Inhibitory nucleic acids that target α-synuclein expression

Abstract

The present invention is characterized by a polynucleotide construct comprising an α-synuclein mRNA targeting sequence. Constructs comprising an α-synuclein mRNA targeting sequence and / or coding sequence can be used, for example, to inhibit α-synuclein mRNA expression and / or to treat diseases or disorders of synucleinopathy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 591,879 filed on 20 October 2023, U.S. Provisional Patent Application No. 63 / 565,101 filed on 14 March 2024, and U.S. Provisional Patent Application No. 63 / 696,066 filed on 18 September 2024, the disclosures of each application being incorporated herein by reference in their entirety.

[0002] Reference to electronically submitted sequence listings The contents of the electronic sequence listing (065830.21WO1; size: 338,301 bytes; and creation date: October 11, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] Synucleinopathy is a neurodegenerative disease or disorder characterized by neuronal inclusions and / or glial inclusions. Pathologically, synucleinopathy can be divided into two main groups: Lewy body diseases or disorders and multiple system atrophy (MSA). Lewy body diseases and disorders are characterized by aggregated α-synuclein and include Parkinson's disease, Parkinson's dementia, Lewy body dementia, infant axonal dystrophy, atypical axonal dystrophy, adult-onset dystonia-parkinsonism, autosomal recessive early-onset parkinsonism, POLG-associated neurodegeneration, Niemann-Pick type C1, and Krabbe disease. (Koga et al. Molecular Neurodegeneration (2021) 16:83).

[0004] Parkinson's disease is an age-related progressive neurodegenerative disorder. It is characterized by the abnormal accumulation of misfolded alpha-synuclein protein aggregates in various regions of the brain. Loss of dopaminergic neurons in the substantia nigra is a pathological feature of Parkinson's disease. (Lee et al. Neuroimmunol. Neuroinflammation (2021) 8:222-44; and Koga et al. Molecular Neurodegeneration (2021) 16:83).

[0005] Various Parkinson's disease treatments, such as dopamine replacement therapy, are used to treat motor symptoms associated with Parkinson's disease. In the early stages of the disease, motor symptoms can generally be adequately controlled with symptomatic treatment. As Parkinson's disease progresses, such treatments become less effective and are associated with debilitating side effects (e.g., dyskinesia), leading to a deterioration in function and quality of life. (Poewe et al., Nat Rev Dis Primers 3, 17013 (2017)).

[0006] Various inhibitory RNAs have been proposed to reduce α-synuclein expression. (See, for example, McCormack et al., PLoS One. 2010 Aug 11;5(8):e12122; Kim et al., Front.Neurol. 2017 Jan 31;8:13; Alarcon-Aris et al., Mol.Ther. 2018 Feb 7;26(2):550-567; Bohn and Sapru USPatent No.8,389,487; and Menon et al., Brain Communications, Volume 3, Issue 4, 2021, fcab247,hypetext: / / doi.org / 10.1093 / braincomms / fcab247). [Overview of the project]

[0007] The present invention is characterized by polynucleotide constructs comprising α-synuclein mRNA targeting sequences. Examples of polynucleotide constructs include inhibitory RNA polynucleotides comprising α-synuclein mRNA targeting sequences, such as pri-miRNA, pre-miRNA, short hairpin RNA (shRNA), microRNA (miRNA), and optionally modified miRNA. Constructs comprising α-synuclein mRNA targeting sequences and / or coding sequences can be used, for example, in methods for inhibiting α-synuclein expression and / or in methods for treating diseases or disorders of synucleinopathy.

[0008] α-synuclein mRNA "targeting" sequences can hybridize to substantially complementary α-synuclein mRNA target sequences. The α-synuclein mRNA target sequences described herein include any of sequence numbers 129-135 and their complements, any of sequence numbers 32-62.

[0009] The reference to "inhibitory" RNA polynucleotides indicates that the polynucleotide contains mRNA-targeting sequences that can inhibit mRNA activity. Inhibition of mRNA activity results in a decrease in protein expression from the target mRNA. Examples of inhibitory RNAs include miRNAs and miRNA precursors, such as pre-miRNA, sh-RNA, and pri-miRNA.

