant AAV vector comprises sequences derived from AAV8 and AAV9. In one embodiment, the recombinant AAV vector comprises sequences derived from AAV2, AAV8 and AAV9.

[0091] In further embodiments, the AAV vector comprises a capsid derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, Rh10, PHP.B, PHP.eB, or PHP.S, as well as variants thereof (e.g., capsid variants, e.g., amino acid insertions, additions, and substitutions). The AAV capsid may comprise a VP1 protein, a VP2 protein, and / or a VP3 protein, and VP2 and VP3 may be amino-terminal truncations of VP1.

[0092] Retrograde infection spreads the virus in the opposite direction of the nerve impulse, from the axon terminal to the parent neuron. Retrograde AAV (AAV-retro) can be used to study specific neuronal populations.

[0093] Regulatory elements In one embodiment, the rAAV virion contains a regulatory element comprising a nucleotide sequence corresponding to the genome sequence. The regulatory element can be a nucleic acid or a small molecule required to turn a gene on or off. Examples of regulatory elements include, but are not limited to, promoters, repressors, activators, silencers, or enhancers. The regulatory element can be a promoter. The promoter can be necessary for binding to enzymes or other factors that initiate transcription of DNA into mRNA. Alternatively, the promoter can be sufficient for binding to enzymes or other factors that initiate transcription of DNA into mRNA.

[0094] Regulatory elements may be located close to a gene along a chromosome. Alternatively, regulatory elements may be located close to a gene due to spatial folding of DNA within the nucleus. Regulatory elements (e.g., promoters or enhancers) may be located near the transcription start site (TSS) of a gene. Regulatory elements may be found 5' to the start site of a gene. Alternatively, regulatory elements may be found 3' to the start site of a gene. Regulatory elements may be found partially or completely within an intron region of a gene. In certain embodiments, the gene is GPR88. The TSS of GPR88 is a guanine, corresponding to positions 1431 of SEQ ID NO: 44, 582 of SEQ ID NO: 45, and 68 of SEQ ID NO: 46.

[0095] The regulatory element can be located less than about 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 nucleotides 3' to the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0096] The regulatory element can be located more than about 100, more than about 200, more than about 300, more than about 400, more than about 500, more than about 600, more than about 700, more than about 800, more than about 900, more than about 1,000, more than about 2,000, more than about 3,000, more than about 4,000, or more than about 5,000 nucleotides 3' to the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0097] The regulatory element can be located less than about 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 nucleotides 5' to the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0098] The regulatory element can be located more than about 100, more than about 200, more than about 300, more than about 400, more than about 500, more than about 600, more than about 700, more than about 800, more than about 900, more than about 1,000, more than about 2,000, more than about 3,000, more than about 4,000, or more than about 5,000 nucleotides 5' to the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0099] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 39. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 39, at least about 82% sequence identity to SEQ ID NO: 39, at least about 84% sequence identity to SEQ ID NO: 39, at least about 86% sequence identity to SEQ ID NO: 39, at least about 88% sequence identity to SEQ ID NO: 39, at least about 90% sequence identity to SEQ ID NO: 39, at least about 91% sequence identity to SEQ ID NO: 39, at least about 92% sequence identity to SEQ ID NO: 39, at least about 93% sequence identity to SEQ ID NO: 39, at least about 94% sequence identity to SEQ ID NO: 39, at least about 95% sequence identity to SEQ ID NO: 39, at least about 96% sequence identity to SEQ ID NO: 39, at least about 97% sequence identity to SEQ ID NO: 39, at least about 98% sequence identity to SEQ ID NO: 39, or at least about 99% sequence identity to SEQ ID NO: 39.

[0100] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 40. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 40, at least about 82% sequence identity to SEQ ID NO: 40, at least about 84% sequence identity to SEQ ID NO: 40, at least about 86% sequence identity to SEQ ID NO: 40, at least about 88% sequence identity to SEQ ID NO: 40, at least about 90% sequence identity to SEQ ID NO: 40, at least about 91% sequence identity to SEQ ID NO: 40, at least about 92% sequence identity to SEQ ID NO: 40, at least about 93% sequence identity to SEQ ID NO: 40, at least about 94% sequence identity to SEQ ID NO: 40, at least about 95% sequence identity to SEQ ID NO: 40, at least about 96% sequence identity to SEQ ID NO: 40, at least about 97% sequence identity to SEQ ID NO: 40, at least about 98% sequence identity to SEQ ID NO: 40, or at least about 99% sequence identity to SEQ ID NO: 40.

[0101] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 41. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 41, at least about 82% sequence identity to SEQ ID NO: 41, at least about 84% sequence identity to SEQ ID NO: 41, at least about 86% sequence identity to SEQ ID NO: 41, at least about 88% sequence identity to SEQ ID NO: 41, at least about 90% sequence identity to SEQ ID NO: 41, at least about 91% sequence identity to SEQ ID NO: 41, at least about 92% sequence identity to SEQ ID NO: 41, at least about 93% sequence identity to SEQ ID NO: 41, at least about 94% sequence identity to SEQ ID NO: 41, at least about 95% sequence identity to SEQ ID NO: 41, at least about 96% sequence identity to SEQ ID NO: 41, at least about 97% sequence identity to SEQ ID NO: 41, at least about 98% sequence identity to SEQ ID NO: 41, or at least about 99% sequence identity to SEQ ID NO: 41.

[0102] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 42. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 42, at least about 82% sequence identity to SEQ ID NO: 42, at least about 84% sequence identity to SEQ ID NO: 42, at least about 86% sequence identity to SEQ ID NO: 42, at least about 88% sequence identity to SEQ ID NO: 42, at least about 90% sequence identity to SEQ ID NO: 42, at least about 91% sequence identity to SEQ ID NO: 42, at least about 92% sequence identity to SEQ ID NO: 42, at least about 93% sequence identity to SEQ ID NO: 42, at least about 94% sequence identity to SEQ ID NO: 42, at least about 95% sequence identity to SEQ ID NO: 42, at least about 96% sequence identity to SEQ ID NO: 42, at least about 97% sequence identity to SEQ ID NO: 42, at least about 98% sequence identity to SEQ ID NO: 42, or at least about 99% sequence identity to SEQ ID NO: 42.

[0103] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 43. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 43, at least about 82% sequence identity to SEQ ID NO: 43, at least about 84% sequence identity to SEQ ID NO: 43, at least about 86% sequence identity to SEQ ID NO: 43, at least about 88% sequence identity to SEQ ID NO: 43, at least about 90% sequence identity to SEQ ID NO: 43, at least about 91% sequence identity to SEQ ID NO: 43, at least about 92% sequence identity to SEQ ID NO: 43, at least about 93% sequence identity to SEQ ID NO: 43, at least about 94% sequence identity to SEQ ID NO: 43, at least about 95% sequence identity to SEQ ID NO: 43, at least about 96% sequence identity to SEQ ID NO: 43, at least about 97% sequence identity to SEQ ID NO: 43, at least about 98% sequence identity to SEQ ID NO: 43, or at least about 99% sequence identity to SEQ ID NO: 43.

[0104] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 44. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 44, at least about 82% sequence identity to SEQ ID NO: 44, at least about 84% sequence identity to SEQ ID NO: 44, at least about 86% sequence identity to SEQ ID NO: 44, at least about 88% sequence identity to SEQ ID NO: 44, at least about 90% sequence identity to SEQ ID NO: 44, at least about 91% sequence identity to SEQ ID NO: 44, at least about 92% sequence identity to SEQ ID NO: 44, at least about 93% sequence identity to SEQ ID NO: 44, at least about 94% sequence identity to SEQ ID NO: 44, at least about 95% sequence identity to SEQ ID NO: 44, at least about 96% sequence identity to SEQ ID NO: 44, at least about 97% sequence identity to SEQ ID NO: 44, at least about 98% sequence identity to SEQ ID NO: 44, or at least about 99% sequence identity to SEQ ID NO: 44.

[0105] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 45. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 45, at least about 82% sequence identity to SEQ ID NO: 45, at least about 84% sequence identity to SEQ ID NO: 45, at least about 86% sequence identity to SEQ ID NO: 45, at least about 88% sequence identity to SEQ ID NO: 45, at least about 90% sequence identity to SEQ ID NO: 45, at least about 91% sequence identity to SEQ ID NO: 45, at least about 92% sequence identity to SEQ ID NO: 45, at least about 93% sequence identity to SEQ ID NO: 45, at least about 94% sequence identity to SEQ ID NO: 45, at least about 95% sequence identity to SEQ ID NO: 45, at least about 96% sequence identity to SEQ ID NO: 45, at least about 97% sequence identity to SEQ ID NO: 45, at least about 98% sequence identity to SEQ ID NO: 45, or at least about 99% sequence identity to SEQ ID NO: 45.

[0106] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 46. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 46, at least about 82% sequence identity to SEQ ID NO: 46, at least about 84% sequence identity to SEQ ID NO: 46, at least about 86% sequence identity to SEQ ID NO: 46, at least about 88% sequence identity to SEQ ID NO: 46, at least about 90% sequence identity to SEQ ID NO: 46, at least about 91% sequence identity to SEQ ID NO: 46, at least about 92% sequence identity to SEQ ID NO: 46, at least about 93% sequence identity to SEQ ID NO: 46, at least about 94% sequence identity to SEQ ID NO: 46, at least about 95% sequence identity to SEQ ID NO: 46, at least about 96% sequence identity to SEQ ID NO: 46, at least about 97% sequence identity to SEQ ID NO: 46, at least about 98% sequence identity to SEQ ID NO: 46, or at least about 99% sequence identity to SEQ ID NO: 46.

[0107] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 48. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 48, at least about 82% sequence identity to SEQ ID NO: 48, at least about 84% sequence identity to SEQ ID NO: 48, at least about 86% sequence identity to SEQ ID NO: 48, at least about 88% sequence identity to SEQ ID NO: 48, at least about 90% sequence identity to SEQ ID NO: 48, at least about 91% sequence identity to SEQ ID NO: 48, at least about 92% sequence identity to SEQ ID NO: 48, at least about 93% sequence identity to SEQ ID NO: 48, at least about 94% sequence identity to SEQ ID NO: 48, at least about 95% sequence identity to SEQ ID NO: 48, at least about 96% sequence identity to SEQ ID NO: 48, at least about 97% sequence identity to SEQ ID NO: 48, at least about 98% sequence identity to SEQ ID NO: 48, or at least about 99% sequence identity to SEQ ID NO: 48.

[0108] The regulatory element may comprise the nucleic acid sequence set forth in SEQ ID NO: 47. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO: 47, at least about 82% sequence identity to SEQ ID NO: 47, at least about 84% sequence identity to SEQ ID NO: 47, at least about 86% sequence identity to SEQ ID NO: 47, at least about 88% sequence identity to SEQ ID NO: 47, at least about 90% sequence identity to SEQ ID NO: 47, at least about 91% sequence identity to SEQ ID NO: 47, at least about 92% sequence identity to SEQ ID NO: 47, at least about 93% sequence identity to SEQ ID NO: 47, at least about 94% sequence identity to SEQ ID NO: 47, at least about 95% sequence identity to SEQ ID NO: 47, at least about 96% sequence identity to SEQ ID NO: 47, at least about 97% sequence identity to SEQ ID NO: 47, at least about 98% sequence identity to SEQ ID NO: 47, or at least about 99% sequence identity to SEQ ID NO: 47.

[0109] The regulatory element can be a promoter. The promoter can be tissue-specific. Alternatively, the promoter can be cell-type specific. Non-limiting examples of cell-type specific promoters include neuron-specific promoters, muscle-specific promoters, blood cell-specific promoters, skin cell-specific promoters, endothelial cell-specific promoters, or epithelial cell-specific promoters.

[0110] The cell type-specific promoter may be neuron-specific. A neuron-specific promoter is a promoter that functions only in neurons to turn on and / or off genes specific to neurons. The neuron-specific promoter may be a synapsin I (SYN) promoter (e.g., hSYN1), a calcium / calmodulin-dependent protein kinase II (CamKII) promoter, a tubulin alpha I, a neuron-specific enolase, a platelet-derived growth factor beta chain promoter, an astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, a cerebellar Purkinje cell-specific L7-6 promoter, a dopamine receptor D1 (DRD1) promoter, a dopamine receptor D2 (DRD2) promoter, a parvalbumin (Pvalb) promoter, or a distal-less homeobox (Dlx) promoter.

[0111] The regulatory elements (e.g., promoters and / or enhancers) can increase expression of a heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 (human synapsin I receptor) gene. The regulatory elements can increase expression of a heterologous gene of interest in neurons of the striatum by at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 90-fold, at least or about 100-fold, at least or about 200-fold, at least or about 300-fold, at least or about 400-fold, at least or about 500-fold, or at least or about 1,000-fold compared to the promoter of the hSYN1 gene.

[0112] The regulatory element can increase expression of the heterologous gene of interest in neurons of the striatum by up to or about 1,000-fold, up to or about 500-fold, up to or about 400-fold, up to or about 300-fold, up to or about 200-fold, up to or about 100-fold, up to or about 90-fold, up to or about 80-fold, up to or about 70-fold, up to or about 60-fold, up to or about 50-fold, up to or about 40-fold, up to or about 30-fold, up to or about 20-fold, up to or about 10-fold, up to or about 9-fold, up to or about 8-fold, up to or about 7-fold, up to or about 6-fold, up to or about 5-fold, up to or about 4-fold, up to or about 3-fold, up to or about 2-fold, or up to or about 1-fold compared to the promoter of the hSYN1 gene.

[0113] The regulatory element can increase expression of the heterologous gene of interest by at least or about 1%, at least or about 2%, at least or about 3%, at least or about 4%, at least or about 5%, at least or about 6%, at least or about 7%, at least or about 8%, at least or about 9%, at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, at least or about 200%, at least or about 300%, at least or about 400%, at least or about 500%, or at least or about 1,000% in neurons of the striatum compared to the promoter of the hSYN1 gene.

