Neuron promoter and application thereof

Abstract

The present invention provides a heterologous target gene coupled to a regulatory element, wherein the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located at the 3'end of the translation initiation site of an endogenous GPR88 gene.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 437,220, filed January 5, 2023, and U.S. Provisional Application No. 63 / 589,864, filed October 12, 2023, which are incorporated herein by reference.

[0003] Sequence Listing Incorporated by Reference

[0004] This application contains a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on December 20, 2023, is named 062692-502001WO_SL.xml, and is 69,865 bytes in size. Background of the Invention

[0006] Adeno-associated viruses (AAVs) are small (25 nm) viruses belonging to the Parvovirus family that infect humans and other primate species. AAVs are used as delivery vectors for gene therapy because they are able to establish latent infection, integrating the AAV genome into host chromosomes without eliciting a destructive T cell immune response. Approximately 13 AAV serotypes have been isolated from the wild. SUMMARY OF THE INVENTION

[0008] Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are usually treated with levodopa or dopamine receptor agonists, but because dopamine receptors are widely distributed in the central nervous system and periphery, their effects lack specificity. The present disclosure includes the development of gene therapy strategies to selectively manipulate circuits affected by PD. For striatal D1 medium spiny neurons (MSNs), whose activity may be suppressed for a long time in PD, we have designed such a treatment strategy, which may include efficient new retrograde AAVs, promoter elements with strong D1-MSN activity, and chemical genetic effectors to enable precise D1-MSN activation after systemic ligand administration. Application of this treatment method can rescue movement, tremor, and motor skill deficits in PD, which supports the usefulness of targeted circuit modulation tools in the treatment of human PD.

[0009] The present disclosure provides heterologous target genes with increased gene expression coupled to regulatory elements. The present disclosure provides promoter sequences for use in methods for treating Parkinson's disease. The present disclosure provides designer receptors (DREADDs) specifically activated by designer drugs, which are used in methods for treating Parkinson's disease.

[0010] The present disclosure provides promoters and promoter sequences with increased gene expression, especially in neuronal cells. Promoters allow gene therapy, therapeutic proteins and / or designed receptors to be expressed in neuronal tissue (e.g., dopaminergic medium spiny neurons). This improved expression can be used to treat Parkinson's disease.

[0011] In some embodiments, the present disclosure provides a nucleic acid comprising a heterologous gene of interest operably coupled 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 an endogenous GPR88 gene.

[0012] In some embodiments, the genomic sequence 3' to the translation start site of the endogenous GPR88 gene is located partially within an intron. In some embodiments, the genomic sequence 3' to the translation start site of the endogenous GPR88 gene is located within an intron.

[0013] 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' from 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.

[0014] 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' from 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: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.

[0015] In some embodiments, the regulatory element comprises a nucleotide sequence corresponding to a genomic sequence located 5' to the translation start site of an endogenous GPR88 gene.

[0016] 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' from 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: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.

[0017] 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' from 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: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.

[0018] 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' from 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.

[0019] 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.

[0020] In some embodiments, the present disclosure provides a nucleic acid comprising a heterologous gene of interest operably coupled 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 shown in SEQ ID NO:47.

[0021] In some embodiments, the heterologous gene of interest is 3' to the regulatory element. In some embodiments, the heterologous gene of interest has therapeutic utility.

[0022] In some embodiments, the target gene comprises a neurotrophic factor, an RNA-guided nuclease, an enzyme, or a DREADD. In some embodiments, the nucleic acid exhibits increased expression of the heterologous target gene compared to the hSYN1 gene promoter in neurons of the striatum. In some embodiments, the target gene comprises a DREADD. In some embodiments, the DREADD is selected from a list consisting of one or more of rM3Ds, hM3Ds, or hM3Ds(A147S-F349Y). In some embodiments, the DREADD is an rM3Ds. In some embodiments, the DREADD comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99% identical or identical to SEQ ID NO: 38. In some embodiments, the heterologous target gene 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 that is at least about 90%, 95%, 97%, 98%, 99% identical 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 that is at least about 90%, 95%, 97%, 98%, 99% identical or identical to SEQ ID NO: 50.

[0023] In some embodiments, the nucleic acid is included in a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is a retrograde AAV (AAVretro) virion.

[0024] In some embodiments, the gene of interest exhibits at least 2-fold expression of the heterologous gene of interest compared to the hSYN1 gene promoter in neurons of the striatum. In some embodiments, the gene of interest exhibits at least 2-fold expression of the heterologous gene of interest compared to the hSYN1 gene promoter in neurons of the striatum. In some embodiments, the gene of interest exhibits at least 2-fold expression of the heterologous gene of interest compared to the hSYN1 gene promoter in neurons of the striatum.

[0025] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent and a viral vector. In some embodiments, the viral vector or pharmaceutical composition is used in a method for expressing a polypeptide in neurons of the striatum. In some embodiments, the neurons of the striatum are D1 dopaminergic medium spiny neurons.

[0026] In some embodiments, the viral vector or pharmaceutical composition is used in a method for genetically modifying neurons in the striatum. In some embodiments, the neurons in the striatum are D1 dopaminergic medium spiny neurons.

[0027] In some embodiments, the viral vector or pharmaceutical composition is used in a method of treating a neurodegenerative disease in a subject. In some embodiments, the neurodegenerative disease comprises Parkinson's disease.

[0028] In some embodiments, the present disclosure provides a method of expressing a polypeptide in neurons of the striatum of an individual, the method comprising administering a nucleic acid or a pharmaceutical composition to the individual, thereby expressing the polypeptide in neurons of the striatum. In some embodiments, the neurons of the striatum are D1 dopaminergic medium spiny neurons.

[0029] In some embodiments, the present disclosure provides a method for genetically modifying neurons in the striatum of an individual, the method comprising administering a nucleic acid or pharmaceutical composition to the individual, thereby genetically modifying the neurons in the striatum. In some embodiments, the neurons in the striatum are D1 dopaminergic medium spiny neurons.

[0030] In some embodiments, the present disclosure provides a method of treating an individual suffering from a neurodegenerative disease, the method comprising administering a nucleic acid or a pharmaceutical composition to an individual suffering from a 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 a human.

[0031] 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 administering to the individual a retrograde AAV or a pharmaceutical composition and a ligand that activates the DREADD, thereby activating the DREADD in the individual's central nervous system. 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 individual's central nervous system treats a neurodegenerative disease. In some embodiments, the neurodegenerative disease 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 retrograde AAV and the ligand that activates the DREADD are administered separately. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features described herein and advantages of said features will be obtained by reference to the following detailed description setting forth illustrative examples in which the principles of the features described herein are utilized and the following drawings:

[0034] Figure 1A Figure 3 shows the mutations introduced into the AAV8 capsid protein at three sites to create AAV8R, including: N385D; at position 588, the amino acid sequence from rAAV2 retro. The insertion of SEQ ID NOS 33, 61, and 61 is shown in bold, with the additional 10-aa insertion indicated; and TS720-721IN. Two additional mutations, V183E and N411S, were incorporated into the AAV8R12 capsid protein. The accompanying figures disclose SEQ ID NOS 33, 61, and 61, respectively, in the order of appearance. Figure 1B The diagram shows the mutations made to generate AAV1R, AAV5R, and AAV6R. To generate AAV1R, the amino acid sequence of SSSTDP (SEQ ID NO: 53) starting at position 586 of AAV1 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 695-698DPQF 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 generated. The accompanying drawings disclose SEQ ID NOS 62-66, 57, 62, and 66, respectively, in the order of appearance. Figure 1C The illustration shows that AAV1R, AAV5R, and AAV6R are inefficient in labeling stripe projection neurons, as barely any neurons are labeled. Figure 1D Shown are images and percentages of retrogradely labeled neurons in the SNr and upstream brain regions following intra-substantial nigral injection of AAV8R12. Brain regions with EYFP+ cells are shown. SNr: substantia nigra pars reticulata. STN: subthalamic nucleus. SC: superior colliculus. OFC: orbitofrontal cortex. ACAv: anterior cingulate cortex, ventral. ILA: infralimbic cortex. PrL: prelimbic cortex. FrA: frontal association cortex. Scale bar, 1 mm. Figure 1EFigure 3 shows images and percentages of retrogradely labeled neurons in the SNr and its upstream brain regions by substantia nigra injection of AAV8R12, with quantification of EYFP+ cells shown. n = 3 mice per group, data are presented as mean ± SEM.

[0035] Figure 2A Illustration of how virus was injected into the substantia nigra pars reticulata (SNr) of wild-type mice. Figures 2B-2C Figure 2 shows how AAV8R and AAV8R12 showed significantly improved efficiency in labeling striatal projection neurons compared to rAAV2-retro. Scale bar (2B) 100 μm. n = 3 mice per group (2C), error bars represent standard error (SEM), one-way ANOVA with post hoc Tukey test (F(2,6) = 48.66; rAAV2-retro vs. AAV8R: 2078 ± 262.4 vs. 10092 ± 463.9, P = 0.0032; rAAV2-retro vs. AAV8R12: 2078 ± 262.4 vs. 16026 ± 1654, P = 0.0002; AAV8R vs. AAV8R12: 10092 ± 463.9 vs. 16026 ± 1654, P = 0.0137). Figures 2D-2E Shown is a robust improvement in retrograde labeling efficiency of accumbal MSNs compared to rAAV2-retro. Figure 2D Shown are representative images of mouse accumbens projection neurons labeled with rAAV2-retro, AAV8R, and AAV8R12 after delivery into the dorsolateral ventral globus pallidus (scale bar, 100 μm. aca, anterior commissure; AcbC, nucleus accumbens core). Figure 2E Shown are representative images of mouse accumbens projection neurons labeled with rAAV2-retro, AAV8R, and AAV8R12 after delivery into the lateral hypothalamus (LH) (scale bar, 100 μm. AcbSh is the nucleus accumbens shell).

[0036] Figures 3A-3C Schematic diagram of the strategy used to identify highly active MSN promoters. A series of genes enriched in the striatum were 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 opposite terminal repeat.

[0037] Figures 4A-4BFigure 4 illustrates how the G88P2, 2G88P3, and G88P7 promoters showed improved efficiency in driving reporter gene expression in MSNs compared to several commonly used promoters, including human synapsin-1 (hSyn), CMV early enhancer / chicken β-actin (CAG), and elongation factor 1α (EF1α), as evidenced by proliferation of labeled neurons. Scale bar (4A) 100 μm, n = 3 mice per group (4B), error bars represent 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 ± 7 07.7, P = 0.2824; hSyn and G88P2: 16052 ± 1630 and 36290 ± 3705, P = 0.0258; hSyn and G88P3: 16052 ± 1630 and 37026 ± 6646, P = 0.0209; hSyn and G88P7: 16052 ± 1630 and 39460 ± 7281, P = 0.0104).

[0038] Figure 5A Figure 3 shows co-labeling (arrows) of striatal neurons for AAV8R12-G88P3-EYFP and Drd1, but not Drd2, after viral injection into the SNr. Scale bar, 5 μm. Figure 5B is a graph showing the quantification of Drd 1+ and Drd 2+ cells among EYFP+ cells; n=3 mice per group, data are expressed as mean±SEM.

[0039] Figure 6A Figure 3. Intraperitoneal (ip) delivery of CNO induced ipsilateral rotations, whereas intracranial (ic) CNO infusion into the dorsomedial striatum induced contralateral rotations following substantia nigra delivery of AAV8R12-G88P3-HA-hM3Dq. n = 8 mice per group, error bars represent mean ± SEM, unpaired t-test, (saline ip vs. CNO i.p.: 45.25 ± 4.128 vs. 87.84 ± 4.666, t = 6.836, P < 0.0001; saline ic vs. CNO ic: 49.63 ± 5.879 vs. 15.08 ± 3.435, t = 5.074, P = 0.0002). Figure 6B Figure 3. Co-labeling of substantia nigra neurons with AAV8R12-G88P3-HA-hM3Dq and c-Fos following intraperitoneal, but not intracranial, infusion of CNO. Scale bar, 20 μm. Figure 6CFigure 1 shows that intraperitoneal (ip) delivery of CNO and intracranial (ic) CNO infusion into the dorsomedial striatum following substantia nigra delivery of AAV8R12-G88P3-EYFP showed no effect on rotational behavior in mice. n = 5 mice per group, error bars represent 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 Included are representative images of retrogradely labeled neurons in the SNr and upstream brain regions by substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq, where the scale bar represents 1 mm. Figure 6E Shown are retrograde labeling of mouse striatal neurons with Drd1 ISH (left panel, arrows) and Drd2 ISH (right panel, arrows) after injection of AAV8R12-G88P3-HA-hM3Dq into the SNr, where the scale bar represents 10 μm. Figure 6F is a graph showing the quantification of Drd1+ and Drd2+ cells among all HA+ cells, where n=3 mice per group and data are expressed as mean±SEM. Figure 6G Figure 2. Electrophysiological responses to CNO in retrogradely labeled D1-MSNs following injection of AAV8R12-G88P3-HA-hM3Dq-2A-EYFP into the SNr. Whole-cell patch clamp recordings were performed on EYFP+ cells in isolated slices. Figure 6G 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, two-tailed paired t-test, and ***p < 0.001. Figures 6H-6I Depicts the delivery of AAV8R12-G88P3-HA-hM3Dq to the substantia nigra and intraperitoneal (ip) injection ( Figure 6H ) or intracranial (ic) infusion targeting the dorsomedial striatum ( Figure 6I ) Chemogenetic BG direct pathway manipulation in mice 3 weeks after CNO delivery. Percentage of contralateral rotation was quantified (left panel), n = 8 mice per group, data are presented as mean ± SEM, two-tailed unpaired t-test, ***p < 0.001. HA and c-Fos antibody staining in the SNr of mice after CNO delivery (right panel). Scale bar, 20 μm.

[0040] Figure 7AFigure 1 shows that both intraperitoneal and intracranial delivery of CNO induced contralateral rotations following substantia nigra delivery of AAV8R12-G88P7-rM3Ds-2A-EYFP. n = 8 mice per group, error bars represent mean ± SEM, unpaired t-test, (saline ip vs. CNO ip: 45.95 ± 4.972 vs. 80.05 ± 5.168, t = 4.754, P = 0.0003; saline ic vs. CNO i.c.: 58.31 ± 4.742 vs. 80.77 ± 4.187, t = 3.551, P = 0.0032). Figure 7B Figure 3. Co-labeling of substantia nigra neurons without AAV8R12-G88P7-rM3Ds-2A-EYFP and c-Fos following intraperitoneal or intracranial infusion of CNO. Scale bar, 20 μm. Figure 7C Figure 1 shows that intraperitoneal and intracranial delivery of CNO after substantia nigra delivery of AAV8R12-G88P7-EYFP showed no effect on rotational behavior in mice. n = 5 mice per group, error bars represent mean ± SEM, unpaired t-test, (saline ip vs. CNO i.p.: 48.68 ± 7.203 vs. 46.88 ± 7.497, t = 0.1733, df = 8, P = 0.8667; saline ic vs. CNO ic: 41.11 ± 12.73 vs. 34.55 ± 7.536, t = 0.4435, df = 8, P = 0.6691). Figure 7D Included are representative images of retrogradely labeled neurons in the SNr and upstream brain regions by substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP, where the scale bar represents 1 mm. Figure 7E Included are images of retrograde labeling of mouse striatal neurons with Drd1 ISH (left, arrows) and Drd2 ISH (right, arrows) after injection of AAV8R12-G88P7-rM3Ds-2A-EYFP into the SNr, where the scale bar represents 10 μm. Figure 7F is a graph showing the quantification of Drd1+ and Drd2+ cells among EYFP+ cells, wherein n=3 mice per group, and the data are expressed as mean±SEM. Figure 7G Figure 2. Electrophysiological responses to CNO in retrogradely labeled D1-MSNs following injection of AAV8R12-G88P7-rM3Ds-2A-EYFP into the SNr. Whole-cell patch clamp recordings were performed on EYFP+ cells in isolated slices. Figure 7G Representative traces (left panel) and quantification (right panel) of action potentials induced by somatic current injection at baseline and after CNO administration are included. n = 10 cells from 6 mice, data are presented as mean ± SEM, two-tailed paired t-test, ****p < 0.0001. Figures 7H-7IDepicts the substantia nigra delivery of AAV8R12-G88P7-rM3Ds-2A-EYFP and i.p. injection ( Figure 7H ) or targeted dorsomedial striatum ic infusion ( Figure 7I ) Chemical genetic manipulation of the BG direct pathway in mice 3 weeks after CNO delivery. The percentage of contralateral rotation was quantified (left panel). n = 8 mice per group. Data are presented as mean ± SEM, two-tailed unpaired t-test, **p < 0.01, ***p < 0.001. EYFP and c-Fos antibody staining in the SNr of mice after CNO delivery (right panel). Scale bar, 20 μm.

[0041] Figures 8A-8B Figure 3. AAV8R12-G88P7-HA-hM3Dq ( Figure 8A ) or AAV8R12-G88P7-rM3Ds-2A-EYFP ( Figure 8B Representative heatmap of macaques injected with CNO in the substantia nigra. Animals administered CNO spent less time observing the higher parts of the cage. Figures 8C-8H Figure 3. AAV8R12-G88P3-HA-hM3Dq (AAV8R12-G88P3-HA-hM3Dq) injected into the substantia nigra after saline or CNO infusion. Figures 8C-8E ) or AAV8R12-G88P7-rM3Ds-2A-EYFP ( Figures 8F-8H Quantification of ipsilateral rotational velocity in macaques ( Figure 8C 、 8F ), the total distance traveled ( Figure 8D 、 8G ) and immobility time ( Figure 8E 、 8H Each group had n=3 monkeys ( Figures 8C-8E ), n = 6 monkeys per group ( Figures 8F-8H ), data are expressed as mean ± SEM, two-tailed paired t test, ns, not significant.

[0042] Figure 9A AAV8R12-G88P3-mCherry was injected into the SNr of a cynomolgus monkey; labeled neurons were found throughout the caudate nucleus and putamen. The position of the coronal section along the anterior-posterior axis is indicated as the distance from the ear bar zero (EBZ). The scale bar is 5 mm. Figure 9B Labeled neurons in the caudate nucleus and putamen are shown. Scale bar, 100 μm. Figure 9C Figure 1 shows retrograde labeling of striatal neurons by DRD1 ISH (upper panel, arrows) and DRD2 ISH (lower panel, arrows) following injection of AAV8R12-G88P3-mCherry into the macaque SNr. Co-labeling was observed only for DRD1. Scale bar, 20 μm. Figures 9D-9EFigure 9: Activation of the BG direct pathway in mice 12 months after substantia nigra delivery of AAV8R12-G88P7-rM3Ds-2A-EYFP and CNO delivery by i.p. injection (9D). The percentage of rotational behavior (ipsilateral and contralateral rotations) was quantified (9E). n = 6 mice per group. Data error bars represent mean ± SEM. Two-tailed unpaired t-test. **p < 0.01. Figure 9F Figure 9 shows strong labeling of D1-MSNs following substantia nigra delivery of AAV8R12 in cynomolgus monkeys, with retrogradely labeled neuronal somata extracted from fluorescent images (9A) distributed throughout the caudate and putamen. The position of coronal sections along the anterior-posterior axis is represented as the distance from the EBZ, with a scale bar of 2 mm. Figure 9G is a graph showing the quantification of DRD1+ and DRD2+ cells among mCherry+ cells, where n=6 sections from one macaque, and the data are expressed as mean±SEM.