[0010] Therefore, a first aspect of the present invention describes an RNA polynucleotide comprising an α-synuclein mRNA-targeting RNA sequence. The RNA polynucleotide comprises a targeting RNA sequence having at least 90% sequence identity with any of the sequences of SEQ ID NOs. 32 to 62.

[0011] A second aspect of the present invention relates to an optionally modified inhibitory RNA duplex comprising (a) a guide strand that can hybridize to any of the target sequences of SEQ ID NOs: 129 to 135, and (b) a substantially complementary passenger sequence, wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA.

[0012] A third aspect of the present invention relates to a polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide containing an α-synuclein mRNA targeting sequence.

[0013] A fourth aspect of the present invention relates to an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide containing an α-synuclein mRNA targeting sequence, and one or more expression regulatory elements operably linked to the encoding nucleic acid sequence.

[0014] A fifth aspect of the present invention relates to a recombinant viral vector nucleic acid comprising (a) an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide containing an α-synuclein mRNA targeting sequence and one or more expression regulatory elements operably ligated to the encoding nucleic acid sequence, and (b) 5' and / or 3' viral elements providing viral packaging and / or replication.

[0015] A sixth aspect of the present invention relates to a delivery vehicle comprising a viral vector or a non-viral vector and (a) an RNA polynucleotide comprising an α-synuclein mRNA targeting sequence, (b) an optionally modified inhibitory RNA duplex that targets α-synuclein mRNA, or (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding an RNA polynucleotide comprising an α-synuclein mRNA targeting sequence.

[0016] A seventh aspect of the present invention relates to a pharmaceutical composition comprising (a) an RNA polynucleotide containing an α-synuclein mRNA targeting sequence, (b) an optionally modified inhibitory RNA duplex that targets α-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid containing a sequence encoding an α-synuclein mRNA targeting sequence, or (d) a delivery vehicle containing (a), (b), or (c), and a pharmaceutically acceptable carrier.

[0017] An eighth aspect of the present invention relates to a method for reducing α-synuclein expression in a subject and / or treating a disease or disorder of synucleinopathy, comprising administering (a) an RNA polynucleotide comprising an α-synuclein mRNA targeting sequence, (b) an optionally modified inhibitory RNA duplex that targets α-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding an α-synuclein mRNA targeting sequence, or (d) a delivery vehicle comprising (a), (b), or (c), and a pharmaceutically acceptable carrier.

[0018] A ninth aspect of the present invention relates to a method for treating a disease or disorder associated with low dopamine, comprising administering (a) an RNA polynucleotide comprising an α-synuclein mRNA targeting sequence, (b) an optionally modified inhibitory RNA duplex targeting α-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding an α-synuclein mRNA targeting sequence, or (d) a delivery vehicle comprising (a), (b), or (c), and a pharmaceutically acceptable carrier.

[0019] Further aspects of the present invention include (a) an RNA polynucleotide comprising an α-synuclein mRNA targeting sequence for use in pharmaceuticals, for reducing α-synuclein, or for treating diseases or disorders of synucleinopathy; (b) an optionally modified inhibitory RNA duplex that targets α-synuclein mRNA; (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding an α-synuclein mRNA targeting sequence; or (d) a delivery vehicle comprising (a), (b), or (c) and a pharmaceutically acceptable carrier; and the use of (a), (b), (c), or (d) in the preparation of pharmaceuticals (for example, for use in pharmaceuticals, for reducing α-synuclein, or for treating diseases or disorders of synucleinopathy).

[0020] Other features and advantages of the present invention will become apparent from the additional descriptions provided herein, including different examples. The provided examples illustrate different components and methodologies useful for carrying out the present invention. Such examples do not limit the claimed invention. Based on this disclosure, those skilled in the art can identify and use other components and methods useful for carrying out the present invention. [Brief explanation of the drawing]

[0021] [Figure 1] This study demonstrates the ability of various small interfering RNAs (siRNAs) to reduce the expression of the human gene encoding α-synuclein (SCNA) in HEK293 cells. siRNAs were provided at concentrations of 1 nM, 10 nM, or 100 nM. GAPDH refers to glyceraldehyde 3-phosphate dehydrogenase.