[0114] The regulatory element can increase expression of the heterologous gene of interest in neurons of the striatum by up to or about 1,000%, up to or about 500%, up to or about 400%, up to or about 300%, up to or about 200%, up to or about 100%, up to or about 90%, up to or about 80%, up to or about 70%, up to or about 60%, up to or about 50%, up to or about 40%, up to or about 30%, up to or about 20%, up to or about 10%, up to or about 9%, up to or about 8%, up to or about 7%, up to or about 6%, up to or about 5%, up to or about 4%, up to or about 3%, up to or about 2%, or up to or about 1% compared to the promoter of the hSYN1 gene.

[0115] Regulatory element (for example, promoter) can be delivered to cell by methods such as but not limited to CRISPR knock-in method, lentivirus method, adenovirus method, plasmid gene transfer method or cell disruption method.The nucleic acid comprising regulatory element can be contained in virus vector.Virus vector can be adeno-associated virus (AAV) vector.

[0116] AAV capsid polypeptide AAV vectors have a protective protein shell called a capsid. The AAV capsid polypeptide is the main interface between the host and the vector genome. The specificity and efficiency of transduction of AAV particles depend on the AAV capsid polypeptide.

[0117] In one embodiment, the rAAV virion may comprise a variant capsid polypeptide, which comprises an amino acid modification. The amino acid modification may include an amino acid addition, deletion, substitution, or alteration, or a combination thereof. The variant capsid polypeptide may comprise an amino acid modification relative to SEQ ID NO: 1. The modification may enable retrograde transport of AAV along neuronal axons, thus allowing AAV to reach neuronal bodies, where it may be able to transcribe heterologous nucleic acids contained therein. The bold and underlined portions in the sequence IDs are retroinsertions. The bolded portions in the sequence IDs are capsid mutations.

[0118] In another embodiment, the variant capsid polypeptide comprises an alteration comprising an insertion of SEQ ID NO: 31, an aspartic acid substitution at the amino acid residue corresponding to position 385 of SEQ ID NO: 1, an isoleucine and asparagine (IN) substitution at the amino acid residues corresponding to positions 721 and 722 of SEQ ID NO: 1, or a combination thereof.

[0119] In certain embodiments, the variant capsid polypeptide comprises an amino acid modification that increases retrograde transport of the rAAV virion. In some embodiments, the modification to increase retrograde transport of the rAAV may comprise SEQ ID NO: 31. An amino acid alteration that increases retrograde transport of rAAV virions may have at least about 80% sequence identity to SEQ ID NO:31, at least about 82% sequence identity to SEQ ID NO:31, at least about 84% sequence identity to SEQ ID NO:31, at least about 86% sequence identity to SEQ ID NO:31, at least about 88% sequence identity to SEQ ID NO:31, at least about 90% sequence identity to SEQ ID NO:31, at least about 91% sequence identity to SEQ ID NO:31, at least about 92% sequence identity to SEQ ID NO:31, at least about 93% sequence identity to SEQ ID NO:31, at least about 94% sequence identity to SEQ ID NO:31, at least about 95% sequence identity to SEQ ID NO:31, at least about 96% sequence identity to SEQ ID NO:31, at least about 97% sequence identity to SEQ ID NO:31, at least about 98% sequence identity to SEQ ID NO:31, or at least about 99% sequence identity to SEQ ID NO:31. The sequence identity may preserve the infectivity of the retrograde AAV of SEQ ID NO:31.

[0120] Modifications to increase retrograde transport of rAAV may include SEQ ID NO:32. An amino acid alteration that increases retrograde transport of rAAV virions may have at least about 80% sequence identity to SEQ ID NO:32, at least about 82% sequence identity to SEQ ID NO:32, at least about 84% sequence identity to SEQ ID NO:32, at least about 86% sequence identity to SEQ ID NO:32, at least about 88% sequence identity to SEQ ID NO:32, at least about 90% sequence identity to SEQ ID NO:32, at least about 91% sequence identity to SEQ ID NO:32, at least about 92% sequence identity to SEQ ID NO:32, at least about 93% sequence identity to SEQ ID NO:32, at least about 94% sequence identity to SEQ ID NO:32, at least about 95% sequence identity to SEQ ID NO:32, at least about 96% sequence identity to SEQ ID NO:32, at least about 97% sequence identity to SEQ ID NO:32, at least about 98% sequence identity to SEQ ID NO:32, or at least about 99% sequence identity to SEQ ID NO:32. The sequence identity may preserve the infectivity of the retrograde AAV of SEQ ID NO:32.

[0121] In certain embodiments, a sequence may correspond in sequence identity to one or more of sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 31, or 32. Corresponding sequences are sequences that have a high degree of identity but also have certain deletions, insertions, etc., that prevent them from strictly aligning with the sequence being compared.

[0122] Retrograde transport can be increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to that of the rAAV control. In certain embodiments, the increase in retrograde transport is in medium spiny neurons. In certain embodiments, the increase in retrograde transport is in D1 medium spiny neurons.

[0123] In another embodiment, the variant capsid polypeptide comprises an alteration that reduces retrograde transport, which may be reduced by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to the retrograde transport of the rAAV control.

[0124] In another embodiment, the cell may be a neuronal cell. Non-limiting examples of neuronal cells include neurons and glial cells. In one embodiment, the neuronal cell may be a neuron. In one embodiment, the variant capsid polypeptide comprises a modification that increases retrograde transport along the axon of the neuron, retrograde transport along the dendrites of the neuron, retrograde transport through the neuronal cell body, or a combination thereof.

[0125] In some embodiments, the modified variant capsid polypeptide can increase the infectivity of rAAV virions to neurons, non-limiting examples of which include excitatory neurons (e.g., dopaminergic neurons or acetylcholinergic neurons) and inhibitory neurons (e.g., GABAergic neurons or medium spiny neurons).

[0126] In some embodiments, the modified variant capsid polypeptide can increase the infectivity of rAAV virions by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to the baseline activity of an rAAV2-retro control. In certain embodiments, the increase in infectivity is in medium spiny neurons, such as D1 medium spiny neurons.

[0127] heterologous nucleic acid In one embodiment, the rAAV may further comprise a heterologous nucleic acid (e.g., DNA or RNA). In another embodiment, the heterologous nucleic acid may comprise one or more sequences that direct integration into a genomic location of the cell. The cell may be a bacterial cell, an archaeal cell, a plant cell, a fungal cell, or an animal cell. The animal cell may be an amphibian cell, a reptilian cell, a mammalian cell, a bird cell, or a fish cell. The mammalian cell may be any cell from or derived from any mammal (e.g., human, hamster, mouse, monkey, rat, pig, cow, or rabbit).

[0128] The heterologous nucleic acid may include a sequence containing a regulatory element (e.g., a promoter). Alternatively, or in addition, the heterologous nucleic acid sequence may include an open reading frame of a gene of interest. The gene of interest may be a non-coding region (e.g., a UTR or a promoter). Alternatively, the gene of interest may be a polypeptide. The gene of interest may be an endogenous gene. Alternatively, the gene of interest may be an exogenous gene. The gene of interest may have therapeutic utility (e.g., can aid in treating neurological disorders). The gene of interest may include a neurotrophic factor, an RNA-guided nuclease, an enzyme, or a DREADD.

[0129] The gene of interest may be the G protein-coupled receptor 88 (GPR88) gene. GPR88 encodes a receptor found almost exclusively in the striatum, a brain structure that controls motor function and cognition. Deficiency of GPR88 has been associated with chorea, speech delay, learning difficulties, and some neuropsychiatric disorders.

[0130] The polypeptide may be an antibody, a contractile protein, an enzyme, a hormone protein, a structural protein, a storage protein, a small molecule, or a transport protein. Non-limiting examples of enzymes include hydrolases, isomerases, nucleases, ligases, transferases, and oxidoreductases. In one embodiment, the gene of interest is a nuclease or a neurotrophic factor (e.g., brain-derived neurotrophic factor (BDNF)). The nuclease may be an RNA-guided nuclease. The RNA-guided nuclease may be a programmable endonuclease (e.g., Cas or Cas9) that can be used to perform targeted genome editing. The programmable endonuclease can interact with a guide RNA to form a CRISPR / Cas or CRISPR / Cas9 complex. In certain embodiments, the gene of interest may be a DREADD (e.g., hM3Dq, hM1Dq, hMD5q, hM4Di, or hM2Di).

[0131] Methods for expressing genes and for delivery to the CNS Gene therapy is a technique that modifies the target gene to treat or cure disease.Gene therapy can work by several mechanisms, including but not limited to, replacing disease-causing genes with healthy copies of genes, inactivating disease-causing genes that do not function properly, or introducing new or modified genes into the body to help treat disease.Gene therapy can use plasmid DNA, viral vectors, bacterial vectors, gene editing technology, or gene therapy products of patient-derived cells to introduce genetic material into cells.

[0132] Delivery of the gene therapy to a subject can be by intravenous (IV) injection, oral administration, intramuscular injection, subcutaneous injection, intrathecal therapy, rectal administration, intravaginal administration, or inhalation.

[0133] The nervous system is composed of two main parts: the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS is the body's processing center and consists of the brain and spinal cord. To reach the CNS, therapeutics may need to bypass the blood-brain barrier. Strategies for delivering therapeutic agents to the CNS include, but are not limited to, intra-arterial chemotherapy, direct injection of therapeutic agents into intracranial lesions, and the use of nanoparticles for drug delivery.

[0134] In certain embodiments, the rAAV virion of the present disclosure can be injected into a subject. In certain embodiments, the injection can be performed directly into the brain. In some embodiments, the pharmaceutical composition is directly injected into the striatum, a nucleus in the subcortical basal ganglia of the forebrain.

[0135] In some embodiments, the injected gene may target a specific type of neuron or group of neurons in the brain. In some embodiments, the neuron may be a neuron in the striatum. In some embodiments, the neuron may be a medium spiny neuron. In some embodiments, the neuron may be a dopaminergic medium spiny neuron. In some embodiments, the dopaminergic medium spiny neuron is a D1 dopaminergic medium spiny neuron.

[0136] Injections can be performed using stereotactic surgery or intracerebral injection. Stereotactic surgery is a minimally invasive surgical intervention that uses a three-dimensional coordinate system to locate small targets within the body and perform a procedure on them, such as ablation, biopsy, lesion, injection, stimulation, implantation, or radiosurgery (SRS). Intracerebral injection, such as intraventricular injection, is an invasive technique for injecting a substance directly into the cerebrospinal fluid within the ventricles of the brain, bypassing the blood-brain barrier.

[0137] In some embodiments, the gene therapy of the present disclosure is used to genetically engineer neurons. In some embodiments, the genetic engineering method results in the expression of a polypeptide by the neuron. In some embodiments, the peptide expressed by the neuron is translated to form a therapeutic protein. In some embodiments, the therapeutic protein delivered by the AAV of the present disclosure is useful for targeting and treating neurodegenerative diseases such as Parkinson's disease. In certain embodiments, described herein are methods for preparing Parkinson's disease treatment, the methods comprising administering one or more pharmaceutically acceptable excipients, carriers, or diluents and rAAV virions of the present disclosure.

[0138] Parkinson's disease (PD) is a progressive nervous system disorder that affects movement. Symptoms begin gradually and may begin with a barely noticeable tremor in only one hand. Although tremor is common, the disorder also commonly causes stiffness or slowed movement. PD symptoms include muscle rigidity, posture and balance problems, loss of involuntary movements, or changes in speech.

[0139] Tremor, also known as shaking, is an involuntary, rhythmic muscle contraction that results in shaking movements of one or more parts of the body. The disclosed PD treatment methods can reduce tremor in patients by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0140] Bradykinesia is the impairment of voluntary motor control and slow movement or standing still.Bradykinesia can be manifested as the reduction of involuntary movements, such as blinking or swinging arms while walking, or as the difficulty of starting intentional movements or simply slowness of action.PD treatment method of the present disclosure can reduce bradykinesia in patients by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0141] Muscle rigidity, also known as muscle tightness, stiffness, or stiffness, is characterized by the inability of muscles to relax normally. This condition can affect any muscle in the body and cause sharp pain that makes movement difficult. The disclosed PD treatment method can reduce muscle rigidity in patients by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0142] Postural instability, or posture and / or balance disorders, is the inability to maintain balance under dynamic and static conditions, such as preparation for movement, perturbation, and quiet posture. Postural instability can manifest as a tendency to become unsteady when standing. Postural instability can manifest as a tendency to fall or an inability to prevent oneself from falling. The PD treatment method of the present disclosure can reduce postural instability in patients by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0143] Involuntary movements are movements that people often make without conscious thought (e.g., blinking an eye or swinging an arm while walking). The disclosed PD treatment methods can reduce the loss of involuntary movements in patients by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0144] Speech alterations can include dysfluency (abnormal repetition of sounds or rhythms), phonation (unusual vocal tone), or dysarthria (distortion of certain sounds). The disclosed PD treatment methods can reduce speech pattern alterations in patients by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0145] In some embodiments, the present disclosure provides a method for expressing and activating a DREADD in the central nervous system of an individual, the method comprising activating the DREADD in the central nervous system of an individual by administering to the individual a retro-AAV or pharmaceutical composition and a ligand that activates the DREADD. In some embodiments, the DREADD is expressed and activated in neurons of the striatum. In some embodiments, the neurons of the striatum are D1 dopaminergic medium spiny neurons. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. In some embodiments, activating a DREADD in the central nervous system of an individual treats a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder includes Parkinson's disease. In some embodiments, the ligand that activates the DREADD includes quetiapine or clozapine. In some embodiments, the ligand that activates the DREADD includes quetiapine. In some embodiments, the ligand that activates the DREADD includes clozapine. In some embodiments, the retro-AAV and the ligand that activates the DREADD are administered separately.