[0043] Figure 10A Depicted are representative top-view locomotion tracking images of observation cages of macaques housed therein that received substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq following icCNO infusion into the dorsomedial caudate nucleus of macaques. Figure 10BRepresentative top-view tracings of macaques receiving substantia nigra injections of AAV8R12-NP3-rM3Ds-2A-EYFP in observation cages following intramuscular (im) CNO injections are shown. Macaques that received substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq (monkey IDs: CM045, CM049) or AAV8R12-G88P7-rM3Ds-2A-EYFP (monkey IDs: CM048, CM051) showed significantly increased contralateral rotations (10C) following intracranial or systemic CNO infusion, respectively, as quantified by time spent observing the top compartment of the cage. n = 4 monkeys per group, error bars represent mean ± SEM, two-tailed paired t-test (t = 3.276, P = 0.0469, saline vs. CNO: 1.004 ± 0.2667 vs. 6.649 ± 1.662). Macaques that received AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP injections in the substantia nigra spent less time observing the top portion of the cage (10D). Quantification of contralateral rotation speed after CNO infusion in macaques is shown, n = 4 monkeys per group, error bars represent mean ± SEM, two-tailed paired 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 speed during contralateral rotation (10G) after intracranial or systemic CNO infusion, 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 plot (10F) (paired t-test, t = 2.272, P = 0.1077, saline vs. CNO: 71.53 ± 15.4 vs. 97.37 ± 19.41), contralateral rotation speed (10G), or ipsilateral rotation speed (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 Representative images of retrograde labeling of the entire basal ganglia following substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. The position of coronal sections along the anterior-posterior axis is represented as distance from the EBZ. Scale bar, 5 mm. Figure 10J High-magnification images of labeled hM3Dq+ neurons in the macaque caudate and putamen are included. Scale bar, 20 μm. Figure 10KIncludes retrograde labeling of hM3Dq+ striatal neurons (green) using DRD1 ISH (left panel, magenta, arrows) and DRD2 ISH (right panel, magenta, arrows). Scale bar, 20 μm. Figure 10L Quantification of DRD1+ and DRD2+ cells among all HA+ cells is shown. n = 6 sections from 1 macaque per group, data are presented as mean ± SEM. Figure 10M Representative images of retrograde labeling of the entire basal ganglia following substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP. Coronal sections are positioned along the anterior-posterior axis as distance from the EBZ. Scale bar, 5 mm. Figure 10N High-magnification images of labeled rM3Ds+ neurons in the macaque caudate and putamen. Scale bar, 20 μm. Figure 10O Figure 3 shows retrograde labeling of rM3Ds+ striatal neurons (green) using DRD1 ISH (left panel, magenta, arrows) and DRD2 ISH (right panel, magenta, arrows). Scale bar, 20 μm. Figure 10P Quantification of DRD1+ and DRD2+ cells among all EYFP+ cells is shown. n = 6 sections from 1 macaque per group, data are presented as mean ± SEM. Figure 10Q Depicted are representative top-view locomotion tracking images of an observation cage housing macaques that received icCNO infusion into the dorsomedial caudate nucleus 8 weeks after substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq. Figure 10R Is displayed relative to Figure 10Q , Graph showing quantification of the ratio of contralateral to ipsilateral rotations after CNO infusion. n = 3 monkeys per group, data are expressed as mean ± SEM, two-tailed paired t-test, *p < 0.05. Figure 10S Is displayed relative to Figure 10B , Graph showing quantification of the ratio of contralateral to ipsilateral rotations after CNO infusion. n = 6 monkeys per group, data are expressed as mean ± SEM, two-tailed paired t-test, *p < 0.05. Figure 10T-10U Depicted are quantifications of time spent observing the top compartment of the cage following icCNO infusion in macaques receiving substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq ( Figure 10T ) and the speed of contralateral rotation ( Figure 10U ). Each group had n=3 monkeys, and the data were expressed as mean±SEM, two-tailed paired t-test, *p<0.05. Figure 10V-10W Depicted are quantifications of time spent observing the top compartment of the cage following imCNO infusion in macaques receiving substantia nigra injection of AAV8R12-G88P7-rM3Ds-2A-EYFP ( Figure 10V ) and the speed of contralateral rotation ( Figure 10W). n = 6 monkeys per group, data are expressed as mean ± SEM, two-tailed paired t test, **p < 0.01, ***p < 0.001.

[0044] Figure 11 is a top view of the observation cage ( Figure 11A ) and side view ( Figure 11B ) shows representative images of macaques undergoing the first trial after saline or CNO infusion. The animals showed no differences in rotational behavior or time spent in the upper part of the observation cage. The percentage of contralateral 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 upper 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), immobility time ( Figure 11E )(Paired t test, t = 0.02596, df = 3, P = 0.9816, saline vs. CNO: 149.9 ± 21.61 vs. 149.8 ± 22.65), total distance ( Figure 11F )(paired t test, t = 1.505, df = 2, P = 0.2713, saline vs. CNO: 53.12 ± 15.3 vs. 59.48 ± 18.65), contralateral rotation speed ( Figure 11G )(paired t test, t = 0.134, df = 2, P = 0.9057, saline vs. CNO: 39.07 ± 5.889 vs. 39.63 ± 7.092) or ipsilateral rotational speed ( Figure 11H ) (paired t-test, t = 1.129, df = 2, P = 0.3762, saline vs. CNO: 41.49 ± 3.96 vs. 42.79 ± 4.705). No significant differences were found after intracranial or systemic CNO infusion, respectively. n = 3 monkeys per group. Error bars represent mean ± SEM. Paired t-test.

[0045] Figures 12A-12F Shown are electrophysiological analyses in macaque monkeys following chemogenetic activation of the direct pathway of the basal ganglia. Figure 12A Schematic diagram of AAV8R12-G88P3-HA-hM3Dq injection in the SNr and electrophysiological recordings in the caudate nucleus following CNO infusion into the dorsomedial caudate nucleus (monkey ID: CM045). Figure 12BThe response time course of a typical neuron in the caudate nucleus after CNO infusion into the dorsomedial caudate nucleus is illustrated. The x-axis represents the period (in minutes) of the count spikes, while the y-axis represents the normalized spike counts. Each inset shows the original spike trajectory (top of the inset) and waveform (bottom of the inset) at the specific time point represented by the arrow. Figure 12C The diagram shows that a total of 34 cells were recorded in the caudate nucleus of monkey CM045, of which 70.6% of the cells (n=24 cells) were activated, 8.8% of the cells (n=3 cells) were inactivated, and 20.6% of the cells (n=7 cells) remained unchanged. Figure 12D Schematic diagram of AAV8R12-G88P7-rM3Ds-2A-EYFP injection in the SNr and electrophysiological recordings in the caudate nucleus after intramuscular CNO injection (monkey ID: CM048). Figure 12E The response time course of a typical neuron in the caudate nucleus after intramuscular CNO injection is illustrated. The x-axis represents the period (in minutes) of counting spikes, while the y-axis represents the normalized spike counts. Each inset shows the original spike trajectory (top of the inset) and waveform (bottom of the inset) at the specific time point represented by the arrow. Figure 12F The diagram shows that a total of 38 cells in the caudate nucleus were recorded from monkey CM048, of which 65.8% of the cells (n=25 cells) were activated, 7.9% of the cells (n=3 cells) were inactivated, and 26.3% of the cells (n=10 cells) remained unchanged. Figure 12G Schematic diagram of electrophysiological recordings in the caudate / putamen combined with light stimulation in the caudate / putamen following intramuscular CNO injection in anesthetized macaques (left). Raw spike traces and waveforms of a representative neuron in the caudate nucleus under blue light (473 nm) illumination (right). Figure 12H Pictured Figure 12G Spike counts for repetitive optogenetic stimulation of a typical neuron shown in . Figure 12I Pictured Figure 12G Raw spike traces and waveforms of a representative neuron at baseline and after intramuscular CNO administration are shown in . Figure 12J The time course of responses of retrogradely labeled caudate / putamen neurons (n=5) identified by optical labeling after intramuscular CNO injection is plotted. The x-axis represents the period of counted spikes (in minutes) and the y-axis represents the normalized population response. Figure 12KSchematic diagram of electrophysiological recordings in the caudate nucleus after injection of AAV8R12-G88P3-HA-hM3Dq into the SNr and CNO infusion into the dorsomedial caudate nucleus (left panel) in anesthetized macaques. The right panel shows the response time course of activated caudate neurons (n=23) after CNO infusion into the dorsomedial caudate nucleus. The x-axis represents the period of counted spikes (in minutes) and the y-axis represents the normalized population response. The inset shows the raw spike traces and waveforms of a representative neuron in the caudate nucleus at baseline and 50-60 minutes after CNO infusion. Figure 12L The figure shows that: for CNO infusion, a total of 34 cells were recorded in the caudate nucleus, of which 67.6% of the cells (n=23 cells) were activated, 2.9% (n=1 cell) were deactivated, and 29.4% (n=10 cells) remained unchanged; for saline infusion, a total of 32 cells were recorded in the caudate nucleus, of which 18.8% of the cells (n=6 cells) were activated, 40.6% (n=13 cells) were deactivated, and 40.6% (n=13 cells) remained unchanged. Figure 12M Schematic diagram of electrophysiological recordings in the caudate nucleus / putamen after AAV8R12-G88P7-rM3Ds-2A-EYFP injection into the SNr and intramuscular CNO injection in anesthetized macaques (left panel). The right panel shows the response time course of activated caudate nucleus / putamen neurons (n=19) after CNO injection. The x-axis represents the period of counted spikes (in minutes) and the y-axis represents 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 minutes after CNO infusion. Figure 12N The graph shows that for CNO administration, a total of 38 cells were recorded in the caudate nucleus / putamen, of which 50% of the cells (n=19 cells) were activated, 13.2% (n=5 cells) were deactivated, 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% of the cells (n=6 cells) were activated, 53.3% (n=16 cells) were deactivated, and 26.7% (n=8 cells) remained unchanged.

[0046] Figures 13A-13F Figure 3. Chemogenetic activation of D1 MSNs reverses PD symptoms in mice. Figure 13A Schematic diagram of the protocol for stereotactic injection and behavioral analysis in PD mice. Figure 13B Representative images of tyrosine hydroxylase (TH) staining in control and PD animals are shown. Dopaminergic neurons in the SNc (bottom) and their terminals in the Cpu (top) undergo robust degeneration. Scale bars: 1000 μm (top), 500 μm (bottom). Figure 13C Shown are representative tracking images of mice in the open field test. Figures 13D-13E The figure shows the total distance ( Figure 13D ) and immobility time ( Figure 13E Quantification of 6-OHDA lesions showed a significant decrease in motor behavior. Chemogenetic activation of D1 MSNs significantly rescued motor deficits in PD model mice (n = 8, error bars represent mean ± SEM, paired t-test, **p < 0.01). Figure 13F Figure 3. Motor skills were significantly reduced after 6-OHDA lesion. Chemogenetic activation of D1 MSNs partially rescued motor deficits in PD model mice. n = 8. Error bars represent mean ± SEM. Paired t-test. *p < 0.05, **p < 0.01. Figure 13G Included are graphs illustrating the quantification of SNc TH+ cell numbers in control and 6-OHDA-treated mice (n=4 mice per group, data are presented as mean ± SEM, two-tailed unpaired t-test, ****p<0.0001). Figure 13H Included are additional representative traces of mice before and after 6-OHDA lesion in the open field test, and after saline or CNO treatment in lesioned animals. Figure 13L Illustration of stereotaxic injection of AAV8R12-G88P7-EYFP into the SNr in a mouse model of Parkinson's disease mediated by 6-OHDA-mediated dopaminergic cell death, followed by CNO administration. Figure 13M Included is a graph quantifying the total distance traveled in the open field test by mice injected with AAV8R12-G88P7-EYFP in the substantia nigra. n = 8 mice per group, data are presented as mean ± SEM, one-way ANOVA with post hoc Tukey test, ***p < 0.001, ****p < 0.0001, ns, not significant. Figure 13N Included is a graph quantifying the immobility time in the open field test in mice injected with AAV8R12-G88P7-EYFP in the substantia nigra. n = 8 mice per group, data are presented as mean ± SEM, one-way ANOVA with post hoc Tukey test, ***p < 0.001, ****p < 0.0001, ns, not significant. Figure 13O Graph quantifying the latency to fall in the rotator rod test in mice injected with AAV8R12-G88P7-EYFP in the substantia nigra. 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 13PFigure 2 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 on EYFP+ cells in isolated 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 expressed as mean ± SEM, two-tailed paired t-test, **p < 0.01, ns, not significant. Figure 13P Figure 2 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 on EYFP+ cells in isolated 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 (M) from 3 mice, data are expressed as mean ± SEM, two-tailed paired t-test, **p < 0.01, ns, not significant.

[0047] Figure 14 Schematic diagram of the scheme for stereotaxic injection and behavioral analysis in PD monkeys.

[0048] Figure 15A Representative images of tyrosine hydroxylase (TH) staining in control and MPP+ injected animals are shown. Dopaminergic neurons show severe degeneration in the SNc and their terminals in the CPu. Scale bars are 5000 μm (Cd and Put), 50 μm (SNc). Figure 15B Graph showing quantification of SNc TH+ cell numbers in control and MPP+-treated macaques. n = 6 sections from 1 macaque per group, data are presented as mean ± SEM, two-tailed unpaired t-test, ***p < 0.001. Figures 15C-15F The graph shows: baseline and CNO ( Figure 15C ) or DCZ( Figure 15E ) in anesthetized macaques. Figure 15D , n = 19 cells) or DCZ ( Figure 15F Response time course of activated caudate / putamen neurons after administration of α(R), n=13 cells. The x-axis represents the period of count spikes (in minutes), and the y-axis represents the normalized population response. Figure 15GThe graph shows that for CNO administration, a total of 36 cells were recorded in the caudate nucleus / putamen, of which 52.8% of cells (n=19 cells) were activated, 13.9% (n=5 cells) were deactivated, and 33.3% (n=12 cells) remained unchanged. For DCZ administration, a total of 31 cells were recorded in the caudate nucleus / putamen, of which 41.9% of cells (n=13 cells) were activated, 9.7% (n=3 cells) were deactivated, and 48.4% (n=15 cells) remained unchanged. For saline administration, a total of 39 cells were recorded in the caudate nucleus / putamen, of which 17.9% of cells (n=7 cells) were activated, 33.4% (n=13 cells) were deactivated, and 48.7% (n=19 cells) remained unchanged. Figure 15H Included are graphs 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 The total activity of macaques in the observation cage is plotted, categorized as low, medium, and high mobility. Quantification of the percentage of high mobility fractions indicates changes in activity in macaques 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.

[0049] Figures 16A-16JFigure 1 shows that chemogenetic activation of D1-MSNs reversed Parkinson's disease symptoms in macaques; representative traces of macaques' distance traveled in the observation cage (16A and 16C) and activity maps showing time quantification (16B and 16D) are shown. Macaques that received MPP+ showed significant reductions in total activity (E), distance traveled (16G), and immobility time (16H). DCZ treatment successfully rescued motor deficits. n = 4 monkeys per group, error bars represent mean ± SEM, one-way ANOVA with post hoc Dunnett test (16C), two-tailed paired t-test (16D), *p < 0.05, **p < 0.01, ns not significant. Macaques' total activity was categorized as low, medium, and high mobility. Macaques that received MPP+ lesions showed motor balance deficits and rarely resided in the top portion of the observation cage, a phenotype significantly reversed by DCZ treatment. n = 4 monkeys per group, error bars represent mean ± SEM, paired t-test, *p < 0.05, **p < 0.01 (16F). Total and isolated PD scores in PD macaques before and after DCZ treatment. n = 4 monkeys per group, error bars represent mean ± SEM, paired t-test, *p < 0.05, **p < 0.01 (16I-16J). Representative EMG images of the macaque biceps. After DCZ treatment, the typical PD-related 4-6 Hz tremor signal was removed (16K). The dyskinesia phenotype was significantly reversed in macaques receiving DCZ, as shown by representative quantification of dyskinesia scores 2 weeks and 1 month after treatment with DCZ or levodopa (L-Dopa). n = 3 monkeys per group, data are represented as mean ± SEM, two-tailed unpaired t-test, *p < 0.05, ****p < 0.0001 (L).

[0050] Figure 17A The graph shows the quantification of the percentage of tremor episodes every 10 minutes. EMG was recorded continuously for 120 minutes after intramuscular administration of DCZ. n = 3 monkeys per group, error bars represent mean ± SEM, one-way ANOVA with post hoc Dunnett's test, **p < 0.01, ns, not significant. Figure 17B Figure quantification of the success rate of hand-to-mouth movements. DCZ treatment partially restored this motor skill. n = 3 monkeys per group, error bars represent mean ± SEM, one-way ANOVA with post hoc Dunnett's test, **p < 0.01. Figure 17C Graph showing quantification of distance traveled after MPP+lesion and DCZ treatment compared to 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 17DGraph showing quantification of immobility time after MPP+lesion and DCZ treatment compared to 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 Graph showing quantification of time spent on the top compartment of the observation cage 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, ns, not significant.

[0051] Figures 18A-18C Figure 18: Total PD scores in Parkinson's macaques before and after L-Dopa treatment. n = 3 monkeys per group, error bars represent mean ± SEM, paired t-test, *p < 0.05, **p < 0.01 (18A). Comparison of the efficacy of DCZ and L-Dopa. DCZ achieved an efficacy comparable to that of L-Dopa (18B). DCZ reached a stable therapeutic effect faster than L-Dopa, and its effect lasted for more than 24 hours (18C). n = 3 monkeys per group, error bars represent SEM, paired t-test, *p < 0.05.

[0052] Figure 19A Depicted is an 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). Alignment was performed using ClustalOmega multiple sequence alignment (www.ebi.ac.uk / Tools / msa / clustalo / ). The "*" symbol indicates a perfect alignment; the ":" symbol indicates that the site belongs to a group with strong similarity, while the "." symbol indicates that the site belongs to a group with weak similarity. Figure 19B Depicted is an alignment of the Cap protein sequences of AAV2, rAAV2-retro, AAV8, AAV8R, and AAV8R12, which were aligned using Clustal Omega and graphically displayed using MViewer 1.63. The figures disclose SEQ ID NOS 20-21 and 67-69, respectively, in order of appearance.

[0053] Figure 20 Graph depicts Seroquel (quetiapine; QTP) stimulation of locomotion in mice with SNr expression of DREADD rM3Ds.

[0054] Figure 21 Graph depicting that Seroquel (quetiapine; QTP) does not stimulate locomotion in mice with SNr expression of DREADD hM3Ds.