[0022] [Figure 2A] Figures 2A, 2B, and 2C show the survival percentages of HEK293 cells administered with different amounts of siRNA. Figure 2A shows the results using 1 nM. Figure 2B shows the results using 10 nM. Figure 2C shows the results using 100 nM. GAPDH refers to glyceraldehyde 3-phosphate dehydrogenase. [Figure 2B] Figures 2A, 2B, and 2C show the survival percentages of HEK293 cells administered with different amounts of siRNA. Figure 2A shows the results using 1 nM. Figure 2B shows the results using 10 nM. Figure 2C shows the results using 100 nM. GAPDH refers to glyceraldehyde 3-phosphate dehydrogenase. [Figure 2C] Figures 2A, 2B, and 2C show the survival percentages of HEK293 cells administered with different amounts of siRNA. Figure 2A shows the results using 1 nM. Figure 2B shows the results using 10 nM. Figure 2C shows the results using 100 nM. GAPDH refers to glyceraldehyde 3-phosphate dehydrogenase.

[0023] [Figure 3] This study demonstrates the ability of different siRNAs to inhibit the expression of α-synuclein-GFP fusion protein in HEK293 cells. Plotted activity was normalized to green fluorescent protein (GFP) / vinculin relative to a naive control. Provided controls were siGLO (a scrambled control with red fluorescence), mismatch 11 (an untargeted control), and naive conditions.

[0024] [Figure 4A] Figures 4A and 4B show the effects of different siRNAs targeting SNCA expression on relevant genes in HEK293 cells. Figure 4A shows the expression of SNCB (the gene encoding β-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM. Figure 4B shows the expression of SNCG (the gene encoding γ-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM. GAPDH refers to glyceraldehyde 3-phosphate dehydrogenase. [Figure 4B]Figures 4A and 4B show the effects of different siRNAs targeting SNCA expression on relevant genes in HEK293 cells. Figure 4A shows the expression of SNCB (the gene encoding β-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM. Figure 4B shows the expression of SNCG (the gene encoding γ-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM. GAPDH refers to glyceraldehyde 3-phosphate dehydrogenase.

[0025] [Figure 5] This demonstrates the ability of different siRNAs to inhibit expression in SH-SY5Y cells at different concentrations.

[0026] [Figure 6] This graph shows differential expression (DE) of genes caused by different siRNAs.

[0027] [Figure 7] This study demonstrates the ability of different pre-miRNAs to reduce normalized GFP signaling (as a measure of SCNA expression) compared to pCC41. Pre-miRNAs are denoted as miR. Statistical analysis was performed using two-way ANOVA along with Dunnett's multiple comparison analysis.

[0028] [Figure 8A] Figures 8A and 8B illustrate the ability of different rAAV vectors containing nucleic acids encoding pre-miRNAs that inhibit α-synuclein expression and α-synuclein aggregation at two different infection multiplicities. Figure 8A shows human α-synuclein normalized to AAV-α-synuclein (AAV-α-syn). Figure 8B shows pS129 intensity normalized to AAV-α-synuclein. pS129 intensity is a measure of α-synuclein aggregation. Statistical analysis was performed using two-way ANOVA against AAV-αSyn. [Figure 8B]Figures 8A and 8B illustrate the ability of different rAAV vectors containing nucleic acids encoding pre-miRNAs that inhibit α-synuclein expression and α-synuclein aggregation at two different infection multiplicities. Figure 8A shows human α-synuclein normalized to AAV-α-synuclein (AAV-α-syn). Figure 8B shows pS129 intensity normalized to AAV-α-synuclein. pS129 intensity is a measure of α-synuclein aggregation. Statistical analysis was performed using two-way ANOVA against AAV-αSyn.