[0146] Designer receptors exclusively activated by designer drugs (DREADDs) The rAAV virion can contain a designer receptor exclusively activated by designer drugs (DREADD) as the gene of interest. DREADDs, also known as receptors activated only by synthetic ligands, can be a class of artificially engineered protein receptors that can be selectively activated by certain ligands. The DREADD can be rM3Ds.

[0147] The DREADD can comprise the amino acid sequence set forth in SEQ ID NO: 38. The variant capsid polypeptide can have at least about 80% sequence identity to SEQ ID NO: 38, at least about 82% sequence identity to SEQ ID NO: 38, at least about 84% sequence identity to SEQ ID NO: 38, at least about 86% sequence identity to SEQ ID NO: 38, at least about 88% sequence identity to SEQ ID NO: 38, at least about 90% sequence identity to SEQ ID NO: 38, at least about 91% sequence identity to SEQ ID NO: 38, at least about 92% sequence identity to SEQ ID NO: 38, at least about 93% sequence identity to SEQ ID NO: 38, at least about 94% sequence identity to SEQ ID NO: 38, at least about 95% sequence identity to SEQ ID NO: 38, at least about 96% sequence identity to SEQ ID NO: 38, at least about 97% sequence identity to SEQ ID NO: 38, at least about 98% sequence identity to SEQ ID NO: 38, or at least about 99% sequence identity to SEQ ID NO: 38.

[0148] The DREADD may be HM3Ds. The DREADD may comprise an amino acid sequence represented by: The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO:49, at least about 82% sequence identity to SEQ ID NO:49, at least about 84% sequence identity to SEQ ID NO:49, at least about 86% sequence identity to SEQ ID NO:49, at least about 88% sequence identity to SEQ ID NO:49, at least about 90% sequence identity to SEQ ID NO:49, at least about 91% sequence identity to SEQ ID NO:49, at least about 92% sequence identity to SEQ ID NO:49, at least about 93% sequence identity to SEQ ID NO:49, at least about 94% sequence identity to SEQ ID NO:49, at least about 95% sequence identity to SEQ ID NO:49, at least about 96% sequence identity to SEQ ID NO:49, at least about 97% sequence identity to SEQ ID NO:49, at least about 98% sequence identity to SEQ ID NO:49, or at least about 99% sequence identity to SEQ ID NO:49.

[0149] A DREADD for use with the methods and systems described herein can be HM3Ds(A147S-F349Y). The DREADD can comprise the amino acid sequence set forth in SEQ ID NO:50. The variant capsid polypeptide may have at least about 80% sequence identity to SEQ ID NO:50, at least about 82% sequence identity to SEQ ID NO:50, at least about 84% sequence identity to SEQ ID NO:50, at least about 86% sequence identity to SEQ ID NO:50, at least about 88% sequence identity to SEQ ID NO:50, at least about 90% sequence identity to SEQ ID NO:50, at least about 91% sequence identity to SEQ ID NO:50, at least about 92% sequence identity to SEQ ID NO:50, at least about 93% sequence identity to SEQ ID NO:50, at least about 94% sequence identity to SEQ ID NO:50, at least about 95% sequence identity to SEQ ID NO:50, at least about 96% sequence identity to SEQ ID NO:50, at least about 97% sequence identity to SEQ ID NO:50, at least about 98% sequence identity to SEQ ID NO:50, or at least about 99% sequence identity to SEQ ID NO:50.

[0150] DREADD can be used with a certain ligand that activates DREADD and produces desired physiological effects.The ligand can be clozapine or quetiapine.In certain embodiments, the DREADD ligand is clozapine.In certain embodiments, the DREADD ligand is quetiapine.The DREADD ligand can be administered separately from the retro-AAV that encodes DREADD.For FDA or EMA approved drugs, the DREADD ligand can be administered at or below the approved dosage.For FDA or EMA approved drugs, the DREADD ligand can be administered at the same or different schedule from the approved schedule.

[0151] Pharmaceutical Composition In certain embodiments, the rAAV virions of the present disclosure are contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, stabilizers, dispersants, suspending agents, thickeners, and / or diluents. The pharmaceutical composition facilitates administration of the compound to an organism. The pharmaceutical composition can be administered in a therapeutically effective amount as a pharmaceutical composition in a variety of forms and routes, including, for example, intravenous, subcutaneous, intramuscular, inhalation, oral, parenteral, ophthalmic, otic, subcutaneous, transdermal, intranasal, intravitreal, intratracheal, intrapulmonary, transmucosal, intravaginal, and topical administration.

[0152] The formulation can be modified depending on the route of administration selected. Pharmaceutical compositions containing the compounds described herein can be prepared, for example, by mixing, dissolving, emulsifying, encapsulating, entrapping, or compressing processes.

[0153] Pharmaceutical compositions can be formulated by combining active compounds with pharmaceutically acceptable carriers or additives.Non-limiting examples of pharmaceutically acceptable additives suitable for use in the methods disclosed herein include granulating agents, binders, lubricants, disintegrants, sweeteners, glidants, anti-adherents, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifiers, antimicrobial agents, plant cellulose-based materials, and spheronizing agents, and any combination thereof.Pharmaceutically acceptable additives include, but are not limited to, liquids such as water, saline, glycerol, and ethanol.Pharmaceutically acceptable salts, such as inorganic acid salts, such as hydrochloride, bromate, phosphate, sulfate, etc.; and organic acid salts, such as acetate, propionate, malonate, benzoate, etc., can be included in the pharmaceutical composition. In addition, strengthening materials, such as wetting or emulsifying agents, pH buffering substances, etc., may be present in such vehicles.A wide variety of pharmaceutically acceptable additives are known in the art and do not need to be discussed in detail here.Pharmaceutically acceptable additives are, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., Eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., Eds., 3rd ed. Amer.

[0154] Non-limiting examples of pharmaceutically acceptable carriers include saline solution, Ringer's solution, and dextrose solution. Further carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing a compound disclosed herein, which matrices are in the form of shaped articles, e.g., films, liposomes, microparticles and microcapsules.

[0155] The carbomer in the aqueous pharmaceutical composition serves as an emulsifier and viscosity modifier. In certain embodiments, the pharmaceutically acceptable additive comprises or consists of a carbomer. In certain embodiments, the carbomer comprises or consists of carbomer 910, carbomer 934, carbomer 934P, carbomer 940, carbomer 941, carbomer 1342, or a combination thereof. The cyclodextrin in the aqueous pharmaceutical composition serves as a solubilizer and stabilizer. In certain embodiments, the pharmaceutically acceptable additive comprises or consists of a cyclodextrin. In certain embodiments, the cyclodextrin comprises or consists of alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, or a combination thereof. The lecithin in the pharmaceutical composition serves as a solubilizer. In certain embodiments, the solubilizer comprises or consists of lecithin. The poloxamer in the pharmaceutical composition serves as an emulsifier, solubilizer, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a poloxamer. In certain embodiments, the poloxamer comprises or consists of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or a combination thereof. The polyoxyethylene sorbitan fatty acid ester in the pharmaceutical composition serves as an emulsifier, solubilizer, surfactant, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a polyoxyethylene sorbitan fatty acid ester. In certain embodiments, the polyoxyethylene sorbitan fatty acid ester comprises or consists of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, or a combination thereof. Polyoxyethylene stearate in the pharmaceutical composition serves as an emulsifier, solubilizer, surfactant, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of polyoxyethylene stearate.In certain embodiments, the polyoxyethylene stearate comprises or consists of polyoxyl 2 stearate, polyoxyl 4 stearate, polyoxyl 6 stearate, polyoxyl 8 stearate, polyoxyl 12 stearate, polyoxyl 20 stearate, polyoxyl 30 stearate, polyoxyl 40 stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 150 stearate, polyoxyl 4 distearate, polyoxyl 8 distearate, polyoxyl 12 distearate, polyoxyl 32 distearate, polyoxyl 150 distearate, or a combination thereof. The sorbitan ester in the pharmaceutical composition serves as an emulsifier, solubilizer, nonionic surfactant, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of a sorbitan ester. In certain embodiments, the sorbitan ester comprises or consists of sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitan sesquioleate, or a combination thereof. In certain embodiments, solubility can be achieved by a protein carrier. In certain embodiments, the protein carrier comprises albumin, human albumin.

[0156] In certain embodiments, the polypeptide can be stabilized by a polyuronide. In certain embodiments, the stabilizer comprises or consists of a polyuronide. In certain embodiments, the polyuronide comprises or consists of calcium alginate.

[0157] In certain embodiments, the rAAV virions of the present disclosure are administered suspended in a sterile solution. In certain embodiments, the solution contains about 0.9% NaCl. In certain embodiments, the solution contains about 5.0% dextrose. In certain embodiments, the solution further contains one or more of a buffering agent, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween® 80), polysorbate 20 (Tween® 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine; or a chelating agent, such as EDTA or EGTA.

[0158] In certain embodiments, the rAAV virions of the present disclosure are shipped / stored, lyophilized, and reconstituted prior to administration. In certain embodiments, the lyophilized rAAV virion formulation includes a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation may be contained in a vial constructed of glass or other suitable non-reactive material. When formulated, the rAAV virions, whether reconstituted or not, may be buffered to a specific pH, generally below 7.0. In certain embodiments, the pH may be between 4.5 and 6.5, between 4.5 and 6.0, between 4.5 and 5.5, between 4.5 and 5.0, or between 5.0 and 6.0.

[0159] The pharmaceutical composition can be administered locally or systemically, for example, by injection of the compound directly into an organ, optionally in a depot or sustained-release formulation or implant.The pharmaceutical composition can be provided in the form of a rapid-release formulation, a sustained-release formulation, or an intermediate-release formulation.The rapid-release formulation can provide immediate release.The sustained-release formulation can provide controlled release or sustained delayed release.

[0160] When implementing the treatment method or use method provided herein, the therapeutically effective amount of compound described herein is administered to the subject with the disease or condition to be treated in pharmaceutical composition.The therapeutically effective amount can vary widely depending on the severity of disease, the age and relative health of the subject, the efficacy of the compound used and other factors.Compound can be used alone or can be used in combination with one or more therapeutic agents as a component of mixture.

[0161] In some embodiments, the administration of the agent is to an animal, including but not limited to a vertebrate such as a mammal, bird, or fish, which may be a human or a bovine, canine, caprine, cervine, crescinae, feline, galline, equine, lapine, murine, musteline, or ovine. The animal may be a human or other mammal, including primates (e.g., monkeys), bovines (e.g., cattle or dairy cows), swine (e.g., hogs or pigs), ovines (e.g., goats or sheep), equines (e.g., horses), canines (e.g., dogs), felines (e.g., domestic cats), antelopes, buffalo, camels, cervids (deer), donkeys, rabbits, and rodents (e.g., guinea pigs, squirrels, rats, mice, gerbils, and hamsters). In some embodiments, the agent is administered to a human.

[0162] The pharmaceutically acceptable excipient may be present in the pharmaceutical composition in an amount of between about 0.1% and about 99% by weight of the composition. For example, the pharmaceutically acceptable excipient may be present in the pharmaceutical composition in an amount of between about 0.1% and about 95% by weight, between about 0.1% and about 90% by weight, between about 0.1% and about 85% by weight, between about 0.1% and about 80% by weight, between about 0.1% and about 75% by weight, between about 0.1% and about 70% by weight, between about 0.1% and about 65% by weight, between about 0.1% and about 60% by weight, between about 0.1% and about 5 ... 0% by weight, between about 0.1% and about 45% by weight, between about 0.1% and about 40% by weight, between about 0.1% and about 35% by weight, between about 0.1% and about 30% by weight, between about 0.1% and about 25% by weight, between about 0.1% and about 20% by weight, between about 0.1% and about 15% by weight, between about 0.1% and about 10% by weight, between about 0.1% and about 5% by weight, or between about 0.1% and about 1% by weight.

[0163] Pharmaceutically acceptable additives may be present at about 0.1% by mass, about 0.2% by mass, about 0.3% by mass, about 0.4% by mass, about 0.5% by mass, about 0.6% by mass, about 0.7% by mass, about 0.8% by mass, about 0.9% by mass, about 1% by mass, about 2% by mass, about 3% by mass, about 4% by mass, about 5% by mass, about 6% by mass, about 7% by mass, about 8% by mass, about 9% by mass, about 10% by mass, about 11% by mass, about 12% by mass, about 13% by mass, about 14% by mass, about 15% by mass, about 16% by mass, about 17% by mass, about 18% by mass, about 19% by mass, about 20% by mass, about 21% by mass, about 22% by mass, about 23% by mass, about 24% by mass, about 25% by mass, about 26% by mass, about 27% by mass, about 28% by mass, about 29% by mass, about 30% by mass, about 31% by mass, about 32% by mass, about 33% by mass, about 34% by mass, about 35% by mass, about 36% by mass, about 37% by mass, about 38% by mass, about 39% by mass, about 40% by mass, about 41% by mass, about 42% by mass, about 43% by mass, about 44% by mass, about 45% by mass, about 46% by mass, about 47% by mass, about 48% by mass, about 49% by mass, about 50% by mass, about 51% by mass, about 52% by mass, about 53% by mass, about 54% by mass, about 55% by mass, about 56% by mass, about 57% by mass, about 58% by mass, about 59% by mass, about 60% by mass, about 61% by mass, about 62% by mass, about 63% by mass, about 64% by mass, about 65% by mass, about 66% by mass, about 67% by mass, about 68% by mass, about 69% by mass, about 70% by mass, about 71% by mass, about 72% by mass, about 73% by mass, about 74% by mass, about 75% by mass, about 76% by mass, about 77% by mass, about 78% by mass, about 79% by mass, about 80% by mass, about 81% by mass, about 82% by mass, about 83% by mass, about 84% by mass, about 85% by mass, about 86% by mass, about 87% by mass, about 88% by mass, about 89% by mass, about 90% by mass, about 91% by mass, about 92% by mass, about 93% by mass, about 94% by mass, about 95% by mass, about 96% by mass, about 97% by mass, about 98% by mass, about 99% by mass, about 99.1% by mass, about 99.2% by mass, about 99.3% by mass, about 99.4% by mass, about 99.5% by mass, about 99.6% by mass, about 99.7% by mass, about 99.8% by mass, or about 99.9% by mass of the formulation.