[0055] Figure 22 An alignment of rM3Ds and hM3Ds is shown. The accompanying figures disclose SEQ ID NOS 70-73, respectively, in column order.

[0056] Figure 23 The graph shows that Seroquel increased luciferase levels in hM3Ds-A147S-F349Y to the same level as observed for rM3Ds, but did not increase luciferase levels in wild-type hM3Ds.

[0057] Figure 24A Quantification of total PD scores in macaques before, 3 days, 1 week, and 2 weeks after L-Dopa treatment is shown. 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 Quantification of changes in PD scores 3 days, 1 week, and 2 weeks after initial and continuous administration of DCZ or L-Dopa is plotted. N = 3 monkeys per group, data are expressed as mean ± SEM, two-tailed paired t-test, *p < 0.05, ns, not significant. Figure 24C Quantification of the effect of a single dose of DCZ or L-Dopa on the total PD score after the drug reached steady-state efficacy was plotted. N = 3 monkeys per group, data are presented as mean ± SEM, two-tailed paired t-test, *p < 0.05, ns, not significant. Figure 24D Corticospinal fluid (CSF) concentrations of DCZ measured by LC-MS at 6, 12, and 24 hours after intramuscular delivery (0.3 mg / kg) are plotted. N=3 monkeys per group, data are presented as mean ± SEM. Figure 24E Quantification of motor impairment scores 2 weeks, 1 month, and 4 months after treatment with DCZ or L-Dopa is plotted. N = 3 monkeys per group, data are presented as mean ± SEM, two-tailed unpaired t-test, *p < 0.05, ****p < 0.0001. Figure 24F Schematic representation: For extended L-Dopa treatment, animals were administered L-Dopa once daily for 4 months. A 1-month washout was allowed before DCZ administration. Figure 24G Quantification of total PD scores in macaques before and 1, 2, and 4 months after L-Dopa treatment is shown. 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 Quantification of total PD scores in macaques before, 1 month after, and 2 months after DCZ treatment after prolonged L-Dopa administration and washout is plotted ( Figure 24F). n = 3 monkeys per group, data are presented as mean ± SEM, one-way ANOVA with post hoc Dunnett test, *p < 0.05. Figure 24I Quantification of distance traveled before, 1 month after, and 2 months after DCZ treatment after prolonged L-Dopa application and washout was plotted ( Figure 24F ). n = 3 monkeys per group, data are presented as mean ± SEM, one-way ANOVA with post hoc Dunnett test, *p < 0.05. Figure 24J Quantification of immobility time before, 1 month after, and 2 months after DCZ treatment after prolonged L-Dopa application and washout was plotted (e.g. Figure 24F ). n = 3 monkeys per group, data are presented as mean ± SEM, one-way ANOVA with post hoc Dunnett test, *p < 0.05. Figure 24K Quantification of the time spent observing the cage top compartment before, 1 month after, and 2 months after prolonged L-Dopa administration and DCZ washout was plotted (e.g., Figure 24F ). n = 3 monkeys per group, data are presented as mean ± SEM, one-way ANOVA with post hoc Dunnett test, *p < 0.05. Figure 24L Quantification of dyskinesia scores 1 month and 2 months after prolonged L-Dopa administration and DCZ treatment after washout is plotted ( Figure 24F ). n = 3 monkeys per group, data are expressed as mean ± SEM.

[0058] Figure 25 is a schematic diagram showing the locations of mutations introduced at two or three sites in the AAV1 / 5 / 6 capsid protein to generate AAV1R, AAV5R, and AAV6R. This schematic is associated with a retrograde AAV tracer targeting D1-MSNs, and Figure 1C The accompanying drawings disclose SEQ ID NOS 74, 63, 55, 65-66, 57, 75 and 66, respectively, in the order of appearance.

[0059] Figures 26A-26E Depicted are the characterization of labeling specificity following intravenous delivery of AAV-PHP.eB-G88P7-EYFP. Figure 26A Includes co-staining of transduced neurons (EYFP) with parvalbumin (PV) following intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Arrows indicate double positive cells. Scale bar 50 μm. Figure 26BIncludes co-staining of transduced neurons (EYFP) with somatostatin (SST) following intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Arrows indicate double+ cells. Scale bar 50 μm. Figure 26C Included are co-staining of transduced neurons (EYFP) with ChAT following intravenous delivery of AAV-PHP.eB-G88P7-EYFP or AAVPHP.eB-hSyn-EYFP. Figure 26D Quantification of PV+, SST+, and ChAT+ cells in EYFP+ cells in the striatum following 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 Includes co-staining of transduced neurons (EYFP) with Drd1 and Drd2 following intravenous delivery of AAV-PHP.eB-G88P7-EYFP. Arrows indicate double+ cells. Scale bar 20 μm.

[0060] Figures 27A-27D Depicted are the characterization of striatonigral projection neurons following substantia nigra delivery of AAV8R12-G88P7-EYFP. Figure 27A Diagram shows retrograde labeling by stereotaxic injection of AAV8R12-G88P7-EYFP into the SNr of Drd1-Cre or Drd2-Cre mice and injection of AAV9-G88P7-DIO-tdTomato into the striatum. Figure 27B Figure 3. Retrograde labeling of striatal neurons (EYFP, green, arrows) 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 Figure 2 shows the quantification of tdT+ and tdT- cells from all EYFP+ cells in densely labeled striatal regions following 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 Figure 2 shows the quantification of tdT+ and tdT- cells from all EYFP+ cells in densely labeled striatal regions following injection of AAV8R12-G88P7-EYFP into the substantia nigra and AAV9-G88P7-DIO-tdTomato into the striatum in Drd2-Cre mice. n = 3 mice per group.

[0061] Figures 28A-28B Depicted are the percentages of retrogradely labeled neurons in the SNr and upstream brain regions by substantia nigra injection of AAV8R12-G88P3-HA-hM3Dq and AAV8R12-G88P7-rM3Ds-2A-EYFP. Figure 28A Figure 2 shows the quantification of EYFP+ cells after injection of AAV8R12-G88P3-HA-hM3Dq into the substantia nigra. n = 3 mice per group, data are presented as mean ± SEM. Figure 28B Figure 2 shows the quantification of EYFP+ cells after injection of AAV8R12-G88P7-rM3Ds-2A-EYFP into the substantia nigra. n = 3 mice per group, data are presented as mean ± SEM.

[0062] Figures 29A-29F Depicts electrophysiological recordings from striatal slices following substantia nigra injection of DREADD-expressing AAV8R12. Figure 29A The latency to the first AP after current injection before and after CNO incubation is shown. n = 5 cells from 3 mice, data are presented as mean ± SEM, two-tailed paired t-test, ***p < 0.001. Figure 29B Figure 3 shows basal activity without current injection recorded from slices prepared from mice injected with AAV8R12-G88P3-HA-hM3Dq-2A-EYFP in the substantia nigra before and after CNO incubation. n = 6 cells from 4 mice, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant. Figure 29C Figure 2. Resting membrane potential recorded from slices prepared from mice injected with AAV8R12-G88P3-HA-hM3Dq-2A-EYFP in the substantia nigra before and after CNO incubation. n = 6 cells from 4 mice, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant. Figure 29D The latency to the first AP after current injection before and after CNO incubation is shown. n = 7 cells from 5 mice, data are presented as mean ± SEM, two-tailed paired t-test, ***p < 0.001. Figure 29E Figure 3 shows basal activity without current injection recorded from slices prepared from mice injected with AAV8R12-G88P7-rM3Ds-2A-EYFP in the substantia nigra before and after CNO incubation. n = 8 cells from 7 mice, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant. Figure 29FFigure 3. Resting membrane potential recorded from slices prepared from mice injected with AAV8R12-G88P7-rM3Ds-2A-EYFP in the substantia nigra before and after CNO incubation. n = 8 cells from 7 mice, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant.

[0063] Figures 30A-30D Depicted are chemical genetic manipulations of mice injected with AAV8R12-G88P3 / G88P7-EYFP. Figure 30A Figure 2 shows the effect of CNO on rotational behavior in mice after intraperitoneal (ip) delivery of AAV8R12-G88P3-EYFP injected into the substantia nigra. n = 5 mice per group, data are presented as mean ± SEM, two-tailed unpaired t-test, ns, not significant. Figure 30B Figure 1 shows the effect of CNO on rotational behavior in mice after intracranial (ic) infusion of AAV8R12-G88P3-EYFP into the substantia nigra, followed by intracranial (ic) infusion into the dorsomedial striatum. n = 5 mice per group, data are presented as mean ± SEM, two-tailed unpaired t-test, ns, not significant. Figure 30C Figure 2 shows the effect of CNO on the rotational behavior of mice after intraperitoneal delivery of AAV8R12-G88P7-EYFP injected into the substantia nigra. n = 5 mice per group, data are presented as mean ± SEM, two-tailed unpaired t-test, ns, not significant. Figure 30D Figure 3. Effects of CNO on rotational behavior in mice following intrasubstantial nigral injection of AAV8R12-G88P7-EYFP followed by infusion into the dorsomedial striatum. n = 5 mice per group, data presented as mean ± SEM, two-tailed unpaired t-test, ns, not significant.

[0064] Figures 31A-31E Depicts an optical labeling test in striatonigral projection neurons in mice. Figure 31A Diagram 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 of C57BL / 6J mice. Figure 31B The image shows HA and EYFP staining in striatal sections. Arrows indicate cells positive for both HA and EYFP. Scale bar, 10 μm. Figure 31C Figure 3. Whole-cell patch clamp recordings from EYFP+ cells in isolated slices. Raw spike traces of a typical neuron in the striatum illuminated with blue light (473 nm). Figure 31DFigure 2 shows the electrophysiological response 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 expressed as mean ± SEM, two-tailed paired t-test, *p < 0.05. Figure 31E is a graph comparing baseline and CNO-induced action potentials.

[0065] Figures 32A-32F Depicts electrophysiological recordings from striatal slices following substantia nigra injection of AAV8R12 in Parkinson's disease mice. Figure 32A The graph shows the latency of the first AP after current injection before and after CNO incubation. n = 8 cells from 7 mice, data are expressed as mean ± SEM, two-tailed paired t-test, ****p < 0.0001. Figures 32B-32C Figure 3 shows basal activity without current injection recorded from slices prepared from parkinsonian mice that received AAV8R12-G88P7-rM3Ds-2A-EYFP injection in the substantia nigra before and after CNO incubation. Figure 32B ) and resting membrane potential ( Figure 32C ). n = 12 cells from 8 mice, data are expressed as mean ± SEM, two-tailed paired t test, ns, not significant. Figure 32D Figure 2 shows the latency of the first AP after current injection before and after CNO incubation. n = 6 cells from 3 mice, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant. Figures 32E-32F Figure 3 shows basal activity without current injection recorded from slices prepared from Parkinson's disease mice that received AAV8R12-G88P7-EYFP injection in the substantia nigra before and after CNO incubation. Figure 32E ) and resting membrane potential ( Figure 32F ). n = 7 cells from 3 mice, data are expressed as mean ± SEM, two-tailed paired t test, ns, not significant.

[0066] Figure 33A Illustration of direct BG pathway manipulation in a macaque model of Parkinson's disease by stereotaxic 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 Schematic diagram of injection sites in the SNr of a macaque monkey. A guidance grid mounted above the SN is used to obtain coordinates from MRI images and guide targeting during injection. Nine distributed sites (green dots) were selected for injection to cover as much of the SNr as possible.

[0067] Figures 34A-34DDepicted are the effects of saline administration on Parkinson's disease symptoms in rhesus monkeys. Figure 34A The figure shows the quantification of the total PD score of macaques before, 3 days, 1 week and 2 weeks after saline administration.N=4 monkeys per group, data are expressed as mean±SEM, one-way ANOVA with post hoc Dunnett test, ns, not significant. Figures 34B-34D Shown are the distance traveled in macaques after MPP+ injury and after saline administration compared to the pre-injury state ( Figure 34B )、Immobility time( Figure 34C ), and the time spent in the top compartment of the observation cage ( Figure 34D ). n = 4 monkeys per group, data are expressed as mean ± SEM, one-way ANOVA with post hoc Dunnett test, *p < 0.05, ns, not significant.

[0068] Figures 35A-35G Depicted: DCZ alone does not alter the Movement-related behaviors in macaques. Figure 35A Included are representative top-view locomotor tracings of observation cages housing unvaccinated macaques following intramuscular (im) infusion of saline or DCZ (0.3 mg / kg). Figure 35B Included are representative side view heatmaps of macaques following intramuscular infusion of saline or DCZ (0.3 mg / kg). Figure 35C Figure 2 shows the quantification of the ratio of contralateral to ipsilateral rotations after saline or DCZ (0.3 mg / kg) infusion in macaques. n = 4 monkeys per group, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant. Figure 35D Graph depicts quantification of time on the top compartment of the observation cage following infusion of saline or DCZ (0.3 mg / kg) in cynomolgus monkeys. n=4 monkeys per group, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant. Figures 35E-35F Figure 2 shows the total distance traveled after saline or DCZ (0.3 mg / kg) infusion in macaques. Figure 35E ) and immobility time ( Figure 35F ) Quantification. n = 4 monkeys per group, data are expressed as mean ± SEM, two-tailed paired t test, ns, not significant. Figure 35G Figure 3. Total activity of macaques in cages following saline or DCZ (0.3 mg / kg) infusion was categorized into low, medium, and high mobility. n = 4 monkeys per group, data are presented as mean ± SEM, two-tailed paired t-test, ns, not significant.

[0069] Figures 36A-36F Delineated the effects of D1-MSN activation on dyskinesia-like behaviors and health-state-related blood factors in parkinsonian macaques. Figure 36AThe graph shows the quantification of dyskinesia scores at 2, 4 and 8 months after treatment with DCZ. n = 3 monkeys per group. Figures 36B-36F Figure 3 Alanine aminotransferase (ALT, Figure 36B ), aspartate aminotransferase (AST, Figure 36C ), γ-glutamyl transferase (GGT, Figure 36D ), creatinine (CREA, Figure 36E ), blood urea nitrogen (BUN, Figure 36F ) Blood test results. n = 3 monkeys per group, data are expressed as mean ± SEM.

[0070] Figures 37A-37F Depicted are the effects of L-Dopa treatment on Parkinson's disease symptoms in macaques. Figure 37A Figure 3 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, two-tailed paired t-test, *p < 0.05, **p < 0.01, ns, not significant. Figure 37B Included are representative top-view movement trajectories of macaque monkeys observed in their cages. Figures 37C-37E Shown are the distance traveled in macaques after MPP+ injury and after treatment with L-Dopa compared to the pre-injury state ( Figure 37C ), the time spent on the top compartment of the observation cage ( Figure 37D ) and immobility time ( Figure 37E ). n = 3 monkeys per group, data are expressed as mean ± SEM, one-way ANOVA with post hoc Dunnett test, *p < 0.05, ns, not significant. Figure 37F The figure shows the total activity of macaques in the observation cage, which was categorized into low, medium, and high mobility. Quantification of the percentage of high mobility fractions indicates the change in activity of macaques after MPP+ injury and L-Dopa treatment compared to the pre-injury state. n = 3 monkeys per group. Data are expressed as mean ± SEM. One-way ANOVA with post hoc Dunnett's test. *p < 0.05, ns, not significant. Detailed Description of the Invention

[0072] Genetic material (e.g., transgenic or nuclease) is introduced into the nucleus using a vector, and gene therapy using viral vectors works. Viral vectors are vectors similar to viruses but do not cause viral infection, and are used to provide gene therapy to cells (e.g., mammalian cells) because viral vectors are able to pass through the cell membrane and deliver the genetic material they are loaded into the nucleus of the host cell. The host cell can then utilize the newly introduced genetic material to provide the desired therapeutic effect. This article describes promoters that can be used with viral vectors and non-viral vectors to achieve neuron-specific expression.

[0073] Adeno-associated viruses (AAVs) can be used as delivery vehicles for gene therapy because they are able to establish latent infection, whereby the AAV genome integrates into host chromosomes without stimulating a destructive T cell immune response. Different types of AAVs allow for the targeting of different cells for more precise delivery of gene therapy in vivo.

[0074] Gene therapy relies on the ability to express heterologous target genes in some way to provide the desired therapeutic effect. The present disclosure provides promoters for disease treatment methods that can enhance target gene translation. The present disclosure also provides recombinant AAV (rAAV) coupled to regulatory elements (e.g., promoters), which increases gene expression of target genes in medium spiny neurons. In one embodiment, rAAV coupled to regulatory elements (e.g., promoters) targets medium spiny neurons and is used to treat Parkinson's disease.

[0075] AAV receptors (AAVRs) are essential for AAV entry into cells. Modified AAVRs can be used to create designer receptors (DREADDs) specifically activated by designer drugs, particularly for targeting neuronal tissue. In one embodiment, DREADDs for medium spiny neurons are used to guide AAV gene therapy for Parkinson's disease.

[0076] Parkinson's disease (PD) is a common neurodegenerative disorder that affects more than 6 million people worldwide. The pathophysiology of PD can include the loss of dopaminergic neurons in the midbrain, but the cause may remain unclear. PD symptoms can be treated with the dopamine precursor levodopa (L-Dopa) or dopamine receptor agonists to restore activity in the basal ganglia (BG) motor control pathways. However, due to the widespread distribution of dopamine receptors in the brain and peripheral organs, the effects of these drugs sometimes lack specificity, which may lead to non-BG depletion of the drugs or interference with other central and peripheral dopamine systems. Therefore, there is a need to develop precision therapeutic solutions for PD that can selectively modulate the specific neuronal populations and circuits affected in PD without interfering with other dopaminergic pathways.

[0077] Efficient and precise methods for manipulating unique cell types may involve the use of genetically encoded recombinases that are specifically expressed in the cell type of interest, but this approach is generally not feasible for clinical intervention. Alternative approaches may employ promoters or enhancers of genes expressed by unique cell types to drive cell type-specific expression, but there may be only a few identified neuronal promoters that maintain endogenous gene expression specificity in rodent and primate models. Retrograde AAV tracers have been developed that may differ from conventional AAV vectors in their ability to infect neurons via axonal terminals, and recombinase-free systems for targeting and modulating specialized projection neuron types may be useful or can be constructed with any of the following components: (1) retrograde AAV that can efficiently infect axons of selected projection neurons; (2) a promoter or enhancer that drives high levels of gene expression in 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 therefore may be more applicable for clinical applications in humans. In rodents with Parkinson's disease, dopamine depletion may induce inhibition of direct pathway activity and targeted activation of striatal D1 dopamine receptor-expressing medium spiny neurons (D1-MSNs), effectively rescuing core motor symptoms. Because D1-MSNs are, in some cases, the only major cell type in the striatum projecting to the substantia nigra pars reticulata (SNr), they may represent an ideal target for implementing PD circuit-specific modulation approaches. Therefore, the present disclosure includes the development of a recombinase-free, retrograde AAV-based strategy to precisely isolate and modulate D1-MSNs and study their efficacy in reversing PD symptoms.