[0029] [Figure 9A] Figures 9A–9D demonstrate the ability of different rAAV vectors containing pre-miRNA encoding nucleic acids to inhibit α-synuclein expression. Figure 9A shows the ratio of change to human α-synuclein + mis11. Figure 9B shows inhibition of human α-synuclein production. Figure 9C shows a decrease in mutant α-synuclein (A53T). Figure 9D shows the effect on mouse α-synuclein expression. Statistical analysis was performed using Dunnett's one-way ANOVA with multiple comparisons. [Figure 9B] Figures 9A–9D demonstrate the ability of different rAAV vectors containing pre-miRNA encoding nucleic acids to inhibit α-synuclein expression. Figure 9A shows the ratio of change to human α-synuclein + mis11. Figure 9B shows inhibition of human α-synuclein production. Figure 9C shows a decrease in mutant α-synuclein (A53T). Figure 9D shows the effect on mouse α-synuclein expression. Statistical analysis was performed using Dunnett's one-way ANOVA with multiple comparisons. [Figure 9C] Figures 9A–9D demonstrate the ability of different rAAV vectors containing pre-miRNA encoding nucleic acids to inhibit α-synuclein expression. Figure 9A shows the ratio of change to human α-synuclein + mis11. Figure 9B shows inhibition of human α-synuclein production. Figure 9C shows a decrease in mutant α-synuclein (A53T). Figure 9D shows the effect on mouse α-synuclein expression. Statistical analysis was performed using Dunnett's one-way ANOVA with multiple comparisons. [Figure 9D] Figures 9A–9D demonstrate the ability of different rAAV vectors containing pre-miRNA encoding nucleic acids to inhibit α-synuclein expression. Figure 9A shows the ratio of change to human α-synuclein + mis11. Figure 9B shows inhibition of human α-synuclein production. Figure 9C shows a decrease in mutant α-synuclein (A53T). Figure 9D shows the effect on mouse α-synuclein expression. Statistical analysis was performed using Dunnett's one-way ANOVA with multiple comparisons.

[0030] [Figure 10A] Figures 10A to 10D show the in vivo distribution of miRNAs and the inhibition of mouse α-synuclein. Figure 10A shows the in vivo distribution in the substantia nigra (SN) and striatum (Str). Figure 10B shows the expression of mouse SNCA (mSCNA) in the SN and Str. Figure 10C shows the expression of mSCNA in the substantia nigra. Figure 10D shows the expression of mSCNA in the striatum. [Figure 10B] Figures 10A to 10D show the in vivo distribution of miRNAs and the inhibition of mouse α-synuclein. Figure 10A shows the in vivo distribution in the substantia nigra (SN) and striatum (Str). Figure 10B shows the expression of mouse SNCA (mSCNA) in the SN and Str. Figure 10C shows the expression of mSCNA in the substantia nigra. Figure 10D shows the expression of mSCNA in the striatum. [Figure 10C] Figures 10A to 10D show the in vivo distribution of miRNAs and the inhibition of mouse α-synuclein. Figure 10A shows the in vivo distribution in the substantia nigra (SN) and striatum (Str). Figure 10B shows the expression of mouse SNCA (mSCNA) in the SN and Str. Figure 10C shows the expression of mSCNA in the substantia nigra. Figure 10D shows the expression of mSCNA in the striatum. [Figure 10D]Figures 10A to 10D show the in vivo distribution of miRNAs and the inhibition of mouse α-synuclein. Figure 10A shows the in vivo distribution in the substantia nigra (SN) and striatum (Str). Figure 10B shows the expression of mouse SNCA (mSCNA) in the SN and Str. Figure 10C shows the expression of mSCNA in the substantia nigra. Figure 10D shows the expression of mSCNA in the striatum.

[0031] [Figure 11] Guide strand level and passenger strand level from the rAAV vector are shown.

[0032] [Figure 12] This study demonstrates the effects of different doses on body weight in α-Syn-treated mice. Mice were administered a 7.5e9vg / SN dose of human α-Syn (AAV1 / 2-A53TαSyn) to induce α-synuclein to mediate neuronal loss. Treated animals received rAAV-miR5 at vector genome (vg) doses of 5e7, 5e8, or 5e9. Controls were administered a diluent or 5e9vg of mis11 (missense construct) + α-Syn. Statistics: Standard one-way ANOVA with Tukey's post-hoc analysis.