[0164] Numbered embodiments The following embodiments are disclosed herein: Embodiment 1. A nucleic acid comprising a heterologous gene of interest operably linked to a regulatory element, wherein the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located 3' to the translation start site of the endogenous GPR88 gene. Embodiment 2. The nucleic acid of embodiment 1, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located, in part, within an intron. Embodiment 3. The nucleic acid of embodiment 1, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located within an intron. Embodiment 4. The nucleic acid of any one of embodiments 1 to 3, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located less than about 1,000 nucleotides 3' to the translation start site of the endogenous GPR88 gene. Embodiment 5. The nucleic acid of embodiment 4, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in SEQ ID NO:39. Embodiment 6. The nucleic acid of embodiment 4, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:39. Embodiment 7. The nucleic acid of any one of embodiments 1 to 3, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located less than about 900 nucleotides 3' to the translation start site of the endogenous GPR88 gene. Embodiment 8. The nucleic acid of embodiment 7, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in SEQ ID NO:40. Embodiment 9. The nucleic acid of embodiment 7, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:40. Embodiment 10. The nucleic acid of any one of embodiments 1 to 9, wherein the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located 5' to the translation start site of the endogenous GPR88 gene. Embodiment 11. The nucleic acid of embodiment 10, wherein the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 100 nucleotides 5' to the translation start site of the endogenous GPR88 gene. Embodiment 12. The nucleic acid of embodiment 11, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:41. Embodiment 13 The nucleic acid of embodiment 11, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:41. Embodiment 14. The nucleic acid of embodiment 10, wherein the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 600 nucleotides 5' to the translation start site of the endogenous GPR88 gene. Embodiment 15. The nucleic acid of embodiment 14, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:42. Embodiment 16 The nucleic acid of embodiment 14, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:42. Embodiment 17. The nucleic acid of embodiment 10, wherein the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 1,500 nucleotides 5' to the translation start site of the endogenous GPR88 gene. Embodiment 18. The nucleic acid of embodiment 17, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:43. Embodiment 19. The nucleic acid of embodiment 17, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:43. Embodiment 20. The nucleic acid of any one of embodiments 1 to 19, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 45, or 46. Embodiment 21. The nucleic acid of any one of embodiments 1 to 19, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 45, or 46. Embodiment 22. A nucleic acid comprising a heterologous gene of interest operably linked to a regulatory element, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:47. Embodiment 23. The nucleic acid of any one of embodiments 1 to 22, wherein the heterologous gene of interest is 3' to the regulatory element. Embodiment 24. The nucleic acid of any one of embodiments 1 to 23, wherein the heterologous gene of interest has therapeutic utility. Embodiment 25. The nucleic acid of any one of embodiments 1 to 23, wherein the gene of interest comprises a neurotrophic factor, an RNA-guided nuclease, an enzyme, or a DREADD. Embodiment 26. The nucleic acid of any one of embodiments 1 to 25, which exhibits increased expression of the heterologous gene of interest relative to the promoter of the hSYN1 gene in neurons of the striatum. Embodiment 27. The nucleic acid of any one of embodiments 1 to 26, which is contained in a viral vector. Embodiment 28. The nucleic acid of embodiment 26, wherein the viral vector is an adeno-associated viral (AAV) vector. Embodiment 29. The nucleic acid of embodiment 28, wherein the viral vector is a retroAAV (AAV retro) virion. Embodiment 30. The nucleic acid of any one of embodiments 1 to 29, wherein the gene of interest exhibits at least 2-fold greater expression of the heterologous gene of interest compared to the promoter of the hSYN1 gene in neurons of the striatum. Embodiment 31. The nucleic acid of any one of embodiments 1 to 29, wherein the gene of interest exhibits at least 2-fold higher expression of the heterologous gene of interest compared to the promoter of the hSYN1 gene in neurons of the striatum. Embodiment 32. The nucleic acid of any one of embodiments 1 to 29, wherein the gene of interest exhibits at least 2-fold higher expression of the heterologous gene of interest compared to the promoter of the hSYN1 gene in neurons of the striatum. Embodiment 33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, or diluent and the nucleic acid of any one of Embodiments 1 to 29. Embodiment 34. A nucleic acid according to any one of embodiments 1 to 29 or a pharmaceutical composition according to embodiment 33 for use in a method for expressing a polypeptide in neurons of the striatum. Embodiment 35 The use of embodiment 34, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons. Embodiment 36. The nucleic acid of any one of embodiments 1 to 29 or the pharmaceutical composition of embodiment 33 for use in a method for genetically manipulating neurons of the striatum. Embodiment 37 The use of embodiment 36, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons. Embodiment 38. A nucleic acid according to any one of embodiments 1 to 29 or a pharmaceutical composition according to embodiment 33 for use in a method for treating a neurodegenerative disease in an individual. Embodiment 39 The use of embodiment 38, wherein the neurodegenerative disease comprises Parkinson's disease. Embodiment 40. A method for expressing a polypeptide in neurons of the striatum of an individual, comprising the step of expressing the polypeptide in the neurons of the striatum by administering to the individual a nucleic acid of any one of embodiments 1 to 29 or a pharmaceutical composition of embodiment 33. Embodiment 41 The method of embodiment 40, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons. Embodiment 42. A method of genetically manipulating neurons of the striatum of an individual, comprising the step of genetically manipulating the neurons of the striatum by administering to the individual a nucleic acid of any one of embodiments 1 to 29 or a pharmaceutical composition of embodiment 33. Embodiment 43 The method of embodiment 42, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons. Embodiment 44. A method of treating an individual suffering from a neurodegenerative disease, comprising administering to said individual suffering from a neurodegenerative disease a nucleic acid of any one of Embodiments 1 to 29 or a pharmaceutical composition of Embodiment 33, thereby treating said neurodegenerative disease. Embodiment 45 The method of embodiment 44, wherein the neurodegenerative disease comprises Parkinson's disease. Embodiment 46 The method of any one of embodiments 40 to 45, wherein the individual is a mammal. Embodiment 47 The method of any one of embodiments 40 to 45, wherein the individual is a human. [Example]

[0165] The following examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0166] Example 1 Development of highly efficient retrograde AAV capsids for D1 MSNs In this experiment, a highly efficient retrograde adeno-associated virus (AAV) capsid, AAV8R, was developed for use in D1-type medium spiny neurons (MSNs).

[0167] The standard retrograde AAV tracer, rAAV2-retro, is only moderately efficient in injecting D1 MSNs when injected into the substantia nigra pars reticulata (SNr), and striatal MSNs are only poorly labeled by rAAV2-retro-hSyn-EYFP. To improve efficiency, multiple rounds of mutation of the AAV capsid were performed on a series of different serotypes. Mutations were introduced at three sites in the AAV8 capsid protein to generate AAV8R, which contains N385D, an insertion of RGNLADQDYTKTARQAATAD (SEQ ID NO: 31) at position 588, and TS711-712IN. Two additional mutations, V183E and N411S, were incorporated into the AAV8R12 capsid protein (Figure 1A). The labeling pattern of the AAV8R12 capsid in combination with the G88 promoter was determined. Retrogradely labeled neurons in the SNr and upstream brain regions after substantia nigra injection of AAV8R12-G88P7-EYFP in mice showed 97.68 ± 0.43% labeled neurons in the striatum, but only 1.14 ± 0.16% and 1.18 ± 0.41% labeled neurons in the SNr and other upstream brain regions, respectively (Figures 1D and 1E; n = 3 mice per group).

[0168] First, the polypeptide fragment [ka] Two other point mutations in the AAV2 Cap protein were inserted at positions N587-R588 to develop rAAV2-retro. Similar mutations were then placed at the equivalent positions in four other serotypes (Figures 1A-1C and 25, Table 1), AAV1 / 5 / 6 / 8 (Figure 1A). All four modified AAVs maintained brain infectivity, but only the AAV8 variant, AAV8R, acquired improved retrograde infectivity of D1 MSNs. When administered into the SNr, mice infected with AAV8R-hSyn-EYFP showed 4.86 ± 0.22-fold higher EYFP-positive MSNs than mice infected with rAAV2-retro-hSyn-EYFP (Figures 2A-2C).

[0169] The baseline infectivity of rAAV2-retro was approximately 0.2 ± 0.03 × 10 per striatal hemisphere. 4 These cells were transfected using the hSyn promoter (Table 2). Among the 14 mutants tested, AAV8R12 showed the most improved efficiency. Compared to rAAV2-retro, the current standard retrograde AAV, AAV8R12 labeled 7.72 ± 0.78-fold more MSNs after delivery to the substantia nigra (Figures 2B-2C, Table 1). Furthermore, robust labeling of MSNs was observed in the nucleus accumbens by AAV8R and AAV8R12 after stereotaxic delivery to the ventral pallidum or lateral hypothalamus. This demonstrates the unique ability of our newly developed AAV capsid to infect the axons of basal ganglion MSNs. AAV8R12 was generated using the AAV8R plasmid as a template.

[0170] The sequence of the mutagenesis primer used to introduce the V183E mutation was 5'-TGGCGACTCAGAGTCAGAGCCAGACCCTCAACCTCT-3' (SEQ ID NO: 34). The sequence of the mutagenesis primer used to introduce the N411S mutation was 5'-TGCTGAGAACCGGCAACAGCTTCCAGTTTACTTACACCT-3' (SEQ ID NO: 35). [Table 1-1] [Table 1-2] [Table 2]

[0171] Example 2 Development of robust promoters for D1 MSNs To find promoters that confer high expression levels in MSNs, we scanned gene expression databases to identify a set of eight genes highly enriched for striatal expression compared with other parts of the basal ganglion (BG). Genes that are highly expressed in the striatum but not in other parts of the BG were initially selected as candidates.

[0172] We examined brain profiles of two epigenetic marks for enhancers and promoters, histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac), and identified approximately 2-kilobase sequences surrounding transcription start sites (TSSs) with high levels of H3K4me1 and / or H3K27ac in mouse brain. Homologous sequences at the equivalent positions in the human genome were then cloned into AAV backbones, and the activity of the homologous sequences in directing reporter expression was tested in mice after injection of AAV into the SNr (Figure 3A-C, Table 3).

[0173] Among the 11 promoters tested, the 2259-bp promoter, neural promoter 1 (G88P2) from the gene GPR88, showed the highest activity in driving gene expression in MSNs compared to the commonly used promoters CAG, EF1a, and hSyn (Figures 4A-4B). G88P2 (2259 bp) was cloned using the following primers: 5'-CATCGCAAGGCTACATGATGG (SEQ ID NO: 36) and 3'-CTGGCCAACTCTTCACACCTC (SEQ ID NO: 37). To increase the payload of the AAV genome, the G88P2 promoter was shortened using restriction enzymes to generate two derivatives, G88P3 and G88P7, which were 1395 bp and 896 bp long, respectively (Figures 3A-3C). Comparative efficiencies in MSN labeling were observed for the two shortened promoters. In mice injected with virus into the SNr, viruses expressing EYFP driven by the G88P3 and G88P7 promoters labeled 3.7 ± 0.66 × 10 and 3.95 ± 0.73 × 10 MSNs per mouse, respectively, compared with 1.61 ± 0.16 × 10 MSNs labeled by the hSyn promoter (Figure 4B). The shortest of these strong MSN promoters, G88P7, consisted of a short 67-bp sequence before the TSS, exon 1 (366 bp), intron 1 (391 bp), and a 72-bp fragment of exon 2 of the human GPR88 gene. This suggests that cis-regulatory elements in exon 1 and intron 1 of the GPR88 gene are likely sufficient to initiate strong striatal expression. [Table 3]

[0174] Consistent with the exclusively high levels of Gpr88 gene expression in MSNs but not other striatal cell types in the mouse striatum, co-staining of EYFP with Drd1 and Drd2, but not with ChAT, parvalbumin, or somatostatin, was observed after intravenous delivery of AAV-PHP.eB-G88P7-EYFP in Figures 26A-26E.

[0175] The labeling specificity of the new retrograde AAV tracer was tested in mice. Double labeling of EYFP with Drd1 or Drd2 after substantia nigra injection of AAV8R12-G88P7-EYFP in mice demonstrated that retrogradely labeled neurons were Drd1+, with <3% showing positive Drd2 immunoreactivity (Figures 5A-5B). In some cases, a very small population of SNr-projecting MSNs expressed both Drd1 and Drd2 receptors. Consistently, further testing of Drd1-Cre and Drd2-Cre mice with simultaneous substantia nigra injection of AAV8R12-G88P7-EYFP and striatal injection of AAV9-G88P7-DIO-tdTomato confirmed that a very small percentage (<3%) of SNr-targeted MSNs labeled by AAV8R12 were tdTomato+ in Drd2-Cre mice (Figures 27A-27D). Using this AAV-based retrograde labeling approach, 12.44 ± 2.3% of D1-MSNs were found to be reporter-positive in mice.

[0176] Example 3 Chemogenetic manipulation of the basal ganglion direct pathway in mice Mice were anesthetized with pentobarbital sodium (Nembutal; 80 mg / kg, i.p.) and then placed in a stereotaxic device (KOPF). Eye cream was applied to both corneas to prevent dehydration. The skull above the target area was thinned with a dental drill and carefully removed. Injections were performed using a microsyringe pump (Legato 130, KD Scientific) with a 10 μL syringe (Neuros; Hamilton) connected to a 33-gauge needle. A total volume of 200 nL of virus was injected into the SNr at a rate of 20 nL / min. The coordinates of the SNr were 3.4 mm posterior, 1.3 mm lateral, and 4.8 mm ventral to the bregma.