[0078] In the following description, certain specific details are described in order to provide a thorough understanding of the various embodiments. However, one skilled in the art will appreciate that the embodiments provided can be practiced without these details. Unless the context requires otherwise, throughout the specification and the appended claims, the word "comprise" and its various variations (e.g., "comprises" and "comprising") should be interpreted in an open and inclusive sense, i.e., as "including, but not limited to," unless the context clearly indicates otherwise. It should be noted that, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents, unless the context clearly indicates otherwise. It should also be noted that the term "or" is generally used in its sense including "and / or," unless the context clearly provides otherwise. Further, the subheadings provided herein are for convenience only and do not explain the scope or meaning of the claimed embodiments.

[0079] definition

[0080] As used herein, the term "about" refers to an amount that is approximately 10% or less of the stated amount.

[0081] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk for developing at least one disease for which the compositions and methods are useful. 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.

[0082] 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 antibodies and antibody chains provided, and other peptides (e.g., linkers and binding peptides), can comprise amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptide may contain modifications with respect to the natural or native sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

[0083] Percentage (%) sequence identity about a reference polypeptide sequence is the percentage of the amino acid residue in the candidate sequence that is identical with the amino acid residue in the reference polypeptide sequence after comparing the sequences and, if necessary, introducing a gap to reach maximum percentage sequence identity and not considering any conservative substitution as a part for sequence identity. The comparison for determining amino acid sequence identity percentage can be achieved by a variety of known means, for example, 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 for maximum alignment on the full length of the compared sequences. However, for purposes of this paper, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity percentage values. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington, D.C., 20559, together with user documentation, and it is registered as U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0084] In the case of amino acid sequence comparison using ALIGN-2, the percentage of amino acid sequence identity between a given amino acid sequence A and, or as compared to a given amino acid sequence B (which can also be alternatively expressed as a given amino acid sequence A having or comprising a certain percentage of amino acid sequence identity with, or as compared to 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 that are scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless specifically stated otherwise, all percentage amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0085] The polypeptides described herein can be encoded by nucleic acids. Nucleic acid is a polynucleotide comprising 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, such as complementary DNA (cDNA), genomic DNA (gDNA), etc.), ribonucleic acid (RNA, such as messenger RNA (mRNA), short 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., containing base analogs, sugar analogs and / or non-natural backbones, etc.), RNA / DNA hybrids and polyamide nucleic acids (PNA), all of which can be single-stranded or double-stranded forms. Unless otherwise limited, nucleic acids can include known natural nucleotide analogs, some of which can function in a manner similar to naturally occurring nucleotides. Nucleic acids can be in any form (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.) that can be used to perform the processes herein. Nucleic acid can be or can come from plasmid, phage, autonomously replicating sequence (ARS), centromere, artificial chromosome, chromosome or other nucleic acid that can be replicated or copied in vitro or in host cell, cell, the nucleus of cell or cytoplasm in certain embodiments.In some embodiments, nucleic acid can be from a single chromosome (for example, nucleic acid sample can be from a chromosome of the sample obtained from diploid organism). Nucleic acid also includes derivatives, variants and analogs of RNA or DNA synthesized, replicated or amplified from single-stranded (" sense " or " antisense ", " positive " chain or " negative " chain, " forward " reading frame or " reverse " reading frame) polynucleotide and double-stranded polynucleotide. Deoxyribonucleotide includes deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, base cytosine is replaced by uracil, and sugar 2 ' position includes hydroxyl moiety. Nucleic acid obtained from subject can be used as template to prepare nucleic acid. In some embodiments, nucleic acid is a component of the vector that can be used for transferring the polynucleotide encoding polypeptide into cell. Heterologous nucleic acid is a nucleic acid exogenous to the cell or cell group being modified. Heterologous nucleic acid can comprise a gene or nucleotide sequence modified from an endogenous gene, or can comprise a recombinant gene or nucleic acid sequence. Heterologous nucleic acid can comprise a regulatory sequence, a fusion encoding an endogenous gene, or other modifications that increase the therapeutic or diagnostic potential of a gene or nucleotide sequence.

[0086] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a genomic integration vector, or "integration vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "additional" vector, such as a nucleic acid capable of extrachromosomal replication. The vector capable of directing the expression of genes encoded by a vector is referred to herein as an "expression vector." An expression vector can appropriately initiate the expression of a target gene effectively coupled to a promoter, and such a promoter can be "universal," i.e., active in all or many different cell types (e.g., CMV promoter), or tissue or cell specific, i.e., active in a certain subset of a cell or tissue. Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc. In an expression vector, regulatory elements (e.g., promoters, enhancers, polyadenylation signals) for controlling transcription can be derived from mammals, microorganisms, viruses, or insect genes. The ability to replicate in a host and the selection gene for promoting identification of transformants, which are typically conferred by an origin of replication, can be incorporated in addition. Vectors derived from viruses such as lentivirus, retrovirus, adenovirus, adeno-associated virus, etc. can be used. Plasmid vectors can be linearized to integrate into a chromosomal location. The vector can contain sequences that direct site-specific integration into a defined position or a restricted set of sites in the genome (e.g., AttP-AttB recombination). In addition, the vector can include sequences derived from transposable elements.

[0087] As used herein, "heterologous" 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. A heterologous nucleic acid can encode a gene or RNA (e.g., antisense or siRNA) that is not normally expressed by the AAV described herein, including synthetic, mammalian, or human genes or RNAs.

[0088] As used herein, "operably coupled" refers to placing a promoter or regulatory region on an open reading frame (e.g., a gene of interest or target gene) on a nucleic acid molecule such that transcription of the open reading frame occurs. Typically, the regulatory region will be at the 5' end of the open reading frame and, therefore, may contain one or more intervening nucleotides that do not significantly inhibit transcription of the open reading frame.

[0089] Designed receptors (DREADDs) activated exclusively by designer drugs are a class of artificially engineered protein receptors for the field of chemical genetics that can be selectively activated by certain ligands. They can be used in biomedical research, such as neuroscience, to manipulate the activity of neurons. 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, Annals of Pathology and Toxicology (Annu. Rev. Pharmacol. Toxicol.) 55: 15.1-15.19 and Roth, 2016, DREADDs for Neuroscientists, Neuron 89: 683-694.

[0090] As used herein, the terms "homologous," "homology," or "percent homology" when used herein to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, revised as Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program by Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percent homology of sequences can be determined using the most recent version of BLAST as of the filing date of this application.

[0091] 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 share the same type and number of surface antigens. Serotypes may or may not differ from strains that are isolates from a single culture. Serotypes may or may not differ from genotypes, which have distinct genomes.

[0092] expression vector

[0093] The promoters described herein can be components of different types of expression vectors and used to initiate 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.

[0094] Adeno-associated virus (AAV)

[0095] AAV is a virus composed of a non-enveloped icosahedral capsid protein coat containing a linear, single-stranded DNA genome. The genomes of AAV vectors retain their packaging signals (also known as inverted terminal repeats or ITRs), but replace other viral sequences with the exogenous DNA of choice. The DNA of interest flanking the AAV ITRs is referred to as a transgene expression cassette.

[0096] The transgenic 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. Intracellular transport is carried out through endocytosis and / or proteasome compartments, followed by endosomal escape, nuclear import, virion uncoating, and viral DNA double-strand conversion, resulting in transcription and expression of the transgene. AAV vectors can be produced as described by Kimura et al. for the production of adeno-associated viral vectors for in vitro and in vivo applications (Science and Technology Report (Sci Rep 9), 12601 (2019)).

[0097] 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. AAV vectors may comprise elements from any one serotype, a mixture of serotypes, hybrids or chimeras of different serotypes, or a combination thereof.

[0098] Recombinant AAV (rAAV) is constructed from single-stranded DNA (ssDNA). These ssDNA viral vectors have high transduction rates and have the property of stimulating endogenous homologous recombination, a DNA repair mechanism that does not cause double-stranded DNA breaks in the genome. In various embodiments, the recombinant AAV vector comprises a sequence derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, Rh10, PHP.B, PHP.eB or PHP.S serotype, or a mixture, hybrid or chimera of any of the foregoing AAV serotypes. In one embodiment, the recombinant AAV vector comprises a sequence derived from AAV2. In one embodiment, the recombinant AAV vector comprises a sequence derived from AAV8. In one embodiment, the recombinant AAV vector comprises a sequence derived from AAV9. In one embodiment, the recombinant AAV vector comprises a sequence 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.

[0099] 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 and variants thereof (e.g., capsid variants such as amino acid insertions, additions and substitutions). The AAV capsid can include VP1 protein, VP2 protein and / or VP3 protein, wherein VP2 and VP3 can be amino-terminal truncations of VP1.

[0100] Retrograde infection involves viral transmission from the axon terminal to the parent neuron, in the opposite direction of the nerve impulse. Retrograde AAV (AAV-retro) can be used to study specific neuronal populations.

[0101] Regulatory elements

[0102] In one embodiment, the rAAV virion comprises a regulatory element comprising a nucleotide sequence corresponding to the genomic sequence. The regulatory element can be a nucleic acid or 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 to bind an enzyme or other factor that initiates transcription of DNA into mRNA. Alternatively, the promoter can be sufficient to bind an enzyme or other factor that initiates transcription of DNA into mRNA.

[0103] Regulatory elements can be close to genes along chromosomes. Alternatively, regulatory elements can be close to genes by spatial folding of DNA in the nucleus. Regulatory elements (e.g., promoters or enhancers) can be located near the transcription start site (TSS) of a gene. Regulatory elements can be found at the 5' end of the gene start site. Alternatively, regulatory elements can be found at the 3' end of the gene start site. Regulatory elements can be found partially or completely within the intron region of a gene. In certain embodiments, the gene is GPR88. The TSS of GPR88 is a guanine corresponding to position 1431 of SEQ ID NO:44, position 582 of SEQ ID NO:45, and position 68 of SEQ ID NO:46.

[0104] The regulatory element can be located less than about 5,000 nucleotides, less than about 4,000 nucleotides, less than about 3,000 nucleotides, less than about 2,000 nucleotides, less than about 1,000 nucleotides, less than about 900 nucleotides, less than about 800 nucleotides, less than about 700 nucleotides, less than about 600 nucleotides, less than about 500 nucleotides, less than about 400 nucleotides, less than about 300 nucleotides, less than about 200 nucleotides, or less than about 100 nucleotides 3' from the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0105] The regulatory element can be located more than about 100 nucleotides, more than about 200 nucleotides, more than about 300 nucleotides, more than about 400 nucleotides, more than about 500 nucleotides, more than about 600 nucleotides, more than about 700 nucleotides, more than about 800 nucleotides, more than about 900 nucleotides, more than about 1,000 nucleotides, more than about 2,000 nucleotides, more than about 3,000 nucleotides, more than about 4,000 nucleotides, or more than about 5,000 nucleotides 3' from the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0106] The regulatory element can be located less than about 5,000 nucleotides, less than about 4,000 nucleotides, less than about 3,000 nucleotides, less than about 2,000 nucleotides, less than about 1,000 nucleotides, less than about 900 nucleotides, less than about 800 nucleotides, less than about 700 nucleotides, less than about 600 nucleotides, less than about 500 nucleotides, less than about 400 nucleotides, less than about 300 nucleotides, less than about 200 nucleotides, or less than about 100 nucleotides 5' from the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0107] The regulatory element can be located more than about 100 nucleotides, more than about 200 nucleotides, more than about 300 nucleotides, more than about 400 nucleotides, more than about 500 nucleotides, more than about 600 nucleotides, more than about 700 nucleotides, more than about 800 nucleotides, more than about 900 nucleotides, more than about 1,000 nucleotides, more than about 2,000 nucleotides, more than about 3,000 nucleotides, more than about 4,000 nucleotides, or more than about 5,000 nucleotides 5' from the translation start site of the gene. In certain embodiments, the gene is GPR88.

[0108] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO: 39. NO:39 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:39.

[0109] The regulatory element may comprise the nucleic acid sequence shown 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, at least about 99% sequence identity to SEQ ID NO:40, at least about 100% sequence identity to SEQ ID NO:40, at least about 101% sequence identity to SEQ ID NO:40, at least about 102% sequence identity to SEQ ID NO:40, at least about 103% sequence identity to SEQ ID NO:40, at least about 104% sequence identity to SEQ ID NO:40, at least about 105% sequence identity to SEQ ID NO:40, at least about 106% sequence identity to SEQ ID NO:40, at least about 107% sequence identity to SEQ ID NO:40, at least about 108% sequence identity to SEQ ID NO:40 NO:40 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:40.

[0110] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO: 41. ID NO:41 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:41.

[0111] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO: 42. NO:42 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:42.

[0112] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO: 43. NO:43 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:43.

[0113] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO: 44. NO:44 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:44.

[0114] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO: 45. NO:45 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:45.

[0115] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO:46. NO:46 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:46.

[0116] The regulatory element may comprise the nucleic acid sequence shown 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, at least about 99% sequence identity to SEQ ID NO:48, at least about 100% sequence identity to SEQ ID NO:48, at least about 101% sequence identity to SEQ ID NO:48, at least about 102% sequence identity to SEQ ID NO:48, at least about 103% sequence identity to SEQ ID NO:48, at least about 104% sequence identity to SEQ ID NO:48, at least about 105% sequence identity to SEQ ID NO:48, at least about 106% sequence identity to SEQ ID NO:48, at least about 107% sequence identity to SEQ ID NO:48 NO:48 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:48.

[0117] The regulatory element may comprise the nucleic acid sequence shown 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, or at least about 98% sequence identity to SEQ ID NO: 47. NO:47 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:47.

[0118] 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.

[0119] The cell type specific promoter can be neuron-specific. Neuron-specific promoter is the promoter that only works in neurons to open and / or close neuron-specific genes. Neuron-specific promoter can be synaptobrevin I (SYN) promoter (for example, hSYN1), calcium / calmodulin-dependent protein kinase II (CamKII) promoter, tubulin α I, neuron-specific enolase, platelet-derived growth factor β chain promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, cerebellar Purkinje cell-specific L7-6 promoter, dopamine receptor D1 (DRD1) promoter, dopamine receptor D2 (DRD2) promoter, parvalbumin (Pvalb) promoter or no distal homeobox (Dlx) promoter.

[0120] The regulatory elements (e.g., promoters and / or enhancers) can increase the expression of a heterologous gene of interest compared to the promoter of the hSYN1 (human synapsin 1 receptor) gene in striatal neurons. The regulatory elements can increase the expression of a heterologous gene of interest 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 in striatal neurons.

[0121] The regulatory element can increase expression of the heterologous gene of interest 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 in striatal neurons.

[0122] 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% compared to the promoter of the hSYN1 gene in striatal neurons.

[0123] The regulatory element can increase expression of the heterologous gene of interest by at most or about 1,000%, at most or about 500%, at most or about 400%, at most or about 300%, at most or about 200%, at most or about 100%, at most or about 90%, at most or about 80%, at most or about 70%, at most or about 60%, at most or about 50%, at most or about 40%, at most or about 30%, at most or about 20%, at most or about 10%, at most or about 9%, at most or about 8%, at most or about 7%, at most or about 6%, at most or about 5%, at most or about 4%, at most or about 3%, at most or about 2%, or at most or about 1%, compared to the promoter of the hSYN1 gene in striatal neurons.

[0124] Regulatory elements (e.g., promoters) can be delivered to cells by methods such as, but not limited to, CRISPR knock-in methods, lentiviral methods, adenoviral methods, plasmid gene transfer methods, or cell disruption methods. Nucleic acids containing regulatory elements can be included in viral vectors. The viral vector can be an adeno-associated virus (AAV) vector.

[0125] AAV capsid polypeptide

[0126] AAV vectors have a protective protein shell called a capsid. The AAV capsid polypeptide is the primary interface between the host and the vector genome. The specificity and efficiency of AAV particle transduction depend on the AAV capsid polypeptide.

[0127] In one embodiment, the rAAV virion may comprise a variant capsid polypeptide, wherein the variant capsid polypeptide comprises a modification to an amino acid. The modification to the amino acid may comprise an amino acid addition, an amino acid deletion, an amino acid substitution, an amino acid change, or a combination thereof. The variant capsid polypeptide may comprise an amino acid change to SEQ ID NO: 1. The change may be one that allows the AAV to retrogradely transport along the axon of the neuron, thereby allowing the AAV to reach the body of the neuron, where it may allow transcription of the heterologous nucleic acid included therein. In the sequence ID, the bold and underlined portions are reverse insertions. In the sequence ID, the bold portions are capsid mutations.

[0128] In another embodiment, the variant capsid polypeptide comprises an alteration comprising an insertion into 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 positions 721 and 722 corresponding to SEQ ID NO:1, or a combination thereof.

[0129] In certain embodiments, the variant capsid polypeptide comprises amino acid changes to increase retrograde transport of rAAV virions. In some embodiments, the changes that increase rAAV retrograde transport may comprise SEQ ID NO:31. The amino acid changes that increase retrograde transport of rAAV virions can 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, at least about 99% sequence identity to SEQ ID NO: 31, at least about 100% sequence identity to SEQ ID NO: 31, at least about 101% sequence identity to SEQ ID NO: 31, at least about 102% sequence identity to SEQ ID NO: 31, at least about 103% sequence identity to SEQ ID NO: 31, at least about 104% sequence identity to SEQ ID NO: 31 NO: 31 has at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 31. The sequence identity can preserve the infectivity of the retrograde AAV of SEQ ID NO: 31.

[0130] The alteration that increases rAAV retrograde transport may comprise SEQ ID NO 32. The amino acid changes that increase retrograde transport of rAAV virions can 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, at least about 99% sequence identity to SEQ ID NO: 32, at least about 100% sequence identity to SEQ ID NO: 32, at least about 101% sequence identity to SEQ ID NO: 32, at least about 102% sequence identity to SEQ ID NO: 32, at least about 103% sequence identity to SEQ ID NO: 32 NO: 32 has at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 32. The sequence identity can preserve the infectivity of the retrograde AAV of SEQ ID NO: 32.

[0131] In certain embodiments, the sequence can correspond to the sequence identity of 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 do not completely align with the compared sequence.

[0132] 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 the retrograde transport of the rAAV control. In some embodiments, the increased retrograde transport is in medium spiny neurons. In some embodiments, the increased retrograde transport is in D1 medium spiny neurons.

[0133] In another embodiment, the variant capsid polypeptide comprises an alteration that reduces retrograde transport. Retrograde transport can 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 retrograde transport of an rAAV control.

[0134] In another embodiment, the cell can be a neural cell. Non-limiting examples of neural cells include neurons and glial cells. In one embodiment, the neural cell can be a neuron. In one embodiment, the variant capsid polypeptide comprises an alteration that increases retrograde transport along a neuronal axon, along a neuronal dendrite, through a neuronal cell body, or a combination thereof.

[0135] In some embodiments, the modified variant capsid polypeptide can increase the infectivity of rAAV virions to neurons. Non-limiting examples of neurons include excitatory neurons (e.g., dopaminergic neurons or acetylcholinergic neurons) and inhibitory neurons (e.g., GABAergic neurons or medium spiny neurons).