[0033] [Figure 13A] Figures 13A and 13B show the distribution of rAAV-miR5 in the substantia nigra. Treated animals received rAAV-miR5 at a vector genome (vg) dose of 5e7, 5e8, or 5e8. Controls were administered a diluent or 5e9vg of mis11 (missense construct) + αSyn. The presence of vectors in the substantia nigra was assessed using specific primers designed based on BgH and RbG polyA sequences. The AAV1 / 2-A53TαSyn vector possesses BgH polyA, while rAAV-miR5 and rAAV-CAG-mis11 possess RbG polyA. Figure 13A shows the measurement of BgH polyA. Figure 13B shows the measurement of RbG polyA. [Figure 13B]Figures 13A and 13B show the distribution of rAAV-miR5 in the substantia nigra. Treated animals received rAAV-miR5 at a vector genome (vg) dose of 5e7, 5e8, or 5e8. Controls were administered a diluent or 5e9vg of mis11 (missense construct) + αSyn. The presence of vectors in the substantia nigra was assessed using specific primers designed based on BgH and RbG polyA sequences. The AAV1 / 2-A53TαSyn vector possesses BgH polyA, while rAAV-miR5 and rAAV-CAG-mis11 possess RbG polyA. Figure 13A shows the measurement of BgH polyA. Figure 13B shows the measurement of RbG polyA.

[0034] [Figure 14] This figure shows the ability of rAAV-miR5 to knock down αSyn, as measured by qRT-PCR. Treated animals received rAAV-miR5 in 5e7, 5e8, or 5e8 vector genome (vg) doses. Controls were administered a diluent or 5e9vg of mis11 (missense construct) + αSyn. Statistical analysis was performed using Tukey's multiple comparison test with standard one-way Anova. **p<0.01, *p<0.05

[0035] [Figure 15] The ability of rAAV-miR5 to knock down α-Syn in the cortex, as measured by the JESS assay, was demonstrated. Treated animals received rAAV-miR5 in 5e7, 5e8, or 5e8 vector genome (vg) doses. Controls were administered a diluent or 5e9vg of mis11 (missense construct) + α-Syn. Standard one-way ANOVA Tukey multiple comparisons were performed. ***p<0.001. Compared to negative control + ASTα-Syn.

[0036] [Figure 16]This figure shows the ability of rAAV-miR5 to inhibit dopamine loss induced by A53Tα-Syn. Treated animals received rAAV-miR5 in 5e7, 5e8, or 5e8 vector genome (vg) doses. Controls were administered a diluent or 5e9vg of mis11 (missense construct) + α-Syn. Data are expressed in absolute numbers. A standard one-way ANOVA Tukey post-hoc test was performed on the logarithmic scale values ​​of the data points compared to negative control + ASTα-Syn. ****p<0.0001, ***p<0.001, *p<0.05.

[0037] [Figure 17] This figure shows the ability of rAAV-miR5 to inhibit dopamine loss induced by A53Tα-Syn. Treated animals received doses of rAAV-miR5 vector genome (vg) at doses of 5e7, 5e8, or 5e9. Controls were administered a diluent or 5e9vg of mis11 (missense construct) + α-Syn. Multiple comparisons were performed by Tukey using standard one-way ANOVA. Compared to negative control + ASTα-Syn. ****p<0.0001, **p<0.01, *p<0.05.

[0038] [Figure 18] This shows microRNA processing determined by small RNA sequencing after rAAV-miR5 administration. G / P refers to the guide / passenger ratio.

[0039] [Figure 19] This report summarizes the results of small RNA sequencing. The endogenous miRNA mechanism was not disrupted by rAAV-miR5 overexpression. Treated animals received doses of the rAAV-miR5 vector genome (vg) at doses of 5e7, 5e8, or 5e9. At the maximum dose of 5e9vg, exogenous mature miRNAs accounted for 1% of the total endogenous miRNA pool. At the medium and low doses of 5e8 and 5e7vg, exogenous miRNAs accounted for less than 0.1% of the total endogenous miRNA pool. The diluent-treated groups and negative control groups did not have miRNAs.

[0040] [Figure 20] This figure shows the ability of rAAV-miR5 to significantly reduce human SNCA mRNA levels at a dose of 5e9vg / SN compared to the diluent injection group in SN female mice, as measured by RT-qPCR of human SNCA mRNA in SN mice. Statistical comparisons between groups were evaluated by one-way ANOVA with Tukey's multiple comparison test. Data are plotted as individual (symbol) and group mean ± SEM (bar).