[0177] Mice used for behavioral experiments were then unilaterally implanted with a guide cannula (KOPF) aimed at the dorsomedial striatum (0.5 mm anterior, 1.5 mm lateral, and 3.5 mm ventral to bregma). The cannula was fixed to the skull with dental cement. A stainless steel obturator was inserted into the guide cannula and replaced every other day to maintain patency until injection. Mice were allowed to recover from surgery for at least 3 weeks before further study.

[0178] The labeling specificity of retrograde AAV tracers was examined. Double labeling of AAV8R12-G88P3-EYFP and Drd1 or Drd2 in mice demonstrated that retrograde AAV exclusively targeted D1 MSNs (Figure 5A). To explore the functional characteristics of retrograde AAV-labeled cells, AAV8R12-G88P3-HA-hM3Dq, which enables neuronal excitation upon clozapine N-oxide (CNO) administration, was injected into the right SNr of C57BL / 6J mice. Three weeks after injection, CNO was delivered intraperitoneally, and an increase in ipsidirectional rotation and a decrease in antidiagonal rotation were observed compared to saline controls (Figure 6A). This result indicated suppression of the BG behavioral control pathway, contrary to the expected effect of activating D1 MSNs in the right striatum. Based on the observed infection pattern of retrograde AAV, we reasoned that isotropic rotation was a consequence of activation of the right SNr, the major inhibitory output center of the BG and marked by locally injected AAV.

[0179] Immunohistochemical analysis confirmed a significant increase in c-Fos+ cells in the right SNr after CNO administration, but not after saline administration (Figure 6B). CNO or saline was then intracranially administered near the dorsomedial part of the right striatum after AAV delivery to the right SNr. Retrograde rotation was induced by CNO, but not by saline (Figure 6A). No significant change in the number of nigral c-Fos+ cells was observed after intracranial CNO injection (Figure 6B). Furthermore, CNO did not induce rotational behavior in animals receiving AAV8R12-G88P3-EYFP injection into the SNr (Figure 6C). These results demonstrated that pharmacogenetic activation of retrogradely labeled D1 MSNs using the proposed strategy is sufficient to drive behavioral changes in mice.

[0180] To explore the functional characteristics of retrogradely AAV-labeled cells, AAV8R12-G88P3-HA-hM3Dq, which expresses the DREADD effector hM3Dq and can enable neuronal excitation upon clozapine N-oxide (CNO) administration, was injected unilaterally into the SNr of C57BL / 6J mice. Three weeks after injection, brains were harvested, and anatomical analysis revealed that the majority of labeled neurons were located in the striatum and that all labeled neurons were Drd1+ (Figures 6D-6F, 28A). Using whole-cell voltage-clamp recordings in slices, an augmentation of excitability in AAV8R12-G88P3-HA-hM3Dq-2A-EYFP-transduced MSNs was observed upon CNO administration, without affecting basal firing rate or resting membrane potential (Figures 6G, 29A-29C). Surprisingly, a reduction in retrograde rotation was observed after intraperitoneal CNO delivery compared with saline injection (Figure 6H). This result indicated inhibition of the direct pathway or excitation of the indirect pathway, contrary to the predicted outcome of unilateral activation of D1-MSNs in the striatum. Based on the observed infection pattern of retrograde AAV, we reasoned that the retrograde rotation may be a consequence of activation of the ipsilateral SNr, which may be the major inhibitory output center of the BG and was lightly transduced by the locally injected AAV (Figures 1D-1E, 5A-5B, 6D, 28A). Immunohistochemical analysis confirmed a significant increase in c-Fos+ cells in the injected SNr after CNO administration, but not after saline administration (Figure 6H). To further explore this observation, CNO or saline was intracranially administered near the dorsomedial region of the ipsilateral striatum after unilateral AAV delivery. Using this approach, retrograde rotation was induced by CNO but not by saline, and no significant changes in the number of nigral c-Fos+ cells were observed (Figure 6I). Furthermore, CNO did not induce behavioral alterations in animals receiving AAV8R12-G88P3-EYFP injections into the SNr (Figures 30A-30B). These results demonstrated that chemogenetic activation of D1 MSNs labeled with our newly developed retrograde AAV can drive behavioral changes in mice.

[0181] To further optimize the system, it was necessary to identify alternative chemogenetic effectors that could potentially allow specific manipulation of the direct pathway compatible with systemic CNO infusion. Commonly used Gq-coupled effectors have been shown to be more effective than other second messengers in driving neuronal excitation in various neuronal subtypes, including Ca. 2+ Using a different chemogenetic effector, rM3Ds, which can effectively activate striatal MSNs using cAMP as a second messenger, we avoided the activation of SNr neurons during systemic CNO administration.

[0182] To test this, we unilaterally injected AAV8R12-G88P7-rM3Ds-2A-EYFP into the right SNr of adult mice. Both intraperitoneal and intracranial injections of CNO induced retrograde rotation (Figure 7A). Immunohistochemistry and in situ hybridization analyses confirmed that the majority of labeled neurons were located in the striatum and that retrogradely transduced neurons were Drd1+ (Figures 7D-7F, 28B). Slice electrophysiological recordings demonstrated an enhancement of excitability of labeled striatal MSNs upon CNO administration, without affecting basal firing rate or resting membrane potential (Figures 7G, ​​29D-29F). CNO did not increase the number of c-Fos+ cells in the SNr after intraperitoneal CNO administration (Figure 7B). Both intraperitoneal and intracranial injections of CNO induced retrograde rotation without increasing the number of c-Fos+ cells in the SNr (Figures 7H-7I). Furthermore, CNO did not induce rotational behavior in animals receiving AAV8R12-G88P7-EYFP injections into the SNr (Figures 7C, 30C-30D). These results demonstrated the specificity of rM3Ds in activating retrogradely labeled MSNs rather than nigral neurons at the injection site. To assess the durability of the approach, mice receiving substantia nigra AAV8R12-G88P7-rM3Ds-2A-EYFP injections were tested 12 months after the initial viral infection, and consistently demonstrated enhanced retrograde rotation (Figures 7J-7I, 9D-9E). Collectively, these findings confirm that the approach we developed is a durable solution for selectively modulating the activity of D1-MSNs and the BG direct pathway.

[0183] The results of this example confirm that the constructed toolkit, which includes the highly efficient designer retrograde AAV tracer AAV8R12, the strong striatal promoter G88P3 / 3, and the chemogenetic effectors rM3Ds or hM3Dq, comprises a recombinase-free system for selectively isolating neuronal subtypes for functional characterization.

[0184] Example 4 Chemogenetic manipulation of the basal ganglion direct pathway in macaque monkeys Chemogenetic manipulation of neuronal activity can be effective in the macaque brain, even if it does not target specific neural networks. To test the effectiveness of this approach to circuit modulation of the BG direct pathway in a primate model, we injected AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP into the SNr of macaques unilaterally. Anatomical analysis showed that the majority of labeled neurons were found in the caudate nucleus and putamen, and that all striatal DREADD+ neurons were DRD1+ (Figures 10I-10P). Electrophysiological recordings in anesthetized animals confirmed increased neuronal activity in the caudate nucleus / putamen after CNO injection, but not after saline injection (Figures 12K-12N). To directly assess how rM3Ds expression in D1-MSNs affects their activity, simultaneous substantia nigra AAV8R12-G88P7-HA-rM3Ds-2A-Cre and striatal AAV9-EF1α-DIO-ChR2-EYFP injections were performed in mice and macaques. Immunohistochemical analysis and slice recordings in mice confirmed coexpression of ChR2 and rM3Ds in striatonigral projection neurons and that labeled neurons were activated by both light (473 nm) and CNO (Figures 31A-31E). In vivo optotagging recordings in anesthetized macaques revealed that CNO effectively induced increased neuronal activity in retrogradely labeled D1-MSNs (Figures 12G-12J).

[0185] In behavioral studies, intracranial or systemic injection of CNO into the dorsal medial caudate nucleus in monkeys receiving either hM3Dq or rM3Ds effectors, respectively, induced a dramatic increase in retrograde rotation (Figure 10B, 10Q-10S). After CNO treatment, a significant decrease in the time spent in the top compartment of the observation cage and an increase in the speed of retrograde rotation were observed (Figures 8A-8B, 10T-10W). No significant differences were observed in the speed of retrograde rotation, total distance traveled, or immobility time (Figures 8C-8H). CNO did not induce significant behavioral changes in naive monkeys that did not receive viral injections (Figures 11A-11H). Collectively, these results clearly demonstrate that the toolkit we developed can accurately isolate and efficiently activate direct pathway projection neurons in primates.

[0186] To test the efficacy of this newly developed retrograde tool system in a primate model, we injected AAV8R12-G88P3-mCherry into the SNr of cynomolgus macaques (Macaca fascicularis). Because the brain of a cynomolgus macaque is approximately 180 times larger than that of a mouse, we delivered the injections to a grid of nine locations, covering most of the target structures.

[0187] To guide the virus injection into the SNr, a guide grid with multiple holes spaced 1 mm apart was attached vertically above each subject's SN. By filling these holes with vitamin E, precise injection coordinates could be obtained from T1-weighted MRI images (3T Tim Trio scanner, Siemens). Viral injections were performed at nine sites covering the entire SNr, with a total volume of 27 μL of virus injected unilaterally into the right SNr at a rate of 300 nL / min.

[0188] For intracranial drug administration and / or electrophysiological recording, a recording chamber covering the anterior caudate nucleus to the posterior GPi was fixed to the skull with six titanium screws and dental cement. Each subject was allowed to recover from surgery for at least 6 weeks before further study.

[0189] Macaques receiving substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP after CNO injection spent less time in the higher part of the observation cage than animals receiving saline injections (Figures 8A-8B). In situ hybridization analysis showed that labeled neurons were exclusively D1 MSNs, confirming the labeling specificity of retrograde AAV in macaques (Figure 9C). Overall, approximately 20.55% of D1 MSNs were labeled (Figures 9A-9G). Activation of the BG direct pathway (Figure 9D) and the percentage of rotational behavior (i.e., i.e., i.v. and i.p.) in mice 12 months after substantia nigra delivery of AAV8R12-G88P7-rM3Ds-2A-EYFP and CNO delivery via i.p. injection were quantified (Figure 9E), with n = 6 mice per group. The efficacy of the D1-MSN retrograde labeling system in a macaque model was assessed by unilateral injection of AAV8R12-G88P3-mCherry into the SNr of cynomolgus macaques (Macaca fascicularis). Robust labeling of projection neurons was detected in the caudate nucleus and putamen, with little labeling elsewhere (Figure 9K).

[0190] Next, AAV8R12-G88P3-HA-hM3Dq was injected unilaterally into the SNr. Intracranial injection of CNO into the dorsal medial caudate nucleus elicited a dramatic increase in retrograde rotation (Figures 10A and 10C). Interestingly, the increase in retrograde rotation was also observed in macaques that received a unilateral AAV8R12-G88P7-rM3Ds-2A-EYFP injection into the SNr 2.5 years after the substantia nigra injection, but not in control animals after systemic CNO injection (Figures 10A-10C, Figures 11A-11H). This demonstrates the ability of retrograde viral tracers to deliver long-term expression of chemogenetic effectors in primates, providing precise isolation and efficient activation of direct pathway neurons. After CNO treatment, we also observed a significant increase in the rate of retrograde rotation and a decrease in the time the animals spent in the top compartment of the observation cage (Figures 10D and 10G, and Figures 11A-11H). Furthermore, an increase in muscle tone in the contralateral biceps brachii was observed by electromyography after CNO injection. No significant differences were found in immobility time, total distance traveled, or rate of idiographic rotation (Figures 10E, 10F, and 10H). Electrophysiological recordings confirmed increased neuronal activity in the caudate nucleus after CNO injection (Figures 12A-12F). These results clearly demonstrate that the toolkit accurately isolates and activates selective projection neuron subtypes in a primate model.

[0191] Example 5 Chemogenetic activation of the direct pathway reversed Parkinson's disease symptoms in a rodent PD model A rodent Parkinson's disease mouse model was created by bilateral injection of 6-OHDA using the same method as described in Example 3 for viral injection. A total volume of 1 μL of 6-OHDA (5 mg / ml, dissolved in sterile saline containing 0.02% ascorbic acid, Sigma) was injected into the striatum at a rate of 100 nL / min. The coordinates of the striatum were 0.5 mm anterior, 1.5 mm lateral, and 3.2 mm ventral to bregma. Animals were premedicated with desipramine (25 mg / kg, Sigma) before 6-OHDA injection to enhance the selectivity and efficacy of 6-OHDA-induced lesions. Mice were supplemented with DietGel (ClearH2O) for one week after surgery. All staining and behavioral experiments were performed at least 14 days after surgery, when dopamine depletion had reached its maximum and stabilized.

[0192] To investigate the efficacy and safety of D1-MSN-specific neuromodulation strategies, AAV8R12-G88P7-rM3Ds-2A-EYFP was injected into the SNr of adult C57 / BL6 mice. 6-OHDA was administered bilaterally to the striatum (Figure 13A). TH immunohistochemistry revealed a significant reduction in dopamine innervation in the striatum and a significant loss of dopamine neurons in the SNc (Figures 13B, 13G). Analysis of locomotor activity in an open field demonstrated that systemic CNO delivery, which selectively activates D1 MSNs, dramatically reversed the dyskinesia-like phenotype of PD mice (Figures 13C-13D, 13H-13I). Furthermore, CNO partially rescued the motor skill impairment of PD mice in the rotarod test (Figures 13E, 13J). CNO partially rescued the motor skill impairment of 6-OHDA-treated mice in the rotarod test (Figures 13F, 13K). Substantia nigra injection of AAV8R12-G88P7-EYFP failed to alleviate the Parkinsonian phenotype (Figures 13C-13O). Whole-cell patch-clamp recordings in slices revealed increased excitability in D1-MSNs transduced with AAV8R12-G88P7-rM3Ds-2A-EYFP, but not in D1-MSNs not transduced with AAV8R12-G88P7-EYFP (Figures 13P-13Q, 32A, 32D). Transduction with these drugs did not affect the basal firing rate or resting membrane potential in either group (Figures 32B-32C, 32E-32F).