[0136] 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 the rAAV2-retro control. In certain embodiments, the increased infectivity is in medium spiny neurons, for example, in D1 medium spiny neurons.

[0137] Heterologous nucleic acid

[0138] 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 to guide integration into the genomic location of the cell. The cell may be a bacterial, archaebacterial, plant, fungal, or animal cell. The animal cell may be an amphibian cell, a reptile 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, cattle, or rabbit).

[0139] The heterologous nucleic acid can include a sequence comprising a regulatory element (e.g., a promoter). Alternatively or in addition, the heterologous nucleic acid sequence can include an open reading frame of a target gene. The target gene can be a non-coding region (e.g., UTR or promoter). Alternatively, the target gene can be a polypeptide. The target gene can be an endogenous gene. Alternatively, the target gene can be an exogenous gene. The target gene can have therapeutic utility (e.g., can help treat neurological diseases). The target gene can include a neurotrophic factor, an RNA-guided nuclease, an enzyme, or a DREADD.

[0140] The target gene can 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. Deficiencies in GPR88 are associated with chorea, speech delays, learning difficulties, and several neuropsychiatric disorders.

[0141] Polypeptide can be an antibody, contractile protein, enzyme, hormone protein, structural protein, storage protein, small molecule or transporter. Non-limiting examples of enzymes include hydrolases, isomerases, nucleases, ligases, transferases and oxidoreductases. In one embodiment, the target gene is a nuclease or a neurotrophic factor (e.g., brain-derived neurotrophic factor (BDNF)). The nuclease can be an RNA-guided nuclease. The RNA-guided nuclease can be a programmable endonuclease (e.g., Cas or Cas9), which can be used to perform targeted genome editing. The programmable endonuclease can interact with the guide RNA to form CRISPR / Cas or CRISPR / Cas9 complexes. In certain embodiments, the target gene can be a DREADD (e.g., hM3Dq, hM1Dq, hMD5q, hM4Di or hM2Di).

[0142] Methods of gene expression and delivery to the CNS

[0143] Gene therapy is a technique for modifying a subject's genes to treat or cure a disease. Gene therapy can work through a variety of mechanisms, including, but not limited to, replacing a disease-causing gene with a healthy copy, inactivating a malfunctioning disease-causing gene, or introducing a new or modified gene into the body to help treat the disease. Gene therapy can introduce genetic material into cells using plasmid DNA, viral vectors, bacterial vectors, gene editing technologies, or patient-derived cell-based gene therapy products.

[0144] Gene therapy can be delivered to a subject by intravenous (IV) injection, oral administration, intramuscular injection, subcutaneous injection, intrathecal treatment, rectal administration, vaginal administration, or inhalation.

[0145] The nervous system is divided into two main parts: the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS is the processing center of the body and is composed of the brain and spinal cord. Accessing the CNS may require therapies that bypass the blood-brain barrier. Strategies for delivering therapies to the CNS include, but are not limited to, intra-arterial chemotherapy, direct injection of therapeutic substances into intracranial lesions, and drug delivery using nanoparticles.

[0146] In certain embodiments, the rAAV virions of the present disclosure can be injected into a subject. In certain embodiments, the injection can be administered directly into the brain. In some embodiments, the pharmaceutical composition is injected directly into the striatum, which is a nucleus in the basal ganglia beneath the forebrain cortex.

[0147] In some embodiments, the injected gene can target a specific type of neuron or group of neurons in the brain. In some embodiments, the neuron can be a neuron of the striatum. In some embodiments, the neuron can be a medium spiny neuron. In some embodiments, the neuron can be a dopaminergic medium spiny neuron. In some embodiments, the dopaminergic medium spiny neuron is a D1 dopaminergic medium spiny neuron.

[0148] Injections can be given using stereotactic surgery or intracerebral injection. Stereotactic surgery is a minimally invasive form of surgical intervention that uses a three-dimensional coordinate system to locate small targets in the body and perform actions on them, such as ablation, biopsy, lesion, injection, stimulation, implantation, and radiosurgery (SRS). Intracerebral injection, such as intraventricular injection, is an invasive injection technique that injects substances directly into the cerebrospinal fluid of the brain ventricles to bypass the blood-brain barrier.

[0149] In some embodiments, the gene therapy of the present disclosure is used to genetically modify 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 can be used to target and treat neurodegenerative diseases, such as Parkinson's disease. In certain embodiments, described herein is a method for preparing a Parkinson's disease treatment comprising administering one or more pharmaceutically acceptable excipients, carriers or diluents and the rAAV virions of the present disclosure.

[0150] Parkinson's disease (PD) is a progressive neurological disorder that affects movement. Symptoms begin gradually, sometimes starting with a barely noticeable tremor in just one hand. Tremor is common, but the disease often also causes stiffness or slowed movements. Symptoms of PD include tremor, bradykinesia, muscle rigidity, impaired posture and balance, loss of automatic movements, or changes in speech.

[0151] Tremor, also known as shaking, is an involuntary, rhythmic muscle contraction that causes shaking movements in one or more parts of the body. The PD treatment methods disclosed herein can reduce a patient's tremor by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0152] Bradykinesia is an impairment in voluntary motor control and slow movements or freezing. Bradykinesia may manifest as a decrease in automatic movements, such as blinking or swinging the arms while walking, or may manifest as difficulty initiating intentional movements or simply slow movements. The PD treatment methods disclosed herein can reduce a patient's bradykinesia by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0153] Muscle stiffness, also known as muscle tightness, stiffness, or rigidity, is characterized by the inability of muscles to relax normally. This condition can affect any muscle in the body, causing severe pain and difficulty moving. The disclosed PD treatment methods can reduce a patient's muscle stiffness by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0154] Postural instability or impaired posture and / or balance is the inability to maintain balance under dynamic and static conditions, such as preparatory movements, disturbances, and resting postures. Postural instability can manifest as a tendency to become unstable while standing. Postural instability can manifest as a tendency to fall or an inability to prevent oneself from falling. The PD treatment methods disclosed herein can reduce a patient's postural instability by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0155] Automatic movements are movements that people often make without conscious awareness (e.g., blinking or swinging their arms while walking). The disclosed PD treatment methods can reduce the patient's automatic movement loss by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0156] Speech changes can include fluency disorders (unusual repetition of sounds or rhythms), voice disorders (atypical intonation), or articulation disorders (distortion of certain sounds). The PD treatment methods disclosed herein can reduce the patient's speech pattern changes by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0157] 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 administering a retrograde AAV or a pharmaceutical composition and a ligand that activates a DREADD to the individual, thereby activating the DREADD in the individual's central nervous system. 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 individual's central nervous system treats a neurodegenerative disease. In some embodiments, the neurodegenerative disease 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 retrograde AAV and the ligand that activates the DREADD are administered separately.

[0158] Designer receptors specifically activated by designer drugs (DREADDs)

[0159] The rAAV virion can contain a designer receptor specifically activated by a designer drug (DREADD) as a target gene. DREADDs, also known as receptors activated only by synthetic ligands, are a class of artificially engineered protein receptors that can be selectively activated by certain ligands. The DREADD can be rM3D.

[0160] The DREADD may comprise the amino acid sequence shown in SEQ ID NO:38. The variant capsid polypeptide may 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, or at least about 98% sequence identity to SEQ ID NO: 38. NO:38 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:38.

[0161] The DREADD may be HM3Ds. The DREADD may comprise the amino acid sequence shown. 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, or at least about 98% sequence identity to SEQ ID NO: 49. ID NO:49 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:49.

[0162] The DREADD used in the methods and systems described herein can be HM3Ds (A147S-F349Y). The DREADD can comprise the amino acid sequence shown 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, at least about 99% sequence identity to SEQ ID NO:50, at least about 100% sequence identity to SEQ ID NO:50, at least about 101% sequence identity to SEQ ID NO:50, at least about 102% sequence identity to SEQ ID NO:50, at least about 103% sequence identity to SEQ ID NO:50, at least about 104% sequence identity to SEQ ID NO:50, at least about 105% sequence identity to SEQ ID NO:50, at least about 106% sequence identity to SEQ ID NO:50, at least about 107% sequence identity to SEQ ID NO:50 NO:50 has at least about 98% sequence identity, or has at least about 99% sequence identity to SEQ ID NO:50.

[0163] DREADDs can be used with certain ligands that result in activation of the DREADD and the desired physiological effect. 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 retrograde AAV encoding the DREADD. In the case of FDA or EMA approved drugs, the DREADD ligand can be administered at an approved dose or a dose lower than the approved dose. In the case of FDA or EMA approved drugs, the DREADD ligand can be administered on a schedule that is the same as or different from the approved schedule.

[0164] Pharmaceutical composition

[0165] In certain embodiments, the rAAV virions of the present disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, stabilizers, dispersants, suspending agents, thickeners and / or diluents. The pharmaceutical composition facilitates the administration of the compound to an organism. The pharmaceutical composition can be administered as a pharmaceutical composition in a therapeutically effective amount by various forms and routes, including, for example, intravenous, subcutaneous, intramuscular, inhalation, oral, parenteral, ocular, otic, subcutaneous, transdermal, nasal, intravitreal, intratracheal, intrapulmonary, transmucosal, vaginal, and topical administration.

[0166] The formulation can be modified depending on the route of administration chosen.Pharmaceutical compositions containing the compounds described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, embedding or compression processes.

[0167] Pharmaceutical compositions can be formulated by combining active compounds with pharmaceutically acceptable carriers or excipients. Non-limiting examples of pharmaceutically acceptable excipients suitable for methods disclosed herein include granulating agents, binding agents, lubricants, disintegrants, sweeteners, glidants, antiadhesives, antistatic agents, surfactants, antioxidants, glues, coatings, colorants, flavorings, coatings, plasticizers, preservatives, suspending agents, emulsifiers, antimicrobials, plant cellulose materials and spheroidizing agents and any combination thereof. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, normal saline, glycerol and ethanol. Pharmaceutically acceptable salts such as inorganic acid salts (such as hydrochlorides, bromates, phosphates, sulfates, etc.) and organic acid salts (such as acetates, propionates, malonates, benzoates, etc.) may be included in the pharmaceutical composition. In addition, reinforcing materials such as wetting agents or emulsifiers, pH buffer materials, etc. may be present in these carriers. A variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients 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) HC Ansel et al., eds., 7th edition, Lippincott Williams & Wilkins; and Handbook of Pharmaceutical Excipients. Pharmaceutical Excipients) (2000) edited by AH Kibbe et al., 3rd edition, USA.

[0168] 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 the compounds disclosed herein, wherein the matrices are in the form of shaped articles, such as films, liposomes, microparticles, and microcapsules.

[0169] Carbomers in aqueous pharmaceutical compositions serve as emulsifiers and viscosity modifiers. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of 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. Cyclodextrins in aqueous pharmaceutical compositions serve as solubilizers and stabilizers. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of cyclodextrin. In certain embodiments, the cyclodextrin comprises or consists of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a combination thereof. Lecithin in pharmaceutical compositions serves as a solubilizer. In certain embodiments, the solubilizer comprises or consists of lecithin. Poloxamer in pharmaceutical compositions serves as an emulsifier, solubilizer, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises or consists of poloxamer. In certain embodiments, the poloxamer comprises poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or a combination thereof, or consists of the foregoing. The polyoxyethylene sorbitan fatty acid ester in the pharmaceutical composition functions 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, or consists of the foregoing. The polyoxyethylene stearate in the pharmaceutical composition functions as an emulsifier, solubilizer, surfactant, and dispersant. In certain embodiments, the pharmaceutically acceptable excipient comprises polyoxyethylene stearate or is composed of it. In certain embodiments, polyoxyethylene stearate comprises polyethylene glycol stearate 2, polyethylene glycol stearate 4, polyethylene glycol stearate 6, polyethylene glycol stearate 8, polyethylene glycol stearate 12, polyethylene glycol stearate 20, polyethylene glycol stearate 30, polyethylene glycol stearate 40, polyethylene glycol stearate 50, polyethylene glycol stearate 100, polyethylene glycol stearate 150, polyethylene glycol distearate 4, polyethylene glycol distearate 8, polyethylene glycol distearate 12, polyethylene glycol distearate 32, polyethylene glycol distearate 150 or a combination thereof, or is composed of the above. The sorbitan ester in the pharmaceutical composition is used 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 sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan, sorbitan trioleate, sorbitan sesquioleate, or a combination thereof, or consists of the foregoing. In certain embodiments, solubility can be achieved with a protein carrier. In certain embodiments, the protein carrier comprises albumin, human albumin.

[0170] In certain embodiments, the polypeptide can be stabilized by polysaccharide uronides. In certain embodiments, the stabilizer comprises or consists of a polysaccharide uronide. In certain embodiments, the polysaccharide uronide comprises or consists of calcium alginate.

[0171] In certain embodiments, the rAAV virions of the present disclosure are suspended in a sterile solution for administration. In certain embodiments, the solution comprises about 0.9% sodium chloride (NaCl). In certain embodiments, the solution comprises about 5.0% glucose. In certain embodiments, the solution further comprises one or more of the following: a buffer, 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; polyols / disaccharides / polysaccharides, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, such as glycine or arginine; antioxidants, such as ascorbic acid, methionine; or chelating agents, such as EDTA or EGTA.

[0172] In certain embodiments, the rAAV virions of the present disclosure are freeze-dried for transport / storage and reconstituted prior to administration. In certain embodiments, the freeze-dried rAAV virion formulation comprises a filler such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The freeze-dried formulation can be contained in a vial made of glass or other suitable non-reactive material. The rAAV virions, when formulated, whether or not reconstituted, can be buffered at a certain pH value, which is typically less than 7.0. In certain embodiments, the pH can 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.

[0173] The pharmaceutical composition can be used in a local or systemic manner, for example, via direct injection of the compound into an organ, optionally using a depot or sustained-release formulation or implant. The pharmaceutical composition can be provided in the form of a quick-release formulation, in the form of an extended-release formulation, or in the form of an intermediate-release formulation. Quick-release forms can provide immediate release. Sustained-release formulations can provide controlled release or continue to delay release.

[0174] In practicing the methods of treatment or uses provided herein, a therapeutically effective amount of a compound described herein is administered in the form of a pharmaceutical composition to a subject suffering from the disease or condition to be treated. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. The compound can be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0175] In some embodiments, the drug is administered to an animal, including but not limited to vertebrates such as mammals, birds or fish. The animal can be a human or cattle, canine, caprine, cervidae, cricoid, feline, galliformes, equine, lagomorph, murine, mustelid and ovine. The animal can be a human or other mammal, including primates (e.g., monkeys), bovine (e.g., cattle or cows), porcine (e.g., pigs or pigs), ovine (e.g., goats or sheep), equine (e.g., horses), canine (e.g., dogs), feline (e.g., domestic cat), antelope, buffalo, camel, cervidae (e.g., deer), donkey, rabbit and rodent (e.g., guinea pig, squirrel, rat, mouse, gerbil and hamster). In some embodiments, the drug is administered to a human.

[0176] The pharmaceutically acceptable excipient may be present in the pharmaceutical composition at about 0.1% to about 99% by mass of the composition. For example, a pharmaceutically acceptable excipient can be present in the pharmaceutical composition at about 0.1% to about 95%, about 0.1% to about 90%, about 0.1% to about 85%, about 0.1% to about 80%, about 0.1% to about 75%, about 0.1% to about 70%, about 0.1% to about 65%, about 0.1% to about 60%, about 0.1% to about 55%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, or about 0.1% to about 1% by mass of the formulation.

[0177] The pharmaceutically acceptable excipients can be present in an amount of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 9%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about %, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81% , about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or about 99.9% are present.

[0178] Numbered implementation plan

[0179] The following embodiments are disclosed herein:

[0180] 1. A nucleic acid comprising a heterologous gene of interest operably coupled 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 an endogenous GPR88 gene.

[0181] 2. The nucleic acid of embodiment 1, wherein the genomic sequence located 3' to the translation start site of the endogenous GPR88 gene is partially located in an intron.

[0182] 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 in an intron.

[0183] 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' from the translation start site of the endogenous GPR88 gene.

[0184] 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%, or 99% homologous to the nucleotide sequence shown in SEQ ID NO:39.

[0185] 6. The nucleic acid of embodiment 4, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO: 39.

[0186] 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' from the translation start site of the endogenous GPR88 gene.

[0187] 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 shown in SEQ ID NO:40.

[0188] 9. The nucleic acid of embodiment 7, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO:40.

[0189] 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.

[0190] 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.

[0191] 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%, 99% homologous to the nucleotide sequence shown in SEQ ID NO:41.

[0192] 13. The nucleic acid of embodiment 11, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO:41.

[0193] 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.

[0194] 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%, 99% homologous to the nucleotide sequence shown in SEQ ID NO:42.

[0195] 16. The nucleic acid of embodiment 14, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO: 42.

[0196] 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.

[0197] 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%, 99% homologous to the nucleotide sequence shown in SEQ ID NO:43.

[0198] 19. The nucleic acid of embodiment 17, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO: 43.

[0199] 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%, 99% homologous to the nucleotide sequence shown in any one of SEQ ID NOs: 44, 45, or 46.

[0200] 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 shown in any one of SEQ ID NOs: 44, 45, or 46.

[0201] 22. A nucleic acid comprising a heterologous gene of interest operably coupled 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 shown in SEQ ID NO:47.

[0202] 23. The nucleic acid of any one of embodiments 1 to 22, wherein the heterologous gene of interest is 3' to the regulatory element.

[0203] 24. The nucleic acid of any one of embodiments 1 to 23, wherein the heterologous gene of interest has therapeutic utility.

[0204] 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.

[0205] 26. The nucleic acid according to any one of embodiments 1 to 25, wherein said nucleic acid exhibits increased expression of said heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

[0206] 27. The nucleic acid of any one of embodiments 1 to 26, wherein the nucleic acid is comprised in a viral vector.

[0207] 28. The nucleic acid of embodiment 27, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0208] 29. The nucleic acid of embodiment 28, wherein the viral vector is a retrograde AAV (AAVretro) virion.

[0209] 30. The nucleic acid of any one of embodiments 1 to 29, wherein said gene of interest exhibits at least 2-fold higher expression of said heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

[0210] 31. The nucleic acid of any one of embodiments 1 to 29, wherein said gene of interest exhibits at least 2-fold higher expression of said heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

[0211] 32. The nucleic acid of any one of embodiments 1 to 29, wherein said gene of interest exhibits at least 2-fold higher expression of said heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

[0212] 33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent and the nucleic acid according to any one of embodiments 1 to 29.

[0213] 34. The nucleic acid of any one of embodiments 1 to 29 or the pharmaceutical composition of embodiment 33, for use in a method for expressing a polypeptide in neurons of the striatum.

[0214] 35. The use according to embodiment 34, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

[0215] 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 engineering neurons of the striatum.

[0216] 37. The use according to embodiment 36, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

[0217] 38. The nucleic acid of any one of embodiments 1 to 29 or the pharmaceutical composition of embodiment 33 for use in a method of treating a neurodegenerative disease in a subject.

[0218] 39. The use according to embodiment 38, wherein the neurodegenerative disease comprises Parkinson's disease.

[0219] 40. A method of expressing a polypeptide in neurons of the striatum of an individual, comprising administering to the individual a nucleic acid according to any one of embodiments 1 to 29 or a pharmaceutical composition according to embodiment 33, thereby expressing the polypeptide in neurons of the striatum.

[0220] 41. A method according to embodiment 40, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

[0221] 42. A method for genetically modifying neurons in the striatum of an individual, comprising administering to the individual a nucleic acid according to any one of embodiments 1 to 29 or a pharmaceutical composition according to embodiment 33, thereby genetically modifying the neurons in the striatum.

[0222] 43. A method according to embodiment 42, wherein the neurons of the striatum are D1 dopaminergic medium spiny neurons.

[0223] 44. A method of treating an individual suffering from a neurodegenerative disease, comprising administering a nucleic acid according to any one of embodiments 1 to 29 or a pharmaceutical composition according to embodiment 33 to the individual suffering from the neurodegenerative disease, thereby treating the neurodegenerative disease.

[0224] 45. The method of embodiment 44, wherein the neurodegenerative disease comprises Parkinson's disease.

[0225] 46. ​​The method of any one of embodiments 40 to 45, wherein the individual is a mammal.

[0226] 47. The method of any one of embodiments 40 to 45, wherein the individual is a human. Example

[0227] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0228] Example 1 - Development of highly efficient retrograde AAV capsids for D1 MSNs

[0229] In this study, a highly efficient retrograde adeno-associated virus (AAV) capsid, AAV8R, was developed for use in D1 medium spiny neurons (MSNs).

[0230] The standard retrograde AAV tracer, rAAV2-retro, was only moderately efficient in injecting D1 MSNs when injected into the substantia nigra pars reticulata (SNr), and striatal MSNs were only sparsely labeled by rAAV2-retro-hSyn-EYFP. To improve efficiency, multiple rounds of AAV capsid mutagenesis were performed on a range of different serotypes. Mutations were introduced at three sites of the AAV8 capsid protein to generate AAV8R, including 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 combination of AAV8R12 capsid and G88 promoter was determined. After injection of AAV8R12-G88P7-EYFP into the substantia nigra of mice, retrograde labeling of neurons in the SNr and its upstream brain regions revealed 97.68±0.43% labeled neurons in the striatum, while only 1.14±0.16% and 1.18±0.41% labeled neurons were found in the SNr and its other upstream brain regions, respectively ( Figure 1D and Figure 1E ; n = 3 mice per group).

[0231] First, insert the peptide fragment at position N587-R588 Two additional point mutations in the AAV2 Cap protein were used to develop rAAV2-retro. These similar point mutations were then placed at equivalent positions in the other four AAV serotypes ( Figures 1A-1C and 25, Table 1), AAV1 / 5 / 6 / 8 ( Figure 1AAll four modified AAVs maintained brain infectivity, but only the AAV8 mutant, AAV8R, achieved improved retrograde infectivity of D1 MSNs. When administered to the SNr, 4.86 ± 0.22-fold more EYFP-positive MSNs were observed in AAV8R-hSyn-EYFP-infected mice compared to rAAV2-retro-hSyn-EYFP-infected mice ( Figures 2A-2C ).

[0232] The baseline infectivity of rAAV2-retro was approximately 0.2 ± 0.03 × 10 per striatal hemisphere. 4 cells (using the hSyn promoter) (Table 2). Of the 14 mutants tested, AAV8R12 showed the most improved efficiency. Compared with the current standard retrograde AAV, rAAV2-retro, AAV8R12 labeled 7.72 ± 0.78 times more MSNs after substantia nigra delivery ( Figures 2B-2C , Table 1). Furthermore, robust labeling of MSNs by AAV8R and AAV8R12 was observed in the nucleus accumbens following stereotactic delivery to the ventral pallidum or lateral hypothalamus, demonstrating the unique ability of the newly developed AAV capsid to infect basal ganglia MSN axons. AAV8R12 was generated using the AAV8R plasmid as a template.

[0233] The sequence of the mutagenic primer used to introduce the V183E mutation was 5′-TGGCGACTCAGAGTCAGAGCCAGACCCTCAACCTCT-3′ (SEQ ID NO.: 34). The sequence of the mutagenic primer used to introduce the N411S mutation was 5′-TGCTGAGAACCGGCAACAGCTTCCAGTTTACTTACACCT-3′ (SEQ ID NO.: 35).

[0234] Table 1: List of AAV8R mutants and their labeling efficiency for striatal neurons

[0235] name mutation Labeling efficiency in the striatonigral pathway AAV8R1 V183E + AAV8R2 N411S + AAV8R3 Y447F + AAV8R4 R490Q + AAV8R5 V125I + AAV8R6 F536Y + AAV8R7 A606S + AAV8R8 T495A + AAV8R9 V125I+F536Y + AAV8R10 V125I+A606S + AAV8R11 V125I+T495A + AAV8R12 V183E+N411S +++ AAV8R13 V125I+F536Y+T495A ++ AAV8R14 V125I+A606S+T495A ++

[0236] This table shows mutations in 15 AAV8R capsid mutants. "+," "++," and "+++" indicate <5k, 5k-10k, and 10k-25k labeled neurons per animal, respectively.

[0237] Table 2: Means and standard deviations of AAVs with capsids and promoters

[0238] AAV mean SD SEM AAV2R-hsyn 2078 454.5 262.4 AAV8R-hsyn 10092 803.5 463.9 AAV8R12-hsyn 16026 2865 1654 AAV8R12-ef1a 6954 1210 698.7 AAV8R12-cag 4610 1226 707.7 AAV8R12-G88P2 36290 6417 3705 AAV8R12-G88P3 37026 11511 6646 AAV8R12-G88P7 39460 12611 7281

[0239] Example 2—Development of a robust promoter for D1 MSNs

[0240] To find promoters that provide high levels of expression in MSNs, we scanned gene expression databases to identify a list of eight genes that were highly enriched in striatal expression compared to other parts of the basal ganglia (BG). We first selected genes that were highly expressed in the striatum but not other parts of the basal ganglia as candidate genes.

[0241] The researchers examined brain maps of two epigenetic marks at enhancers and promoters, monomethylation of histone H3 lysine 4 (H3K4me1) and acetylation of histone H3 lysine 27 (H3K27ac), and identified approximately 2 kbp-long sequences around the transcription start site (TSS) in the mouse brain that had high levels of H3K4me1 and / or H3K27ac. They then cloned homologous sequences at equivalent locations in the human genome onto an AAV backbone and tested their activity in directing reporter gene expression in mice following AAV injection into the SNr. Figure 3A -C, Table 3).

[0242] Among the 11 promoters tested, the 2259-bp promoter from the gene GPR88, neural promoter 1 (G88P2), showed the highest activity in driving gene expression in MSNs compared with 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, and two derivatives, G88P3 and G88P7, were prepared, which were 1395-bp and 896-bp long, respectively ( Figures 3A-3C Comparative efficiency of MSN labeling by the two shortened promoters was observed. For mice receiving viral injection into the SNr, viruses expressing EYFP driven by the G88P3 and G88P7 promoters labeled 3.7 ± 0.66 × 104 and 3.95 ± 0.73 × 104 MSNs per mouse, respectively, while the hSyn promoter labeled 1.61 ± 0.16 × 104 MSNs ( Figure 4B The shortest of these strong MSN promoters, G88P7, consists of a short 67 bp sequence preceding the TSS of the human GPR88 gene, exon 1 (366 bp), intron 1 (391 bp), and a 72 bp fragment of exon 2, suggesting that possible cis-regulatory elements in exon 1 and intron 1 of the GPR88 gene are sufficient to initiate strong striatal expression.

[0243] Table 3: List of AAv8R mutants and their labeling efficiency on striatal neurons

[0244]

[0245]

[0246] This table shows the location, size, and activity level of the tested promoters. "+," "++," "+++," and "++++" indicate the number of YFP-expressing neurons per animal with <5k, 5k-10k, 10k-25k, and >25k, respectively. SN denotes substantia nigra; STR denotes striatum.

[0247] Consistent with the high level of Gpr88 gene expression only in MSNs but not in other striatal cell types in the mouse striatum, after intravenous delivery of AAV-PHP.eB-G88P7-EYFP, Figures 26A-26E Co-staining of EYFP with Drd1 and Drd2, but not with ChAT, parvalbumin, or somatostatin, was observed.

[0248] The labeling specificity of the novel retrograde AAV tracer was examined in mice. Following intranigral injection of AAV8R12-G88P7-EYFP in mice, dual labeling with EYFP and Drd1 or Drd2 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 with simultaneous nigral injections of AAV8R12-G88P7-EYFP and striatal injections of AAV9-G88P7-DIO-tdTomato in Drd1-Cre and Drd2-Cre mice confirmed that a very small fraction (<3%) of SNr-targeted MSNs labeled by AAV8R12 in Drd2-Cre mice were tdTomato+ ( Figures 27A-27D Using this AAV-based retrograde labeling approach, we found that 12.44 ± 2.3% of D1-MSNs in mice were reporter gene positive.

[0249] Example 3 - Chemical genetic manipulation of the mouse basal ganglia direct pathway

[0250] Mice were anesthetized with sodium pentobarbital (Nembutal; 80 mg / kg, ip) and placed in a stereotactic apparatus (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) using a 10 μL syringe connected to a 33-gauge needle (Neuros; Hamilton). 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 to bregma, 1.3 mm lateral to bregma, and 4.8 mm ventral to bregma.

[0251] Then, a guide cannula (KOPF) was implanted in the mice used in the behavioral experiments, which was unilaterally aimed at the dorsomedial striatum (0.5 mm anterior to the bregma, 1.5 mm lateral to the bregma, and 3.5 mm ventral to the 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 infusion was performed. The mice were allowed to recover from surgery for at least 3 weeks before further study.

[0252] The labeling specificity of retrograde AAV tracers was examined. Dual labeling with AAV8R12-G88P3-EYFP and Drd1 or Drd2 in mice showed that retrograde AAV only labeled D1 MSNs ( Figure 5A To explore the functional characteristics of retrograde AAV-labeled cells, AAV8R12-G88P3-HA-hM3Dq, which is capable of stimulating neurons 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, compared with saline controls, increased ipsilateral rotations and decreased contralateral rotations were observed ( Figure 6A This result suggests that the basal ganglia motor control pathway is inhibited, which is contrary to the predicted action of activated D1MSNs in the right striatum. Based on the observed retrograde AAV infection pattern, we inferred that the ipsilateral rotation was the result of activation of the right SNr, which is the major inhibitory output center of the basal ganglia and was labeled by the locally injected AAV.

[0253] Immunohistochemical analysis confirmed that c-Fos+ cells in the right SNr were significantly increased after CNO administration, not after saline administration ( Figure 6B ). Then, after AAV delivery to the right SNr, CNO or saline was administered intracranially near the dorsomedial portion of the right striatum. Contralateral rotations were induced by CNO but not by saline ( Figure 6A No significant changes in the number of c-Fos+ cells in the substantia nigra were observed after intracranial CNO infusion ( Figure 6BFurthermore, CNO did not induce rotational behavior in animals that received AAV8R12-G88P3-EYFP infusion into the SNr ( Figure 6C These results show that pharmacogenetic activation of retrogradely labeled D1 MSNs via the proposed strategy is sufficient to drive behavioral changes in mice.

[0254] To explore the functional characteristics of retrograde AAV-labeled cells, AAV8R12-G88P3-HA-hM3Dq, which expresses the DREADD effector hM3Dq and can enable neuronal excitation after clozapine N-oxide (CNO) administration, was unilaterally injected into the SNr of C57BL / 6J mice. Three weeks after injection, brains were collected and anatomical analysis revealed that most labeled neurons were located in the striatum and all labeled neurons were Drd1+ ( Figures 6D-6F Using slice whole-cell voltage-clamp recordings, we found enhanced excitability in AAV8R12-G88P3-HA-hM3Dq-2A-EYFP-transduced MSNs after CNO administration, but without affecting basal firing rate or resting membrane potential ( Figure 6G , 29A-29C). Surprisingly, a decrease in contralateral rotation was observed after intraperitoneal CNO delivery compared to saline injection ( Figure 6H This result suggests inhibition of the direct pathway or excitation of the indirect pathway, contrary to the prediction of unilateral activation of D1-MSNs in the striatum. Based on the observed infection pattern of retrograde AAV, it is speculated that ipsilateral rotation may be the result of activation of the ipsilateral SNr, which may be the main inhibitory output center of the BG and is mildly 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 but not saline administration ( Figure 6H To further explore this observation, CNO or saline was administered intracranially near the dorsomedial region of the ipsilateral striatum after unilateral AAV delivery. By this approach, CNO, but not saline, induced contralateral rotations, and no significant changes in the number of c-Fos+ cells in the substantia nigra were observed ( Figure 6I Furthermore, CNO did not induce behavioral changes in animals that received AAV8R12-G88P3-EYFP injections into the SNr ( Figures 30A-30B These results demonstrate that chemogenetic activation of D1-MSNs labeled by a newly developed retrograde AAV could drive behavioral changes in mice.

[0255] To further optimize the system, it is necessary to identify an alternative chemogenetic effector that might allow specific manipulation of the direct pathway compatible with systemic CNO infusion. Commonly used Gq-coupled effectors cause an increase in intracellular Ca2+ concentration, which has been shown to be more effective than other second messengers in driving neuronal firing in various neuronal subtypes. A different chemogenetic effector, rM3D, which uses cAMP as a second messenger and can effectively activate striatal MSNs, was used to avoid activation of SNr neurons after systemic CNO administration.

[0256] To test this, AAV8R12-G88P7-rM3Ds-2A-EYFP was injected unilaterally into the right SNr of adult mice, and both intraperitoneal and intracranial infusions of CNO induced contralateral rotations ( Figure 7A Immunohistochemistry and in situ hybridization analysis confirmed that the majority of labeled neurons were located in the striatum and that the retrogradely transduced neurons were Drd1+ ( Figures 7D-7F , 28B). Slice electrophysiological recordings demonstrated that CNO administration enhanced the excitability of labeled striatal MSNs but had no effect on basal firing rate or resting membrane potential ( Figure 7G , 29D-29F). After intraperitoneal CNO administration, CNO did not increase the number of c-Fos+ cells in the SNr ( Figure 7B Both intraperitoneal and intracranial infusions of CNO induced contralateral rotation and did not increase the number of c-Fos+ cells in the SNr ( Figures 7H-7I Furthermore, CNO did not induce rotational behavior in animals that received AAV8R12-G88P7-EYFP infusion into the SNr ( Figure 7C , 30C-30D). These results demonstrate the specificity of rM3D in activating retrogradely labeled MSNs rather than activating substantia nigra neurons at the injection site. To assess the durability of the approach, mice that received AAV8R12-G88P7-rM3Ds-2A-EYFP infusion into the substantia nigra 12 months after the initial viral infection were tested and sustained elevated contralateral rotations were found (Figures 7J-7I, 9D-9E). Taken together, these findings confirm that the approach we developed is a durable solution for selectively modulating the activity of D1-MSN and BG direct pathways.

[0257] The results of this example demonstrate that the constructed toolkit (including the highly efficient designer retrograde AAV tracer AAV8R12, the strong striatal promoter G88P3 / 3, and the chemogenetic effectors rM3D or hM3Dq) comprises a recombinase-free system that can selectively isolate neuronal subtypes for functional interrogation.

[0258] Example 4 - Chemogenetic manipulation of the macaque basal ganglia direct pathway

[0259] Although chemical genetic manipulation of neuronal activity does not always target specific neural circuits, it may be effective in the macaque brain. To test the effectiveness of the BG direct pathway circuit modulation approach in primate models, AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP was unilaterally injected into the SNr of macaques. Anatomical analysis showed that most labeled neurons were present in the caudate nucleus and putamen, and all striatal DREADD+ neurons were DRD1+ ( Figures 10I-10P Electrophysiological recordings in anesthetized animals confirmed increased neuronal activity in the caudate / putamen following CNO, but not saline, infusion ( Figures 12K-12N To directly assess how rM3D expression in D1-MSNs affects their activity, AAV8R12-G88P7-HA-rM3Ds-2A-Cre and AAV9-EF1α-DIO-ChR2-EYFP injections were performed simultaneously in mice and macaques. Immunohistochemical analysis and slice recordings in mice confirmed the co-expression of ChR2 and rM3D in striatal nigral projection neurons, and the labeled neurons were activated by light (473 nm) and CNO ( Figures 31A-31E In vivo photolabeling recordings in anesthetized macaques revealed that CNO effectively induced an increase in neuronal activity in retrogradely labeled D1-MSNs ( Figures 12G-12J ).

[0260] In behavioral testing, CNO administered intracranially to the dorsomedial caudate nucleus or systemically to monkeys receiving hM3Dq or rM3Ds effectors, respectively, induced a dramatic increase in contralateral rotations ( Figure 10B 、 10Q After CNO treatment, it was observed that the time the animals spent in the top compartment of the observation cage was significantly reduced, and the speed of the counter-rotation was increased ( Figures 8A-8B , 10T-10W). No significant differences were found for the speed of ipsilateral rotation, total distance traveled, or immobility time ( Figures 8C-8H ) and CNO did not induce significant behavioral changes in unvaccinated monkeys that did not receive virus injection ( Figures 11A-11H Together, these results clearly demonstrate that the toolkit we developed can precisely isolate and efficiently activate direct pathway projection neurons in primates.

[0261] To test the effectiveness of the newly developed retrograde tool system in a primate model, AAV8R12-G88P3-mCherry was injected into the SNr of cynomolgus macaques (Macaca fascicularis). Because the cynomolgus macaque brain is approximately 180 times larger than the mouse brain, the injection was delivered in a grid of nine points covering most of the target structure.

[0262] To guide viral injections in the SNr, a guide grid with multiple holes spaced 1 mm apart was installed vertically above the SN of each subject. By filling these holes with vitamin E, accurate injection coordinates could be obtained from T1-weighted MRI images (3T Tim Trio scanner, Siemens). Virus 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.

[0263] For intracranial drug administration and / or electrophysiological recordings, a recording trough covering the area from the anterior caudate nucleus to the posterior GPi was fixed to the skull using six titanium screws and dental cement. Each subject was allowed to recover from surgery for at least 6 weeks before further study.