[0193] These results demonstrated that targeted activation of the basal ganglion (BG) direct pathway in an AAV-mediated retrograde scheme can effectively antagonize parkinsonian-like symptoms in a rodent PD model.

[0194] Example 6 Chemogenetic activation of the direct pathway reversed Parkinson's disease symptoms in a monkey PD model A parkinsonian state was established in macaque monkeys by unilateral injection of 1-methyl-4-phenylpyridinium (MPP+) into the SNc (Figure 14). MPP+ was injected unilaterally using the same method as previously described for virus injection. Drug injections were performed at five sites covering the entire SNc. A total volume of 10 μl of MPP+ was injected into the SNc at a rate of 50 nL / min. After the lesion, the monkeys were continuously monitored by a veterinarian. Parkinsonian symptoms, such as bradykinesia and balance impairment, could be observed immediately after the lesion surgery. Stable parkinsonian symptoms were observed for more than 12 weeks before the animals were used in experiments.

[0195] TH immunohistochemistry confirmed the loss of nigral dopamine neurons and their fibers in the caudate and putamen (Figures 15A-15B). Stereotactic injection of AAV8R12-G88P7-rM3Ds-2A-EYFP was performed at six sites in the SNr of adult cynomolgus macaques (Figure 14). Monkeys receiving MPP+ injections exhibited characteristic PD-like symptoms, including bradykinesia, tremor, rigidity, and postural abnormalities (Figures 16A-16K). However, manipulation approaches targeting D1-MSNs did not elicit dyskinesia-like behavior (Figure 16L). In some cases, retrograde stereotactic injection of AAV8R12 was performed at nine sites in the SNr of adult cynomolgus macaques (Figures 33A-33B). MPP+ was then injected unilaterally into the SNc, resulting in a loss of nigral dopamine neurons in the SNc and their fibers in the caudate and putamen, as observed by TH immunohistochemistry (FIGS. 6B and 6C).

[0196] Interestingly, many of the symptoms were significantly improved after systemic deschloroclozapine (DCZ) administration. DCZ, a potent, brain-penetrant agonist of rM3Ds with reduced off-target binding compared to CNO, was administered to activate the DREADD system in macaque brains. In vivo electrophysiological recordings in anesthetized animals showed that DCZ or CNO, but not saline, induced increased neuronal activity in MPP+-injured macaques (Figures 15C-15G). Monkeys receiving MPP+ injections exhibited characteristic PD-like symptoms, including bradykinesia, tremor, rigidity, and postural abnormalities. Following systemic DCZ treatment, but not saline, we observed reversal of canonical Parkinson's disease symptoms in all monkeys tested (Figures 15H-15I, 16A, 16H-16K, 17A-17D, 34A-34D). First, we observed an increase in spontaneous locomotion in the observation cage, similar to the animal's activity level before MPP+ injection (Figures 15I, 16A–16F, 17C–17D). Second, tremor was significantly reduced or even eliminated after DCZ treatment (Figures 16H–16K, 17A). Third, we observed significant recovery of motor skills (here, activation of D1-MSNs) by chemogenetic manipulation (Figures 16H, 17A–17B). Furthermore, an effective dose of DCZ (0.3 mg / kg) did not alter motor-related behavior in naive monkeys (Figures 35A–35G). Notably, the alleviation of Parkinson's disease symptoms appeared consistent over 8 months of continuous DCZ treatment (Figures 16C–16G). Additionally, animals were free of dyskinesias during treatment, and blood levels of common liver- and kidney-related factors remained stable (Figures 36A-36F). These data strongly suggest that our targeted circuit manipulation approach can effectively and safely reverse core symptoms of Parkinson's disease in primates.

[0197] The efficacy of DCZ was then compared with that of levodopa, a first-line drug for treating PD patients. Similar improvements in Parkinson's disease symptoms were observed with these two chemicals (Figures 18A–18C, 24A, 37A–37F), but DCZ showed a faster improvement than levodopa. Targeted chemogenetic circuit activation mediated by DCZ resulted in a faster improvement than L-dopa during the early phase of drug administration (Figure 24B). Surprisingly, DCZ showed a much longer duration of efficacy compared with levodopa, with symptom relief occurring 24 hours after drug administration (Figure 18C). After the drug reached steady-state efficacy, DCZ extended its efficacy window after each dose of treatment, relieving symptoms for at least 24 hours after drug administration (Figure 24C), which was much longer than the clinically observed therapeutic window of L-dopa. Cerebrospinal fluid samples collected 24 hours after drug injection showed no detectable levels of DCZ (Figure 24D). This suggests a significant effect due to altered neural network dynamics or residual DREADD ligands in the brain. Furthermore, the manipulation approach targeting D1-MSNs did not induce dyskinesia-like behaviors that become evident after chronic L-dopa administration (Figure 24E). Long-term (4 months) prior L-dopa administration did not affect the efficacy of DCZ treatment or the absence of dyskinesias (Figures 24F-24L). Collectively, these results clearly demonstrate the efficacy of our approach in the NHP PD model and strongly support its feasibility for the treatment of PD in humans.

[0198] Example 7 Seroquel, a novel DREADD ligand for the clinic Parkinson's disease patients undergoing gene therapy would benefit from receiving an FDA-approved ligand to activate the DREADD component of the treatment described herein. While clozapine (CNO), the active metabolite of CNO, is a potential candidate for such a molecule, we sought additional molecules with fewer clinical monitoring requirements and a more favorable safety profile. Using chemical structure analysis in addition to safety analysis for use in PD patients, we identified Seroquel (quetiapine; QTP) as a potential candidate. Seroquel's ability to activate rM3Ds was first tested in vivo. After bilateral injection of AAV8R12-G88P7-rM3Ds-EYFP into the SNr of adult mice, either QTP or CNO was delivered by IP injection, and total distance traveled was quantified in an open field. As shown in Figure 20, QTP was able to stimulate behavior in the open field, indicating that QTP is a suitable in vivo ligand for rM3Ds. Next, we tested whether QTP could induce movement in animals injected with AAV8R12-G88P20-hM3Ds. However, compared with CNO, we found that QTP administration did not increase the distance traveled in these animals, as shown in Figure 21. This suggests that the difference in the structure of hM3Ds and rM3Ds renders QTP ineffective in activating hM3Ds.

[0199] As a first step toward designing hM3Ds variants that can be activated by QTP, we aligned the sequences of rM3Ds and hM3Ds to identify differences. In general, rM3Ds and hM3Ds share 96.6% consensus and are 94.5% identical. However, only two mutations were found within the ligand-binding domain (Figure 22), suggesting that these residues account for the difference in the response of rM3Ds and hM3Ds to NQN. Next, we introduced two mutations, A147S and F349Y, into these sites in hM3Ds, reverting them to the rM3Ds sequence, and tested the ability of QTP to activate this receptor in an in vitro luciferase activity assay in HEK293 cells. As can be seen in Figure 23, treatment with 10 μM QTP significantly increased the luciferase level of hM3Ds-A147S-F349Y to the same level as observed for rM3Ds, but did not increase the luciferase level for wild-type hM3Ds.

[0200] Example 8 Additional considerations of the study results One of the challenges of modern neuroscience is translating cutting-edge technological advances into effective therapeutic strategies for human brain disorders. In these examples, we exploit the relatively high-throughput nature of mouse studies for discovery research and further leverage the additional relevance of NHPs to develop and validate gene therapies that manipulate novel circuits to treat core symptoms of Parkinson's disease.

[0201] Some approaches developed herein exploit the distinct axon-targeting properties of different subtypes of projection neurons that can be commonly found in many brain regions and subregions. Thus, some embodiments may provide a viable solution for exploiting both the anatomical and functional properties of a variety of unique projection neuron types, many of which have direct therapeutic utility. Robust and specific targeting may rely on AAV serotypes with enhanced labeling efficiency for different cell types and appropriate cell-type-selective promoters that enable targeting of subsets of neurons within a mixed population of cells. Furthermore, we have shown that the choice of chemogenetic effectors or other modulators of cellular activity can influence the specificity of functional manipulation. Continued advances in viral capsid evolution, promoter and distal regulatory element identification and characterization, and neuromodulation technologies can be further developed to develop a comprehensive toolkit that further enables the rapid development of research strategies and therapeutic approaches based on circuit-specific activity modulation.

[0202] In a macaque model of Parkinson's disease, administration of 0.3 mg / kg / day of DCZ achieved significant correction of some motor symptoms. Considering that the standard clinical dose of clozapine, a DREADD agonist with a similar core structure to DCZ, used to treat schizophrenia is approximately 4.5–9 mg / kg / day, a substantially lower dose of DCZ may significantly reduce the side effects observed in clinical use of clozapine, such as neutropenia and weight gain. Nevertheless, the development and characterization of new clinically safe DREADD ligands is a valuable area for further translation of chemogenetic manipulation approaches to treat brain disorders. In this primate PD model, therapeutic retrograde AAVs were delivered to the SNr before damaging dopamine neurons in the SNc. We chose this reversed experimental order because, in the MPP+SNc injection PD macaque model, large-scale destruction of dopamine neurons, although highly specific and efficient, can lead to strong local inflammation and immune cell activation and penetration in the SNc and adjacent SNr. This change in the local environment could hinder efficient viral transduction into the SNr when therapeutic AAV is subsequently delivered. To verify that the change in experimental order does not affect the evaluation of therapeutic efficacy, retrograde AAV injection was performed before striatal injection of 6-OHDA to ablate SNc dopamine neurons in mice. This resulted in a reversal of Parkinson's disease phenotypes identical to those observed when pre-existing dopamine neuron loss was observed.

[0203] Systemic administration of L-dopa is a treatment method for Parkinson's disease patients. The action of L-dopa on the central non-BG and peripheral dopamine systems can contribute to the occurrence of many side effects. Some approaches described herein can precisely modulate the basal ganglia direct pathway without affecting any other dopamine pathways in the body, potentially preventing the occurrence of most or all L-dopa-induced side effects. Furthermore, L-dopa may in some cases require the survival of at least some nigral dopamine neurons to convert it to dopamine, which may contribute to the fluctuation and decline in its efficacy after long-term use and the progressive death of dopamine neurons in PD patients. On the other hand, in some embodiments, the chemogenetic gene therapy method devised herein does not require the survival of nigral dopamine neurons, and may provide a treatment option for end-stage PD patients who have lost most or all of their nigral dopaminergic neurons. The observation that the approach described herein can reverse Parkinson's disease symptoms in PD primates receiving extended L-dopa treatment indicates that it is a viable treatment candidate for advanced PD. Furthermore, while significant L-dopa-induced dyskinesias were observed in primates with Parkinson's disease, the same group of animals was dyskinesia-free after 8 months of treatment with DCZ. Given that alterations in D1-MSN activity may be a major driver of acute and chronic side effects observed with dopamine replacement therapy, the lack of dyskinesia observed with some approaches described herein may be due to the inability of DREADDs to induce corticostriatal synaptic plasticity or their effects on local striatal circuitry. Another feature of some approaches described herein is their extended efficacy window compared to the standard 6-hour window for L-dopa. Some methods described herein are effective in Parkinson's disease monkeys 24 hours after drug administration, with no signs of off-time due to the significantly extended therapeutic window. In addition, mixed results have been seen in trials applying dopamine agonists to treat depression, a common non-motor symptom of PD.The approach described here, which specifically modulates one of the major dopamine-dependent circuits in the brain, may help differentiate the roles of distinct dopamine systems in emotion modulation and may provide an alternative strategy to alleviate mood symptoms in Parkinson's disease. Overall, the precise gene therapy approach developed here may be useful for treating neurological disorders such as PD.

[0204] Example 9 Further methodological details of the study AAV capsid modification AAV1 / 5 / 6 / 8R (retro) Rep-Cap plasmids were chemically synthesized (Genewiz) by replacing the Cap sequence in the rAAV2-retrohelper plasmid (Addgene, 81070) with a sequence containing the intended sequence modifications. AAV8R1-14 Cap variants were generated by introducing one to three mutations into the AAV8R backbone. Mutagenesis was performed using PCR with PrimeSTAR HS DNA polymerase (Takara, R010A) and a pair of primers for each site. For example, the sequences of the mutagenesis primers used to induce the V183E mutation were 5'-TGGCGACTCAGAGTCAGAGCCAGACCCTCAACCTCT-3' (SEQ ID NO: 34) and 5'-AGAGGTTGAGGGTCTGGCTCTGACTCTGAGTCGCCA-3' (SEQ ID NO: 58). The PCR products were purified, digested with DpnI (NEB, R0176S) to remove the template, and transformed into competent E. coli cells. DNA was then extracted from individual colonies using a Miniprep kit (Qiagen, 27106) and subjected to Sanger sequencing to confirm the introduction of the intended mutations.

[0205] AAV8R Cap protein sequence [ka]

[0206] AAV8R12 Cap protein sequence [ka]

[0207] Promoter design and screening Region-selective promoter identification was based on Allen Brain Atlas in situ hybridization (ISH) data (mouse.brain-map.org / ). Genes highly expressed in the striatum but not in other parts of the basal ganglia were initially selected as candidates. H3K4me1 and H3K27ac are known epigenetic marks of active promoters and enhancers. Therefore, we identified regions in the mouse brain with high levels of these two chromatin modification marks as candidate regions based on the ENCODE annotation data in the UCSC Genome Browser (genome.ucsc.edu / ). To further expand the potential application of the promoter to primates, the sequence of interest was PCR amplified from human genomic DNA. Based on the above strategy, GPR88 was selected as a candidate gene. Nucleotide sequences upstream of the start codon with high levels of H3K4me1 and H3K27ac were selected as candidate promoter sequences. G88P2 (2259 bp) was cloned using the following primers: 5'-CATCGCAAGGCTACATGATGG-3' (SEQ ID NO: 36), 5'-CTGGCCAACTCTTCACACCTC-3' (SEQ ID NO: 60). G88P3 (1395 bp) and G88P7 (896 bp) were further shortened by subcloning.

[0208] G88P2 / 3 / 7 promoter sequence G88P2 promoter [ka] [ka]

[0209] G88P3 promoter [ka]

[0210] G88P7 promoter [ka] [ka]

[0211] AAV vector construction The promoter was subcloned into the pAAV-hSyn-EYFP vector derived from pAAV-hSyn-EGFP (Addgene, 50465), replacing the hSyn promoter using the appropriate restriction enzyme combination. To generate pAAV-G88P7-DIO-tdTomato, the DIO-tdTomato cassette was subcloned into pAAV-G88P7-EYFP, replacing the EYFP by restriction enzyme digestion. To generate pAAV-G88P3-HA-hM3Dq, the G88P3 promoter was subcloned into pAAV-hSyn-HA-hM3Dq-IRES-mCitrine (Addgene, 50463) via the EcoRI / BamHI restriction sites, replacing the hSyn promoter, followed by removal of the IRES-mCitrine by restriction enzyme digestion. To generate pAAV-G88P3-HA-hM3Dq-2A-EYFP, the 2A-EYFP fragment was subcloned into pAAV-G88P3-HA-hM3Dq after hM3Dq. To generate pAAV-G88P7-rM3Ds-2A-EYFP, the G88P7 promoter was subcloned into pAAV-hSyn-DIO-rM3Ds-mCherry (Addgene, 50458) to replace the hSyn promoter, followed by replacement of mCherry with 2A-EYFP. The DIO construct was subsequently removed by restriction enzyme digestion. To generate pAAV-G88P7-HA-rM3Ds-2A-Cre, EYFP was replaced with Cre in pAAV-G88P7-HA-rM3Ds-2A-EYFP by restriction enzyme digestion.

[0212] AAV production and titration HEK293T cells (ATCC) were co-transfected with AAV vector plasmids, AAV Rep-Cap plasmids (rAAV2-retro, AAV1R, AAV5R, AAV6R, AAV8R, and AAV8R1-14), and the pAdDeltaF6 helper plasmid (Addgene, 112867) carrying the adenoviral genes required for the AAV life cycle using calcium phosphate. HEK293T cells grown in 15 cm cell culture dishes were co-transfected at 80% confluency with a mixture of the three plasmids (1:1:1). 48–72 h after transfection, cells were harvested and resuspended in a buffer containing 150 mM NaCl and 100 mM Tris-HCl (pH 8.0). Cells were lysed by repeated freeze-thaw cycles in liquid nitrogen and a 37°C water bath. AAV particles were purified and concentrated using Millipore Amicon 100K columns (Merck Millipore, UFC910008). Encapsidated viral DNA was quantified by qPCR (Thermo Fisher) using primers recognizing viral WPRE and / or ITR sequences after denaturation of AAV particles with proteinase K. Titers were calculated as genome copies per milliliter.

[0213] Surgery and virus injection About the mouse: For retrograde labeling in Drd1-Cre or Drd2-Cre mice, a total volume of 200 nL of AAV8R12-G88P7-EYFP was injected into one side of the SNr. Simultaneously, a total volume of 300 nL of AAV9-G88P7-DIO-tdTomato was injected into the ipsilateral side of the striatum (0.5 mm anterior, 1.5 mm lateral, and 3.5 mm ventral to bregma). For optotagging in mice, a total volume of 200 nL of AAV8R12-G88P7-HA-rM3Ds-2A-Cre was injected into one side of the SNr. AAV9-EF1α-DIO-ChR2-EYFP was injected ipsilaterally at two sites to maximize striatal coverage (250 nL / site; site 1: 1.2 mm anterior, 1.5 mm lateral, and 3.2 mm ventral to bregma; site 2: 0.4 mm anterior, 1.6 mm lateral, and 3.3 mm ventral to bregma).

[0214] About monkeys: All neurosurgical procedures were performed using aseptic technique while the subject was anesthetized. For general anesthesia, monkeys received atropine (0.05 mg / kg, intramuscular) to reduce bronchial secretions before ketamine administration (15 mg / kg, intramuscular). Anesthesia was maintained using propofol (6 mg / kg, intravenous). The level of anesthesia was adjusted to eliminate movement when assessed by toe pinch. Corneal reflexes were consistently absent. The subject was placed on a permanently heated standard operating table, and the subject's head was firmly fixed in a stereotactic frame (David Kopf Instruments). Electrocardiograms, heart rate, oxygen saturation (SpO2) (range 95–100%), and rectal temperature (37.5–38.5°C) were continuously monitored using a physiological monitor (Mindray, uMEC7).

[0215] Striatal virus injections were performed at a rate of 300 nL / min into both the caudate nucleus (12 uL virus, 4 sites) and putamen (18 uL virus, 6 sites).

[0216] For intracranial drug administration and / or electrophysiological recording, a recording chamber covering the anterior caudate nucleus to the posterior GPi was fixed to the skull with six titanium screws and dental cement. Each subject was allowed to recover from surgery for at least 6 weeks before further study.

[0217] Generation of a mouse model of Parkinson's disease 6-OHDA was injected bilaterally using the same method as described for viral injections. A total volume of 1 μL of 6-OHDA (5 mg / mL, dissolved in sterile saline containing 0.02% ascorbic acid, Sigma) was injected into the striatum at a rate of 100 nL / min. The coordinates of the striatum were 0.5 mm anterior, 1.5 mm lateral, and 3.2 mm ventral relative to bregma. Animals were premedicated with desipramine (25 mg / kg, Sigma) before 6-OHDA injection to enhance the selectivity and efficacy of the 6-OHDA-induced lesion. Mice were supplemented with DietGel (ClearH2O) for 1 week after surgery. All staining and behavioral experiments were performed at least 14 days after surgery, when dopamine depletion had reached its maximum and stabilized.

[0218] PD score Two experienced observers blindly assessed the monkeys for Parkinson's disease symptoms three days a week throughout the observation period. Parkinson's disease symptoms were quantified according to the well-established Kurlan scale (Part I. Parkinson's Disease Characteristics), a widely used scale for quantifying PD symptoms in Old World monkeys. A score of zero indicates a normal monkey, whereas a maximum score of 29 indicates an animal with severe PD symptoms. For individual behavioral categories, scores for the upper and lower limbs were summed. In addition, action or intention tremor and resting tremor were summed.

[0219] Dyskinesia score Two experienced observers blindly assessed the monkeys' dyskinesia symptoms 3 days a week throughout the observation period. The dyskinesia score criteria were as follows: 0: Absent; 1: Mild, fleeting dyskinesia present <30% of the observation period; 2: Moderate, dyskinesia present >30% of the observation period and not interfering with normal activity; 3: Marked, dyskinesia present <70% of the observation period and occasionally interfering with normal activity; 4: Severe, persistent dyskinesia present >70% of the observation period and displacing normal activity.

[0220] Immunofluorescence testing Animals were deeply anesthetized with pentobarbital sodium (Nembutal; 80 mg / kg, i.p.) and perfused with PBS (0.1 M) and 4% paraformaldehyde in PBS (4% PFA / PBS, 30 mL for mice and 500 mL for monkeys at 4°C). Dissected brains were post-fixed in 4% PFA / PBS at 4°C and cryoprotected in 30% sucrose / PBS at 4°C. Coronal sections (40 μm for mice and 50 μm for monkeys) were prepared using a cryostat (Leica, CM1950). All sections were post-fixed for 20 minutes in 4% PFA / PBS at 4°C. Sections were blocked and permeabilized for 1 hour at room temperature in a PBS solution containing 5% bovine serum albumin (BSA) and 0.3% Triton® X-100. Primary antibody application was performed by incubating sections overnight at 4°C in PBS containing 5% BSA and polyclonal anti-GFP (Rockland, 600-101-215M), anti-RFP (Rockland, 600-401-379), anti-c-Fos (Cell Signaling Technology, 2250), anti-HA (Biolegend, 923501), and / or anti-TH (Abcam, ab76442) antibodies. Secondary antibody incubations were performed for 1 hour using Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 594 donkey anti-rabbit IgG, Alexa Fluor 488 goat anti-chicken IgG, and / or Alexa Fluor 488 streptavidin (Thermo Fisher, A32814, A32754, A11039, and S11223, respectively). Nuclei were stained with DAPI (Sigma, D9542). Brain sections were mounted on slides using Fluoromount-G mounting medium (SouthernBiotech, 0100-01).

[0221] Cell counting Images were acquired using a confocal microscope (Carl Zeiss, LSM880) and an AxioImager.Z1 microscope (Carl Zeiss) equipped with an apotome. A mouse brain atlas was used to identify brain structures microscopically and digitally. To analyze the number of EYFP+ cells in the striatum, striatal slices from rostral to caudal were used. One out of every six sections was analyzed, and the number of EYFP+ cells was multiplied by six to obtain the approximate total and average number of cells per animal. Images were processed using ImageJ (NIH, USA), and final quantification was performed manually by two blinded experimenters.

[0222] In situ hybridization Fragments of the coding regions of mouse Drd1 / Drd2 or macaque DRD1 / DRD2 were isolated from brain cDNA using PCR amplification. The amplified fragments were cloned into the pCR4 TOPO vector (Thermo Fisher). In situ hybridization was performed as previously described with minor modifications. Briefly, digoxigenin (DIG)-labeled cRNA probes (riboprobes) were prepared using DIG RNA Labeling Mix (Roche). Brains were frozen in OCT (Tissue-Tek), and 40- to 50-μm-thick coronal cryostat sections were hybridized with the DIG-labeled cRNA probes at 56°C for 15 to 18 hours. After hybridization, sections were washed twice in 0.2X SSC for 30 minutes at 62°C, incubated with peroxidase (POD)-conjugated anti-DIG antibody (Roche, 1207733910) for 2 hours at 37°C, and then processed with the TSA-plus kit (Perkin Elmer). Sections were then incubated with anti-RFP antibody (Rockland, 600-401-379) or anti-GFP antibody (Rockland, 600-101-215M) overnight at 4°C, and finally incubated with Alexa Fluor 594 donkey anti-rabbit IgG (Thermo Fisher, A32754) or Alexa Fluor 488 donkey anti-goat IgG (Thermo Fisher, A32814) for 2 hours at room temperature before mounting with Fluoromount-G (SouthernBiotech, 0100-01).

[0223] Behavioral assays for mice Open field test Mice were individually handled for 10–20 s per day for at least 5 days to allow them to become accustomed to the experimenter. The open-field test was conducted in a testing apparatus (50 cm × 50 cm × 50 cm) with an HD digital camera (Sony) positioned above the arena. On day 1, mice were allowed to acclimate to the apparatus for 10 min. On day 2, mice were administered saline (0.1 mL i.p. or 200 nL intracranially [i.c.] via a guide cannula). On day 3, mice were administered clozapine-N-oxide (CNO, Hello Bio, HB1807; 0.3 mg / kg i.p. or 200 nL i.c. at 100 μM). All behavioral tests were performed 30 min after injection. During testing, mice were allowed to freely explore the apparatus for 10 min. Behavioral data were analyzed using ANY-maze software (Stoelting Co.).

[0224] Rotarod test Mice were handled individually as described for the open field test. Before the test session, mice were transferred to the test room and allowed to acclimate for 15 min. Mice were placed on the rod (Shanghai Xinruan) and the apparatus was set to mode (10-40 rpm). The latency to fall for each trial was automatically recorded by the apparatus. Each mouse was tested for three trials per day on two consecutive days, with a minimum intertrial interval of 15 min. The latency to fall was averaged across the three trials on each day. Behavioral assays for monkeys Mobile Test: Locomotion tests on the monkeys were conducted in a custom-made observation cage (100 cm × 100 cm × 100 cm). The top and front of the cage were made of reinforced glass to allow a clear view for behavioral recording. The monkeys were habituated to the observation cage by being placed inside for 30 minutes on each day for three days.

[0225] Monkeys injected with AAV8R12-G88P3-HA-hM3Dq received intracranial injections of CNO into the dorsomedial caudate nucleus via a recording chamber. To administer the injections while the animals were awake, the monkeys were trained to sit in a specially designed primate chair, with the head secured to the chair by a thermoplastic mask. The injections were performed using a 33-gauge needle connected to a 250 μL syringe via polyethylene tubing (Hamilton, Neuros). A total volume of 3 μL of CNO (100 μM) was infused at a rate of 0.5 μL / min using a microsyringe pump (KD Scientific, Legato 130). The needle was held for 5 minutes to allow for drug diffusion before being removed. Monkeys injected with AAV8R12-G88P7-rM3Ds-2A-EYFP received intramuscular injections of CNO (10 mg / kg). After successful CNO injection, the monkeys were immediately transferred to an observation cage for video recording. Video capturing the target behavior was recorded for at least 90 minutes. Animal behavior was further analyzed and quantified 30–90 minutes after saline / CNO injection.

[0226] Parkinson's disease monkeys were administered DCZ (deschloroclozapine, 0.3 mg / kg, MCE) by intramuscular injection. After successful DCZ injection, the monkeys were immediately transferred to an observation cage for video recording. For levodopa treatment, L-dopa / benserazide (20 / 5 mg / kg / day, 4:1 L-dopa / benserazide ratio, such as Madopar®) was administered orally. For the initial study, DCZ and levodopa treatment trials were separated by 2 weeks to allow for drug washout. For long-term DCZ treatment, Parkinson's disease monkeys were given DCZ by intramuscular injection at a dose of 0.3 mg / kg every other day. For extended L-dopa treatment, animals were administered L-dopa once daily for 4 months. After a 1-month washout, DCZ was administered for 2 months of treatment.