[0264] Macaques that received substantia nigra injections of AAV8R12-G88P3-HA-hM3Dq or AAV8R12-G88P7-rM3Ds-2A-EYFP after CNO infusion spent less time observing the higher parts of the cage than animals administered saline infusion ( Figures 8A-8B In situ hybridization analysis showed that the labeled neurons were only D1 MSNs, confirming the labeling specificity of macaque retrograde AAV ( Figure 9C Overall, approximately 20.55% of D1 MSNs were labeled ( Figures 9A-9G ). AAV8R12-G88P7-rM3Ds-2A-EYFP was delivered in the substantia nigra and CNO was delivered by intraperitoneal injection ( Figure 9D ) 12 months after induced sedation, the activation of the BG direct pathway in mice and the percentage of rotational behaviors (ipsilateral rotation and contralateral rotation) were quantified ( Figure 9E ), n = 6 mice per group. The effectiveness of the D1-MSN retrograde labeling system in the macaque model was assessed by unilateral injection of AAV8R12-G88P3-mCherry into the SNr of cynomolgus macaques (Macaca fascicularis). Strong labeling of projection neurons was detected in the caudate nucleus and putamen, with minimal labeling elsewhere (Figure 9K).

[0265] Next, AAV8R12-G88P3-HA-hM3Dq was injected unilaterally into the SNr. Intracranial injection of CNO into the dorsomedial caudate nucleus induced a dramatic increase in contralateral rotation ( Figure 10A and 10C Interestingly, contralateral rotations were also observed in macaques that received unilateral AAV8R12-G88P7-rM3Ds-2A-EYFP infusion into the SNr for 2.5 years, but not in control animals, following systemic CNO infusion ( Figures 10A-10C , Figures 11A-11H), demonstrating the ability to achieve long-term expression of chemogenetic effectors via retroviral tracers, as well as the precision with which direct pathway projection neurons can be isolated and activated in primates. A significant increase in contralateral rotation speed and a decrease in the time animals spent in the top compartment of the observation cage were also observed after CNO treatment ( Figure 10D and 10G , Figures 11A-11H ). In addition, after CNO infusion, increased muscle tension in the contralateral biceps brachii was observed by electromyographic recording. No significant differences were found for immobility time, total distance traveled, or rotational speed of ipsilateral rotation ( Figure 10E 、 10F and 10H). Electrophysiological recordings confirmed increased neuronal activity in the caudate nucleus after CNO infusion ( Figures 12A-12F These results clearly show that the toolkit accurately isolates and activates selective projection neuron subtypes in primate models.

[0266] Example 5 - Chemogenetic Activation of the Direct Pathway Reverses Parkinsonian Symptoms in a Rodent PD Model

[0267] A rodent Parkinson's disease mouse model was created by bilateral injection of 6-OHDA using the same method as described in the viral injection (Example 3). A total volume of 1 ul 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 to the bregma, 1.5 mm lateral to the bregma, and 3.2 mm ventral to the bregma. Animals were given a premedication of desipramine (25 mg / kg, Sigma) before the injection of 6-OHDA to increase the selectivity and efficacy of the 6-OHDA-induced injury. Mice were supplemented with DietGel (ClearH2O) within one week after surgery. All staining and behavioral experiments were performed at least 14 days after surgery, when dopamine consumption was maximal and stable.

[0268] To investigate the efficacy and safety of a D1-MSN-specific neuromodulatory strategy, AAV8R12-G88P7-rM3Ds-2A-EYFP was injected into the SNr of adult C57 / BL6 mice. 6-OHDA was administered bilaterally into the striatum ( Figure 13A TH immunohistochemistry showed a significant reduction in dopamine innervation in the striatum and a substantial loss of dopamine neurons in the SNc ( Figure 13B , 13G). Analysis of spontaneous locomotion in the open field revealed that systemic CNO delivery, which selectively activated D1MSNs, dramatically reversed the dyskinesia-like phenotype of PD mice ( Figures 13C-13D ,13H-13I). In addition, CNO partially rescued the motor skill deficits of PD mice in the rotor rod test ( Figure 13E , 13J). CNO partially rescued the motor skill deficits of 6-OHDA-treated mice in the rotor rod test ( Figure 13F , 13K). Injection of AAV8R12-G88P7-EYFP into the substantia nigra failed to alleviate the Parkinson's disease phenotype ( Figures 13C-13O Whole-cell patch clamp recordings in slices revealed that D1-MSN excitability was increased in AAV8R12-G88P7-rM3Ds-2A-EYFP-transduced, but not AAV8R12-G88P7-EYFP-transduced, cells. Figures 13P-13Q , 32A, 32D), but did not affect the basal firing rate and resting membrane potential of either group ( Figures 32B-32C ,32E-32F).

[0269] These results suggest that targeted activation of the basal ganglia (BG) direct pathway using an AAV-mediated retrograde approach can effectively antagonize parkinsonian symptoms in rodent PD models.

[0270] Example 6 - Chemogenetic Activation of the Direct Pathway Reverses Parkinsonian Symptoms in a Monkey PD Model

[0271] Parkinson's disease was established in macaques by unilateral injection of 1-methyl-4-phenylpyridine (MPP+) into the SNc ( Figure 14 MPP+ was injected unilaterally using the same method previously described for viral 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. The monkeys were continuously monitored by a veterinarian after injury. Parkinsonian symptoms, such as bradykinesia and impaired balance, were observed immediately after the injury surgery. Stable parkinsonian symptoms were observed for more than 12 weeks before the animals were used in experiments.

[0272] TH immunohistochemistry confirmed the loss of nigral dopamine neurons and their fibers in the caudate nucleus and putamen ( Figures 15A-15B AAV8R12-G88P7-rM3Ds-2A-EYFP was stereotactically injected into six sites of the SNr of adult cynomolgus monkeys ( Figure 14 Monkeys injected with MPP+ showed characteristic PD-like symptoms, including bradykinesia, tremors, rigidity, and abnormal posture ( Figures 16A-16K However, the D1-MSN targeted manipulation approach did not induce dyskinesia-like behavior ( Figure 16L In some cases, stereotactic injections of retrograde AAV8R12 were performed into 9 sites of the SNr of adult cynomolgus monkeys ( Figures 33A-33B ). MPP+ was then injected unilaterally into the SNc, and the loss of substantia nigra dopamine neurons in the SNc and their fibers in the caudate nucleus and putamen was observed by TH immunohistochemistry ( Figure 6B and6C ).

[0273] Interestingly, many of these symptoms were significantly reversed after systemic administration of desclozapine (DCZ). DCZ is a potent, brain-penetrant rM3D agonist with reduced off-target binding compared to CNO, and 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+-lesioned macaques ( Figures 15C-15G Monkeys injected with MPP+ showed characteristic PD-like symptoms, including bradykinesia, tremors, rigidity, and abnormal postures. With systemic DCZ treatment (but not saline treatment), we observed a reversal of typical Parkinsonian symptoms in all tested monkeys ( Figures 15H-15I , 16A, 16H-16K, 17A-17D, 34A-34D). First, an increase in spontaneous locomotion in the observation cage was observed, approaching the activity level of the animals before the injection of MPP+ ( Figure 15I ,16A-16F,17C-17D). Secondly, tremor was greatly reduced or even eliminated after DCZ treatment ( Figures 16H-16K ,17A). Third, we observed that chemogenetic manipulation (in this case, activation of D1-MSNs) significantly restored motor skills ( Figure 16H , 17A-17B). In addition, an effective dose of DCZ (0.3 mg / kg) did not alter motor-related behaviors in unvaccinated monkeys ( Figures 35A-35G Notably, the improvement in Parkinson's disease symptoms appeared consistent during 8 months of continuous treatment with DCZ ( Figures 16C-16G Furthermore, the animals showed no motor impairment, and blood levels of common liver- and kidney-related factors remained stable during treatment ( 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.

[0274] The efficacy of DCZ was subsequently compared with that of levodopa, a first-line drug used to treat PD patients. Similar reversal of Parkinson's symptoms was observed with both chemicals ( Figures 18A-18C ,24A,37A-37F), but DCZ showed faster symptom reversal than L-DOPA. In the initial phase of drug administration, DCZ-mediated activation of the targeted chemogenetic circuit showed faster symptom reversal than L-Dopa ( Figure 24B Surprisingly, DCZ showed a significantly prolonged duration of effectiveness compared to levodopa, demonstrating symptom relief 24 hours after drug administration ( Figure 18C After the drug reaches steady-state efficacy, DCZ prolongs the window of efficacy following each therapeutic dose, providing symptom relief for at least 24 hours after drug administration ( Figure 24C ), which is much longer than the therapeutic window of L-Dopa observed clinically. Cerebrospinal fluid samples were collected 24 hours after drug infusion and no detectable levels of DCZ were found ( Figure 24D ), suggesting that changes in neural network dynamics or residual DREADD ligands in the brain have a significant effect. In addition, the D1-MSN targeted manipulation approach did not induce dyskinesia-like behavior, which was evident after long-term administration of L-Dopa ( Figure 24E The prolonged duration of prior L-Dopa administration (4 months) did not affect the efficacy of DCZ treatment or the absence of dyskinesias ( Figures 24F-24L Together, these results demonstrate the effectiveness of our approach in an NHP PD model and strongly support its feasibility for treating PD in humans.

[0275] Example 7 - Seroquel, a novel DREADD ligand for clinical use

[0276] Parkinson's disease patients receiving gene therapy will benefit from administration of FDA-approved ligands to activate the DREADD component of the therapy described herein. Although clozapine (CNO), the active metabolite of CNO, is a potential candidate for such a molecule, the search is on for other molecules with less demanding clinical monitoring and a better safety profile. Using chemical structure analysis combined with safety analysis for use in PD patients, Seroquel (quetiapine; QTP) was identified as a potential candidate. Seroquel was first tested in vivo for its ability to activate rM3Ds. Following bilateral injection of AAV8R12-G88P7-rM3Ds-EYFP into the SNr of adult mice, QTP or CNO was delivered by IP injection and total distance traveled was quantified in an open field. As Figure 20 As shown, QTP was able to stimulate locomotion in an open field, indicating that it is a suitable in vivo ligand for rM3Ds. Next, we tested whether QTP could induce locomotion in animals injected with AAV8R12-G88P20-hM3Ds. However, compared to CNO, we found that QTP administration did not increase the distance traveled by these animals, as Figure 21 This suggests that the differences between the structures of hM3Ds and rM3Ds render QTP ineffective in activating hM3Ds.

[0277] As a first step toward designing QTP-activatable hM3Ds variants, we aligned the sequences of rM3Ds and hM3Ds to identify differences. Overall, rM3Ds and hM3Ds were 96.6% identical and 94.5% identical. However, only two mutations were located within the ligand-binding domain ( Figure 22), indicating that these residues are responsible for the differences in the responses of rM3Ds and hM3Ds to NQN. Next, we made two mutations, A147S and F349Y, at 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. Figure 23 As shown in , 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 of wild-type hM3Ds.

[0278] Example 8 - Additional Discussion of Research Results

[0279] One of the challenges of modern neuroscience has been translating cutting-edge technological advances into effective therapeutic strategies for human brain disorders. In these examples, the relatively high-throughput nature of mouse studies was exploited for discovery research, and the further relevance of NHPs was further exploited to develop and demonstrate novel circuit-manipulating gene therapies to treat the core symptoms of Parkinson's disease.

[0280] Some of the methods developed herein exploit the unique axon targeting properties of different subtypes of projection neurons, which may be common across many brain regions and subregions. Thus, some embodiments may provide viable solutions to capture the anatomical and functional characteristics of multiple unique projection neuron types, many of which have direct therapeutic uses. Robust and specific targeting may rely on AAV serotypes with enhanced labeling efficiency for different cell types and appropriate cell type-selective promoters that allow targeting of neuronal subsets within mixed cell populations. Furthermore, it is shown herein 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, identification and characterization of promoters and distal regulatory elements, and neuromodulatory technologies are likely to lead to the development of a comprehensive toolkit that further enables the rapid development of research strategies and therapeutic approaches based on circuit-specific activity modulation.

[0281] In the Parkinson's disease macaque model, 0.3 mg / kg / day of DCZ was administered to achieve correction of some important motor symptoms. Given that the standard clinical dose of clozapine (a DREADD agonist with a core structure similar to that of DCZ) in the treatment of schizophrenia is approximately 4.5-9 mg / kg / day, a significantly lower dose of DCZ in the treatment of primate PD animals can significantly reduce the side effects observed in the clinical use of clozapine, such as neutropenia and weight gain. Nevertheless, the development and characterization of novel and clinically safe DREADD ligands is an important area for further transforming chemical genetic manipulation methods in the treatment of brain disorders. In the primate PD model, therapeutic retrograde AAV was delivered to the SNr before damaging dopamine neurons in the SNc. This opposite experimental order was chosen because in the MPP+SNc injection PD macaque model, the massive destruction of dopamine neurons, although highly specific and effective, may lead to strong local inflammation and immune cell activation and infiltration in the SNc and adjacent SNr. If therapeutic AAV is subsequently delivered, this change in the local environment could hinder efficient viral transduction in the SNr. To verify that the change in experimental order did not affect the assessment of therapeutic efficacy, retrograde AAV injection was performed before striatal injection of 6-OHDA to eliminate SNc dopamine neurons in mice and observed a reversal of the parkinsonian phenotype that was identical to those in which existing dopamine neurons were eliminated.

[0282] Systemic administration of L-Dopa is a treatment for patients with Parkinson's disease. The effects of L-Dopa on central non-BG and peripheral dopamine systems may contribute to the occurrence of many side effects. Some of the methods described herein can precisely regulate the basal ganglia direct pathway without affecting any other dopamine pathways in the body, and are likely to prevent the occurrence of most or all L-Dopa-induced side effects. In addition, in some cases, L-Dopa may require the survival of at least some substantia nigra dopamine neurons to convert it into dopamine, which may be the reason for its fluctuation and decline in efficacy after long-term use and the progressive death of dopamine neurons in PD patients. On the other hand, in some embodiments, the chemical genetic gene therapy method designed herein does not require the survival of substantia nigra dopamine neurons and can provide a treatment option for patients with advanced PD who have lost most or all of their substantia nigra dopamine neurons. The observation that the method described herein can reverse Parkinson's symptoms in PD primates that have received extended L-Dopa treatment suggests that it is a viable candidate treatment for advanced PD. In addition, significant L-Dopa-induced dyskinesias were observed in Parkinson's primates, but the same group of animals did not develop dyskinesias after 8 months of treatment with DCZ. Given that changes in D1-MSN activity may be the main driver of acute and chronic side effects observed with dopamine replacement therapy, the lack of dyskinesias in some of the methods described herein may be due to the inability of DREADDs to induce plasticity at corticostriatal synapses or their effects on local striatal circuits. Another feature of some of the methods described herein is an extended efficacy window compared to the standard 6-hour window of L-Dopa. Some of the methods described herein were effective 24 hours after drug administration in Parkinson's monkeys and showed no signs of stalling within the significantly extended therapeutic window. In addition, mixed results have been seen in trials using dopamine agonists to treat depression, a common non-motor symptom of Parkinson's disease. The methods described herein, which specifically regulate one of the major dopamine-dependent circuits in the brain, may help distinguish the roles of different dopamine systems in mood regulation and provide alternative strategies to alleviate Parkinson's mood symptoms. Overall, the precision gene therapy methods developed herein can be used to treat neurological disorders such as Parkinson's disease.

[0283] Example 9 - Additional Methodological Details of the Study

[0284] AAV capsid modification

[0285] The Rep-Cap plasmids for AAV1 / 5 / 6 / 8R (retro) were chemically synthesized (GENEWIZ) by replacing the Cap sequence in the rAAV2-retro helper plasmid (Addgene, 81070) with a sequence carrying the desired sequence modification. AAV8R1-14 Cap variants were generated by introducing 1-3 mutations into the AAV8R backbone. Mutagenesis was performed by PCR using PrimeSTAR HS DNA polymerase (Takara, R010A) and a pair of primers for each site. For example, the sequences of the mutagenic primers used to introduce 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 each colony using the Miniprep kit (Qiagen, 27106) and subjected to Sanger sequencing to confirm the introduction of the intended mutation.

[0286] AAV8R cap protein sequence

[0287]

[0288] AAV8R12 cap protein sequence

[0289]

[0290] Promoter design and screening

[0291] Region-selective promoter identification was based on in situ hybridization (ISH) data from the Allen Brain Atlas (mouse.brain-map.org / ). Genes that were highly expressed in the striatum but not in other parts of the basal ganglia were first selected as candidate genes. H3K4me1 and H3K27ac are known epigenetic markers of active promoters and enhancers. Therefore, we identified regions in the mouse brain with high levels of these two chromatin modification markers as candidate regions based on ENCODE annotation data from the UCSC Genome Browser (genome.ucsc.edu / ). To further expand the potential application of the promoter in primates, the target sequence 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.

[0292] G88P2 / 3 / 7 promoter sequence

[0293] G88P2 promoter

[0294]

[0295] G88P3 promoter

[0296]

[0297] G88P7 promoter

[0298]

[0299] AAV vector construction

[0300] The promoter was subcloned into the pAAV-hSyn-EYFP vector derived from pAAV-hSyn-EGFP (Addgene, 50465) to replace the hSyn promoter by using the appropriate restriction enzyme combination. To generate pAAV-G88P7-DIO-tdTomato, the DIO-tdTomato cassette was subcloned into pAAV-G88P7-EYFP to replace EYFP by restriction enzyme digestion. To generate pAAV-G88P3-HA-hM3Dq, the promoter G88P3 was subcloned into pAAV-hSyn-HA-hM3Dq-IRES-mCitrine (Addgene, 50463) via EcoRI / BamHI restriction sites to replace the hSyn promoter, and then the IRES-mCitrine was removed 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 promoter G88P7 was subcloned into pAAV-hSyn-DIO-rM3Ds-mCherry (Addgene, 50458) to replace the hSyn promoter, followed by replacing mCherry with 2A-EYFP. The DIO construct was then removed by restriction enzyme digestion. To generate pAAV-G88P7-HA-rM3Ds-2A-Cre, the EYFP in pAAV-G88P7-HA-rM3Ds-2A-EYFP was replaced with Cre by restriction enzyme digestion.

[0301] AAV production and titration

[0302] HEK293T cells (ATCC) were co-transfected with AAV vector plasmids, AAV Rep-Cap plasmids (rAAV2-retro, AAV1R, AAV5R, AAV6R, AAV8R, and AAV8R1-14), and pAdDeltaF6 helper plasmids carrying adenoviral genes required for the AAV life cycle (Addgene, 112867) using calcium phosphate. HEK293T cells grown in 15 cm cell culture dishes were co-transfected with a mixture of the three plasmids (1:1:1) at 80% confluence. 48-72 hours 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 a Millipore Amicon 100K column (Merck Millipore, UFC910008). After denaturation of AAV particles with proteinase K, encapsidated viral DNA was quantified by qPCR (Thermo Fisher) using primers recognizing viral WPRE and / or ITR sequences. Titers were calculated as the number of genome copies per ml.