[0227] Motor Skills Test The grasp-and-eat / hand-to-mouth movement test was performed as an index of motor skills in monkeys. First, monkeys were trained to sit in a primate chair with their healthy hand restrained. Then, the monkeys were trained to use their MPP+-affected hand to grasp food (a slice of apple, approximately 1 cm3) from the experimenter. The task included three blocks, with a 20-minute interval between different blocks. Each block included 10 consecutive trials. A trial was defined as successful if the monkey was able to bring the food to its mouth within 1 minute. Before MPP+ lesion, monkeys were trained to reach an 85% success rate.

[0228] Behavioral analysis of monkeys During video recording, the observation cage was recorded using top (x / y) and side (x / z) cameras. Subject activity was analyzed using the VigiePrimate system (Viewpoint) to extract behavioral data from the recorded videos. Detection data were synchronously sampled at 25 Hz for both top and side views and then combined to generate a single dataset displaying 3D space. Detection data were normalized to 0–100 cm for all x, y, and z axes, where x represents right to left, y represents front to back, and z represents bottom to top.

[0229] For rotation analysis, only movements that occurred less than 30 cm on the z-axis were counted. To determine the direction of rotation, we first calculated the center of the rotation data in the xy plane as (x0, y0) by averaging all data points, and then calculated the angle relative to (x0, y0) for each time point using the following formula:

number

[0230] Electrophysiological recordings About the mouse: Mice from different groups were anesthetized with 0.04% isoflurane and then decapitated. Brains were rapidly extracted and placed in ice-cold N-methyl-D-glucamine (NMDG) cutting solution containing (in mM): 92 NMDG, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 4.5 D-glucose, 20 HEPES, 5 L-ascorbic acid, 3 Na-pyruvate, 2 thiourea, 10 MgSO4, and 0.5 CaCl2. The pH was adjusted to 7.2 ± 0.1 using HCl, and the osmolality was adjusted to 305 ± 5 mosmol / L using NMDG. The solution was bubbled with 95% O2 / 5% CO2 before use. Coronal striatal slices were cut at 250-μm thickness using a vibratome slicer (Leica, VT1200 S) and then recorded after incubation for at least 45 min at 37°C in artificial cerebrospinal fluid (aCSF) saturated with 95% O2 / 5% CO2. aCSF contained (in mM): 125 NaCl, 1.25 KCl, 25 NaHCO3, 1.25 KH2PO4, 25 D-glucose, 2 CaCl2, and 1 MgCl2, supplemented with 2 mM Na-pyruvate, 3 mM myo-inositol, and 0.4 mM L-ascorbic acid.

[0231] Acute striatal slices were transferred to a recording chamber and constantly perfused with oxygenated aCSF at a rate of 3 ml / min. Neurons were visualized at room temperature (approximately 25°C) using an upright microscope (Olympus, BX-51) equipped with a 40x water-immersion objective. EYFP-positive neurons in the striatum were selected for whole-cell recording. Neurons were quickly visualized using a fluorescence microscope and then patch-clamped under infrared illumination and a CCD camera. Data were collected using an Axopatch 700B patch-clamp amplifier, a Digidate-1444A data acquisition system, and pCLAMP 10.6 software (Axon CNS). Patch pipette electrodes (OD = 1.5 mm, Sutter Instrument) were pulled with a Model P-1000 puller (Sutter Instrument) to a final tip resistance of 6–7 MΩ. Patched cells were stimulated with a single current step injection (150–200 pA, 150 ms duration) per minute. Stimulus intensity was adjusted to evoke one action potential with a 50% probability. Baseline recordings were performed with 18 current injections, after which the perfusion solution was switched to aCSF containing CNO (10 μM) and allowed to stabilize for 3 min. Current-clamp recordings were performed in the presence of CNO for 20 min using the same current-stimulation protocol. The recording electrode was filled with a potassium methanesulfonate (KMeSO3)-based internal solution containing (in mM): 135 KMeSO3, 10 KCl, 10 HEPES, 5 MgATP, 0.5 NaGTP, and 1 EGTA. The pH was adjusted to 7.2 ± 0.1 using KOH, and the osmolality was adjusted to 305 ± 5 mosmol / L using KMeSO3.

[0232] About monkeys: Electrophysiological recordings were performed using a recording chamber while the monkeys were anesthetized. Neural responses were recorded using a 16-channel linear probe (Plexon Inc., Uprobe) driven by a mechanical microdrive (Alpha Omega, FlexMT). The signal was passed through a headstage (Plexon Inc., HST / 16V-G20 LN) and then split using an amplifier system (Plexon Inc., OmniPlex). Spiking activity was then identified by filtering between 300 Hz and 5 kHz. Detected spikes were then sorted using commercially available software (Plexon Inc., Offline Sorter) for further analysis. To quantify the effects of CNO / DCZ injection, neural activity was recorded for 60 min after CNO / DCZ injection. Spikes within every 10-min window were counted and then normalized to the maximum value to obtain the time course of the response for each channel.

[0233] To determine the effect of CNO / DCZ injection on the direction of signal change, we performed a Pearson correlation between neural response and time using the "corr" function in Matlab®, which generates a correlation coefficient "r" and a significance level "p." Neurons were considered to have "increased" activity if r > 0 and p < 0.05, and "decreased" activity if r < 0 and p < 0.05. Otherwise, neurons were considered "unchanged" in their neural activity. After CNO / DCZ injection, all increased neuronal responses were then averaged after normalization by the maximum response (Crowe et al., 2014; Dai and Wang, 2018; Hirokawa et al., 2019) and plotted as a function of time to generate normalized population responses.

[0234] Optotagging About the mouse: Brain slices were prepared under low-light conditions. 473 nm blue LED light was obtained using an imaging objective (40× / 0.8 water-immersion objective, Olympus, Japan). Recordings were performed in current-clamp mode. For recordings to verify ChR2 function, 200 μM CdCl2 was included in the bath to prevent back-propagated calcium currents from dendrites. ChR2 was activated by 2 ms-long light pulses at 2 Hz with an intensity of 86.89 mW / mm2 (Sanwa-LP10, Japan). Cells that were faithfully stimulated by the light pulses were treated with CNO and further recorded.

[0235] About monkeys: To enable in vivo validation of the strategy using the optotagging approach, an AAV virus encoding opsin ChR2 (AAV9-EF1α-DIO-ChR2-EYFP) was injected into the striatum, and a retrograde AAV encoding Cre and rM3Ds (AAV8R12-G88P7-HA-rM3Ds-2A-Cre) was simultaneously injected into the substantia nigra. Total volumes of 27 μl (3 μl × 9 slices) and 30 μl (3 μl × 10 slices) were injected into the SNr and striatum, respectively. A recording chamber was implanted over the striatum to enable optical stimulation and electrophysiological recording.

[0236] Optogenetic testing was performed 6 weeks after viral injection. Optical stimuli were delivered and neural responses were recorded using a 16-channel linear probe (Plexon Inc., Uprobe) with embedded optical fibers. A mechanical microdrive (FlexMT, Alpha Omega, Nazareth, Israel) was used to mount and drive the probe. Optical stimulation was generated using a blue laser (473 nm, Changchun New Industries Tech, MBL-III-473) and controlled by a DAQ board (National Instruments, PCIe-6321) via the MonkeyLogic toolbox (NIMH version).

[0237] Neuronal responses to optical stimulation were probed using 40 Hz light pulses lasting 500 ms. Once neurons demonstrated repeatable responses to light stimulation, further testing was performed to examine their responses to CNO from 10 minutes before to 60 minutes after ligand administration. Responses from these optically identified cells were grouped to generate normalized population responses as a function of time.

[0238] Electromyography (EMG) recording and data analysis Customized surface EMG electrodes were used for EMG recording. The target skin area was shaved and thoroughly cleaned with alcohol wipes. Two electrodes were placed along the longitudinal axis of the biceps muscle, approximately midway between the two tendon insertions. The monkey was trained to sit in a primate chair in an awake state with free upper limb movement. The recording procedure was performed immediately after DCZ injection. EMG signals from the biceps brachii muscle were collected for at least 120 min. EMG signals were amplified by a signal acquisition and processing system (TECHMAN, BL-420N) collecting at a sampling rate of 1000 Hz. The raw EMG data were then plotted and analyzed using custom Matlab® (MathWorks) code (https: / / github.com / chenyef / PD_ana / blob / main / EMG_FFT_ana.txt).

[0239] Quantitative and statistical analysis Statistics were performed using GraphPad Prism 9.0. Paired t-tests, unpaired t-tests, one-way ANOVA, Tukey's test, and Dunnett's test were used as appropriate. All t-tests were performed as two-tailed tests. For all statistical tests, a P value of <0.05 was considered statistically significant. Sample sizes were selected based on previous publications or experience to ensure sufficient power to detect specific effects. All statistical tests used are listed in the figure legends.

[0240] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will immediately occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention.

[0241] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the incorporated-by-reference texts that conflict with definitions in this disclosure are excluded. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

Claims

1. A nucleic acid comprising a heterologous gene of interest operably linked to a regulatory element, wherein the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located 3' to the translation start site of the endogenous GPR88 gene.

2. 2. The nucleic acid of claim 1, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located, in part, within an intron.

3. 2. The nucleic acid of claim 1, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located within an intron.

4. 2. The nucleic acid of claim 1, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located less than about 1,000 nucleotides 3' to the translation start site of the endogenous GPR88 gene.

5. The nucleic acid of claim 4, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:

39.

6. The nucleic acid of claim 4, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:

39.

7. 2. The nucleic acid of claim 1, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is located less than about 900 nucleotides 3' to the translation start site of the endogenous GPR88 gene.

8. The nucleic acid of claim 7, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:

40.

9. 8. The nucleic acid of claim 7, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:

40.

10. 2. The nucleic acid of claim 1, wherein the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located 5' to the translation start site of the endogenous GPR88 gene.

11. 11. The nucleic acid of claim 10, wherein the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 100 nucleotides 5' to the translation start site of the endogenous GPR88 gene.

12. 12. The nucleic acid of claim 11, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:

41.

13. 12. The nucleic acid of claim 11, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:

41.

14. 11. The nucleic acid of claim 10, wherein the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 600 nucleotides 5' to the translation start site of the endogenous GPR88 gene.

15. 15. The nucleic acid of claim 14, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:

42.

16. 15. The nucleic acid of claim 14, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:

42.

17. 11. The nucleic acid of claim 10, wherein the genomic sequence located 5' to the translation start site of the endogenous GPR88 gene is located less than about 1,500 nucleotides 5' to the translation start site of the endogenous GPR88 gene.

18. 18. The nucleic acid of claim 17, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:

43.

19. 18. The nucleic acid of claim 17, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in SEQ ID NO:

43.

20. The nucleic acid of claim 1, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% homologous to the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 45 or 46.

21. 2. The nucleic acid of claim 1, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 45 or 46.

22. A nucleic acid comprising a heterologous gene of interest operably linked to a regulatory element, wherein the regulatory element comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous to the nucleotide sequence set forth in SEQ ID NO:

47.

23. The nucleic acid of claim 1 , wherein the heterologous gene of interest is 3′ to the regulatory element.

24. The nucleic acid of claim 1 , wherein the heterologous gene of interest has therapeutic utility.

25. 2. The nucleic acid of claim 1, wherein the gene of interest comprises a neurotrophic factor, an RNA-guided nuclease, an enzyme, or a DREADD.

26. 2. The nucleic acid of claim 1, which exhibits increased expression of the heterologous gene of interest relative to the promoter of the hSYN1 gene in neurons of the striatum.

27. The nucleic acid of claim 1 contained in a viral vector.

28. 27. The nucleic acid of claim 26, wherein the viral vector is an adeno-associated viral (AAV) vector.

29. 29. The nucleic acid of claim 28, wherein the viral vector is a retroAAV (AAV retro) virion.

30. 2. The nucleic acid of claim 1, wherein the gene of interest exhibits at least twice the expression of the heterologous gene of interest compared to the promoter of the hSYNl gene in neurons of the striatum.

31. 2. The nucleic acid of claim 1, wherein the gene of interest exhibits at least twice the expression of the heterologous gene of interest compared to the promoter of the hSYNl gene in neurons of the striatum.

32. 2. The nucleic acid of claim 1, wherein the gene of interest exhibits at least twice the expression of the heterologous gene of interest compared to the promoter of the hSYNl gene in neurons of the striatum.

33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent and the nucleic acid of claim 1.

34. 34. The pharmaceutical composition of claim 33 for use in a method for expressing a polypeptide in neurons of the striatum.

35. 35. The use of claim 34, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

36. 34. The pharmaceutical composition of claim 33 for use in a method for genetically manipulating neurons of the striatum.

37. 37. The use of claim 36, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

38. 34. The pharmaceutical composition of claim 33 for use in a method for treating a neurodegenerative disease in an individual.

39. 39. The use of claim 38, wherein the neurodegenerative disease comprises Parkinson's disease.

40. A method for expressing a polypeptide in neurons of the striatum of an individual, comprising expressing the polypeptide in the neurons of the striatum by administering to the individual the pharmaceutical composition of claim 33.

41. 41. The method of claim 40, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

42. 34. A method of genetically manipulating neurons of the striatum of an individual, comprising genetically manipulating said neurons of said striatum by administering to said individual the pharmaceutical composition of claim 33.

43. 43. The method of claim 42, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

44. 34. A method of treating an individual suffering from a neurodegenerative disease, comprising administering to said individual suffering from a neurodegenerative disease the pharmaceutical composition of claim 33, thereby treating said neurodegenerative disease.

45. 45. The method of claim 44, wherein the neurodegenerative disease comprises Parkinson's disease.

46. 41. The method of claim 40, wherein the individual is a mammal.

47. 41. The method of claim 40, wherein the individual is a human.

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