[0303] Surgery and virus injection

[0304] For mice:

[0305] For retrograde labeling of Drd1-Cre or Drd2-Cre mice, AAV8R12-G88P7-EYFP was injected unilaterally into the SNr in a total volume of 200 nL. Simultaneously, AAV9-G88P7-DIO-tdTomato was injected ipsilaterally into the striatum (0.5 mm anterior to bregma, 1.5 mm lateral to bregma, and 3.5 mm ventral to bregma) in a total volume of 300 nL. For optical labeling of mice, AAV8R12-G88P7-HA-rM3Ds-2A-Cre was injected unilaterally into the SNr in a total volume of 200 nL. AAV9-EF1α-DIO-ChR2-EYFP was injected ipsilaterally into two sites to maximize coverage of the striatum (250 nL / site, site 1: 1.2 mm anterior to bregma, 1.5 mm lateral to bregma, 3.2 mm ventral to bregma; site 2: 0.4 mm anterior to bregma, 1.6 mm lateral to bregma, 3.3 mm ventral to bregma).

[0306] For Monkey:

[0307] All neurosurgery procedures were performed aseptically while the subjects were anesthetized. For general anesthesia, monkeys were given atropine (0.05 mg / kg, intramuscular injection) to reduce bronchial secretions before ketamine (15 mg / kg, intramuscular injection). Anesthesia was maintained using propofol (6 mg / kg, intravenous injection). As assessed by pinching the toes, the level of anesthesia was adjusted to eliminate movement. The corneal reflex was always absent. The subjects were placed on a standard operating table that was continuously heated, and the subjects' heads were securely fixed to a stereotactic frame (David Kopf Instruments). Electrocardiogram, heart rate, blood oxygen saturation (SpO2) (range 95-100%) and rectal temperature (37.5-38.5°C) were continuously monitored by a physiological monitor (Mindray, uMEC7).

[0308] Virus injections in the striatum were performed at a rate of 300 nL / min in the caudate nucleus (12 uL virus, 4 sites) and putamen (18 uL virus, 6 sites).

[0309] For intracranial drug administration and / or electrophysiological recordings, 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.

[0310] Generation of a mouse model of Parkinson's disease

[0311] 6-OHDA was injected bilaterally using the same method as for viral injection. A total volume of 1 μL 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 to the bregma, 1.5 mm lateral to the bregma, and 3.2 mm ventral to the bregma. Animals were given a premedication of desipramine (25 mg / kg, Sigma) before the injection of 6-OHDA to increase 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 consumption was maximal and stable.

[0312] PD score

[0313] Throughout the observation period, two experienced observers blindly assessed the monkeys' Parkinsonian symptoms three days a week. Parkinsonian symptoms were quantified using the well-established Kurlan scale (Part I: Parkinsonian features), a widely used scale for quantifying Parkinsonian symptoms in Old World monkeys. A score of zero indicates a normal monkey, while a maximum score of 29 indicates an animal with severe PD symptoms. For each behavioral category, the scores for the upper and lower limbs were summed. In addition, action or intention tremor and rest tremor were summed.

[0314] Movement disorder score

[0315] Throughout the observation period, two experienced observers blindly assessed the monkeys' motor impairments three days a week. The motor impairment scoring scale was as follows: 0: absent; 1: mild, transient, present for <30% of the observation period; 2: moderate, not interfering with normal activities, present for >30% of the observation period; 3: marked, sometimes interfering with normal activities; present for <70% of the observation period; 4: severe, persistent, replacing normal activities, present for >70% of the observation period.

[0316] Immunofluorescence

[0317] Animals were deeply anesthetized with sodium pentobarbital (Nembutal; 80 mg / kg, ip) and perfused with 4% paraformaldehyde in PBS (0.1 M) and PBS solution (4% PFA / PBS, 4°C, 30 mL for mice and 500 mL for monkeys). The 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 in 4% PFA / PBS at 4°C for 20 minutes. The sections were blocked and permeabilized in a PBS solution containing 5% bovine serum albumin (BSA) and 0.3% Triton X-100 at room temperature for 1 hour. 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). Secondary antibody incubation was 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 (ThermoFisher, A32814, A32754, A11039, and S11223, respectively). Cell nuclei were stained with DAPI (Sigma, D9542). Brain sections were mounted on slides using Fluoromount-G mounting medium (Southern Biotech, 0100-01).

[0318] Cell counting

[0319] Images were acquired using a confocal microscope (Carl Zeiss, LSM880) and an AxioImager.Z1 microscope with apotome (Carl Zeiss). Brain structures were identified by microscopy and digital photography using a mouse brain atlas. To analyze the number of EYFP+ cells in the striatum, sections of the striatum from the rostral to the caudal end were used. Every sixth section was analyzed, and the number of EYFP+ cells was multiplied by 6 to obtain the approximate total number of cells and the average number of cells per animal. Images were processed using ImageJ (NIH, USA), and final quantification was performed manually by two blinded experimenters.

[0320] In situ hybridization

[0321] The coding region fragments 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 described previously with minor modifications. Briefly, digoxigenin (DIG)-labeled cRNA probes (riboprobes) were prepared using DIG RNA Labeling Mix (Roche). The brain was frozen in OCT (Tissue-Tek), and 40-50 μm thick coronal cryosections were hybridized with DIG-labeled cRNA probes at 56°C for 15-18 hours. After hybridization, the sections were washed twice in 0.2X SSC at 62°C for 30 minutes, incubated with peroxidase (POD)-conjugated anti-DIG antibody (Roche, 1207733910) at 37°C for 2 hours, and then processed using the TSA-plus kit (Perkin Elmer). The sections were then incubated with anti-RFP antibody (Rockland, 600-401-379) or anti-GFP antibody (Rockland, 600-101-215M) at 4°C overnight 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) at room temperature for 2 hours before mounting with Fluoromount-G (Southern Biotech, 0100-01).

[0322] Behavioral assays in mice

[0323] Open field test

[0324] The mice were manipulated individually for 10-20 seconds every day for at least 5 days to accustom them to the experimenter. The open field test was carried out in a test apparatus (50 cm × 50 cm × 50 cm) with a high-definition digital camera (Sony) located above the venue. On the first day, the mice were accustomed to the apparatus for 10 minutes. On the second day, saline was administered to the mice (0.1 mL ip or intracranial [ic] 200 nL via a guide cannula). On the third day, clozapine-N-oxide (CNO, Hello Bio, HB1807; 0.3 mg / kg ip or 100 μM ic 200 nL) was administered to the mice. All behavioral tests were performed 30 minutes after injection. During the test, the mice were allowed to freely explore the apparatus for 10 minutes. Behavioral data were analyzed using ANY-maze software (Stoelting Co.).

[0325] Rotor bar test

[0326] As described in the open field test, mice are manipulated separately. Before the test phase, mice are transferred to the test room and adapted for 15 minutes. Mice are placed on a rod (Shanghai Xinruan), and the device is set to a mode (10-40rpm). The device automatically records the delay of falling in each test. Each mouse performs 3 tests in one day for two consecutive days, with a minimum interval of 15 minutes between each test. The delay of falling is averaged in three tests per day.

[0327] Behavioral assessment of monkeys

[0328] Exercise test:

[0329] The monkeys were tested for locomotion in custom-made observation cages (100 cm × 100 cm × 100 cm). The top and front of the cages were made of tempered glass to provide a clear view for behavioral recording. The monkeys were placed in the observation cages individually for 30 minutes on three days to allow them to habituate to the cages.

[0330] For monkeys injected with AAV8R12-G88P3-HA-hM3Dq, CNO was infused intracranially into the dorsomedial caudate nucleus through a recording chamber. To perform the infusion while the animals were awake, the monkeys were trained to sit in a specially designed primate chair with their heads fixed to the primate chair via a mask made of thermoplastic material. 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 microinjection pump (KD Scientific, Legato130). The needle remained in place for 5 minutes for drug diffusion before being retracted. For monkeys injected with AAV8R12-G88P7-rM3Ds-2A-EYFP, CNO was infused by intramuscular injection (10 mg / kg). After successful CNO infusion, the monkeys were immediately transferred to an observation cage for video recording. Videos capturing the subjects' behavior were recorded for at least 90 minutes. Animal behavior was further analyzed and quantified 30–90 min after saline / CNO infusion.

[0331] For Parkinson's monkeys, DCZ (desclozapine, 0.3 mg / kg, MCE) was administered intramuscularly. After successful DCZ infusion, 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, e.g., For initial testing, a 2-week interval was set between DCZ and L-DOPA treatment trials to allow for drug washout. For long-term DCZ treatment, parkinsonian monkeys were given DCZ at a dose of 0.3 mg / kg via intramuscular injection every other day. For extended L-DOPA treatment, animals were administered L-DOPA once daily for 4 months. A 1-month washout was allowed before a 2-month DCZ treatment.

[0332] Motor skills test

[0333] A grasping-to-eating / hand-to-mouth movement test was performed as an indicator of the monkey's motor skills. The monkey was first trained to sit in a primate chair with its healthy hand restrained. The monkey was then trained to use the hand affected by MPP+ to grasp food (a piece of apple, approximately 1 cm3) from the experimenter. The task consisted of three blocks with an interval of 20 minutes between blocks. Each block consisted of ten consecutive trials. A trial was defined as a success when the monkey was able to bring the food to its mouth within one minute. Before the MPP+ injury, the monkey was trained to achieve an 85% success rate.

[0334] Monkey behavior analysis

[0335] During video recording, top (x / y) and side (x / z) cameras were used to record the observation cage. The VigiePrimate system (Viewpoint) was used to analyze the subject's activity and extract motion data from the recorded videos. Tracking data from the top and side views were synchronously sampled at 25 Hz and then combined to generate a single dataset representing the 3D space. All x-, y-, and z-axis tracking data were normalized to 0–100 cm, where x represents right to left, y represents front to back, and z represents bottom to top.

[0336] For rotation analysis, only movements below 30 cm in 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. We then calculated the angle relative to (x0, y0) at each time point using the following formula:

[0337]

[0338] Among them, θ i represents the rotation angle at time i, (x i ,y i ) represents the xy coordinates at time i. We then calculate the change in rotation angle as Δθ i =θ i -θ i-1For right substantia nigra injections, when Δθ < 0, the rotation direction is counterclockwise and is labeled as contralateral. When Δθ > 0, the rotation direction is clockwise and is labeled as ipsilateral. Raw data were analyzed and plotted using a custom R package. The code is available on Github (github.com / chenyef / PD_ana / blob / main / Behavior_ana.txt).

[0339] Electrophysiological recordings

[0340] For mice:

[0341] The mice of different groups were anesthetized with 0.04% isoflurane and then decapitated. The brain was quickly extracted and placed in ice-cold N-methyl-D-glucamine (NMDG) cutting solution, which contained (in mM): 92NMDG, 2.5KCl, 25NaHCO 3 , 1.25NaH 2 PO 4 , 4.5D-glucose, 20HEPES, 5L-ascorbic acid, 3-sodium pyruvate, 2 thioureas, 10MgSO 4 , 0.5CaCl 2 . HCl was used to adjust the pH to 7.2 ± 0.1, and NMDG was used to adjust the osmotic pressure to 305 ± 5mosmol / L. 95% O 2 / 5% CO 2 was used to bubble in the solution before use. Coronal striatal slices were cut to 250 μm thickness using a vibratome (Leica, VT1200 S) and then incubated at 37°C for at least 45 minutes in artificial cerebrospinal fluid (aCSF) saturated with 95% O2 / 5% CO2 before recording. aCSF contains (in mM) 125 NaCl, 1.25 KCl, 25 NaHCO3, 1.25 KH2PO4, 25 D-glucose, 2 CaCl2, and 1 MgCl2, and is supplemented with 2 mM sodium pyruvate, 3 mM inositol, and 0.4 mM L-ascorbic acid.

[0342] Acute striatal slices were transferred to a recording chamber and perfused with oxygenated aCSF at a constant rate of 3 ml / min. Neurons were observed at room temperature (~25°C) using an upright microscope (Olympus, BX-51) with a 40X water immersion objective. In the striatum, EYFP-positive neurons were selected for whole-cell recording. Neurons were quickly observed under a fluorescence microscope, and then patch clamping was performed 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 to a final tip resistance of 6-7 MΩ using a Model P-1000 puller (Sutter Instrument). Attached cells were stimulated with a current step injection (150-200 pA, 150 ms long) once per minute. The stimulation intensity was adjusted to induce 1 action potential with a probability of 50%. Baseline recordings were performed with 18 current injections, and then the perfusion solution was switched to aCSF containing CNO (10 μM) and allowed to stabilize for 3 minutes. Current clamp recordings were performed for 20 minutes using the same current stimulation protocol in the presence of CNO. The recording electrode was filled with an internal solution based on potassium methanesulfonate (KMeSO3), which contained (in mM): 135KMeSO3, 10KCl, 10HEPES, 5MgATP, 0.5NaGTP, 1EGTA, and the pH was adjusted to 7.2±0.1 using KOH, and the osmotic pressure was adjusted to 305±5mosmol / L using KMeSO3.

[0343] For Monkey:

[0344] When monkeys were anesthetized, electrophysiological recordings were performed in a recording chamber. Neural responses were recorded using a 16-channel linear probe (Plexon Inc, Uprobe) driven by a mechanical micro-driver (AlphaOmega, FlexMT). The signal was separated and filtered between 300 Hz and 5 kHz by a headstage (Plexon Inc, HST / 16V-G20 LN) to identify spike activity using an amplifier system (Plexon Inc, OmniPlex). The spikes detected were then classified for further analysis using commercially available software (Plexon Inc, Offline Sorter). In order to quantify the effect of CNO / DCZ injections, neural activity was recorded 60 minutes after CNO / DCZ injection. The response time course of each channel was obtained by calculating the spikes in each 10-minute window and then normalizing to the maximum value.

[0345] To determine the effect of CNO / DCZ injection on the direction of signal change, we performed Pearson correlations between neural responses and time using the "corr" function in Matlab, which generates a correlation coefficient 'r' and a significance level 'p'. When r > 0 and p < 0.05, the neuron was considered to have "increased" activity; when r < 0 and p < 0.05, it was considered to have "decreased" activity. Otherwise, the neuron's neural activity was considered "unchanged." After CNO / DCZ injection, the responses of all neurons that increased were averaged after normalization by the maximum response (Crowe et al., 2014; Dai and Wang, 2018; Hirokawa et al., 2019) and then plotted as a function of time to generate a normalized population response.

[0346] Light Marker

[0347] For mice:

[0348] Brain slices were prepared under low light conditions. 473 nm blue LED light was applied through an imaging objective (40x / 0.8 water immersion objective, Olympus, Japan). Recordings were performed in current clamp mode. In the recordings verifying ChR2 function, 200 μM CdCl2 was included in the bath to prevent back propagation of calcium currents from dendrites. 2-ms long light pulses at 2 Hz and 86.89 mW / mm2 (sanwa-LP10, Japan) were administered to activate ChR2. Cells accurately stimulated by light pulses were further recorded by CNO treatment.

[0349] For Monkey:

[0350] To enable in vivo validation of this strategy by optical labeling, an AAV virus encoding the opsin ChR2 (AAV9-EF1α-DIO-ChR2-EYFP) was injected into the striatum, while a retrograde AAV encoding Cre and rM3Ds (AAV8R12-G88P7-HA-rM3Ds-2A-Cre) was infused into the substantia nigra. A total volume of 27 μl (3 μl × 9 sites) and 30 μl (3 μl × 10 sites) was injected into the SNr and striatum, respectively. A recording chamber was implanted above the striatum to allow optical stimulation and electrophysiological recordings.

[0351] Optogenetics validation was performed six weeks after viral injection. A 16-channel linear probe (Plexon Inc., Uprobe) with an embedded optical fiber was used to deliver light stimulation and record neural responses. A mechanical microdrive (FlexMT, Alpha Omega, Nazareth, Israel) was used to mount and drive the probe. A blue laser (473 nm, Changchun New Industries Tech, MBL-III-473) was used to generate light stimulation, which was controlled by a DAQ board (National Instruments, PCIe-6321) through the MonkeyLogic toolbox (NIMH version).

[0352] 40 Hz light pulses of 500 ms duration were used to probe neuronal responses to light stimulation. Once a neuron exhibited a reproducible response to light stimulation, further testing was performed to verify its response to CNO from 10 minutes before to 60 minutes after ligand administration. The responses of these optically identified cells were grouped to generate a normalized population response as a function of time.

[0353] Electromyography (EMG) recording and data analysis

[0354] 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 insertion points. Monkeys were trained to sit in a primate chair while awake with their upper limbs free to move. The recording procedure was performed immediately after DCZ infusion. EMG signals from the biceps brachii were collected for at least 120 minutes. The EMG signals were amplified by a signal acquisition and processing system (TECHMAN, BL-420N) collected 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).

[0355] Quantitative and statistical analysis

[0356] Statistics were performed in GraphPad Prism 9.0. Paired t-tests, unpaired t-tests, one-way analysis of variance, Tukey's test, and Dunnett's test were used where appropriate. All t-tests were performed two-tailed. In all statistical tests, a P value < 0.05 was considered statistically significant. Sample sizes were selected based on previous publications or experience to generate sufficient power to detect specific effects. All statistical tests used are indicated in the figure legends.

[0357] Although 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. Those skilled in the art will now appreciate that numerous variations, changes, and substitutions will be apparent without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention.

[0358] All publications, patent applications, issued patents, and other documents referenced in this specification are herein incorporated by reference 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. To the extent that definitions contained in text incorporated by reference conflict with definitions in this disclosure, such definitions are not included.

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

Claims

1. A nucleic acid comprising a heterologous gene of interest operably coupled 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 an endogenous GPR88 gene.

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

3. 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 an intron.

4. 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 shown in SEQ ID NO:

39.

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

39.

7. 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%, 99% homologous to the nucleotide sequence shown in SEQ ID NO:

40.

9. The nucleic acid according to claim 7, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO:

40.

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

41.

13. The nucleic acid according to claim 11, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO:

41.

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

42.

16. The nucleic acid according to claim 14, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID NO:

42.

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

43.

19. The nucleic acid according to claim 17, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown 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 shown in any one of SEQ ID NO: 44, 45 or 46.

21. The nucleic acid according to claim 1, wherein the regulatory element comprises a nucleotide sequence identical to the nucleotide sequence shown in any one of SEQ ID NO: 44, 45 or 46.

22. A nucleic acid comprising a heterologous gene of interest operably coupled 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 shown 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. 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. The nucleic acid of claim 1, wherein said nucleic acid exhibits increased expression of said heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

27. The nucleic acid of claim 1, wherein the nucleic acid is comprised in a viral vector.

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

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

30. The nucleic acid of claim 1, wherein the gene of interest exhibits at least 2-fold greater expression of the heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

31. The nucleic acid of claim 1, wherein the gene of interest exhibits at least 2-fold greater expression of the heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

32. The nucleic acid of claim 1, wherein the gene of interest exhibits at least 2-fold greater expression of the heterologous gene of interest in neurons of the striatum compared to the promoter of the hSYN1 gene.

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

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

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

36. The pharmaceutical composition according to claim 33, for use in a method for genetically modifying neurons in the striatum.

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

38. The pharmaceutical composition of claim 33, for use in a method of treating a neurodegenerative disease in a subject.

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

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

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

42. A method for genetically modifying neurons in the striatum of an individual, comprising administering the pharmaceutical composition according to claim 33 to the individual, thereby genetically modifying the neurons in the striatum.

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

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

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

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

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

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