[0041] [Figure 21A] Figures 21A and 21B show the in vivo distribution of the vector genome and miR5 expression in the substantia nigra of African green monkeys ("AGM") two months after administration of rAAV-miR5, respectively. The X-axis represents the three tested doses of rAAV-miR5 (3e8vg, 3e9vg, and 3e10vg / substantia nigra), and the Y-axis represents the copy number (CN) / μg of DNA of the vector genome (Figure 21A) or the CN / μg of RNA of miR5 miRNA (Figure 21B). miRNA expression analysis showed the dose-dependent distribution of rAAV-miR5 in the substantia nigra. [Figure 21B] Figures 21A and 21B show the in vivo distribution of the vector genome and miR5 expression in the substantia nigra of African green monkeys ("AGM") two months after administration of rAAV-miR5, respectively. The X-axis represents the three tested doses of rAAV-miR5 (3e8vg, 3e9vg, and 3e10vg / substantia nigra), and the Y-axis represents the copy number (CN) / μg of DNA of the vector genome (Figure 21A) or the CN / μg of RNA of miR5 miRNA (Figure 21B). miRNA expression analysis showed the dose-dependent distribution of rAAV-miR5 in the substantia nigra.

[0042] [Figure 21C]Figure 21C shows the relative expression of SNCA mRNA in the AGM administration groups. The X-axis shows the two control groups (diluent and mis11) and the three doses of rAAV-miR5 administered (3e8vg, 3e9vg, and 3e10vg / substantia nigra). The Y-axis shows the change ratio relative to the diluent group. For all groups, n=2 AGMs. Mis11 refers to untargeted miRNA expressed under the synthetic EF-1α promoter.

[0043] [Figure 21D] Figure 21D shows immunohistochemical (IHC) analysis of cytoplasmic α-synuclein protein (also referred to herein as α-Syn) intensity in tyrosine hydroxylase (TH)-positive dopaminergic (DA) neurons in the substantia nigra, demonstrating a dose-dependent decrease in cytoplasmic α-Syn protein mediated by rAAV-miR5. In each group, two AGMs (n=2 / group) were analyzed, and each data point represents the analyzed DA neurons (2000-2500 cells total / group) from a section containing SNs. The X-axis shows Group 1 (diluent), Group 2 (mis11), Group 3 (rAAV-miR5 administered at 3e8vg / substantia nigra), Group 4 (rAAV-miR5 administered at 3e9vg / substantia nigra), and Group 5 (rAAV-miR5 administered at 3e10vg / substantia nigra). The Y-axis shows α-Syn intensity. Each data point represents the average value per monkey from which two tissue punches were collected for analysis. Error bars represent SEM.

[0044] [Figure 22A]Figures 22A, 22B, and 22C provide graphical representations of the in vivo distribution analysis of vector genome copy number (CN), reported as CN per microgram of genomic DNA, at individual doses of 3e10vg / SN (Figure 21A), 3e9vg / SN (Figure 21B), and 3e8vg / SN (Figure 21C). CN was determined using primer probes targeting the unique sequence of rabbit globin polyA. The X-axis represents the basal ganglia and adjacent brain structures, and the Y-axis represents DNA in CN / μg. A dose-dependent increase in rAAV-miR5 is observed in the substantia nigra, and relatively low to moderate distributions are found in adjacent anatomically connected brain regions of basal ganglia structures (e.g., subthalamic nucleus and globus pallidus). In vivo distribution analysis of other brain regions of basal ganglia and cortical structures indicates that the rAAV-miR5 distribution is primarily contained within the target region. Each group has two AGMs (n=2 / group), and each data point represents the average value per animal (2 punches / SN). [Figure 22B] Figures 22A, 22B, and 22C provide graphical representations of the in vivo distribution analysis of vector genome copy number (CN), reported as CN per microgram of genomic DNA, at individual doses of 3e10vg / SN (Figure 21A), 3e9vg / SN (Figure 21B), and 3e8vg / SN (Figure 21C). CN was determined using primer probes targeting the unique sequence of rabbit globin polyA. The X-axis represents the basal ganglia and adjacent brain structures, and the Y-axis represents DNA in CN / μg. A dose-dependent increase in rAAV-miR5 is observed in the substantia nigra, and relatively low to moderate distributions are found in adjacent anatomically connected brain regions of basal ganglia structures (e.g., subthalamic nucleus and globus pallidus). In vivo distribution analysis of other brain regions of basal ganglia and cortical structures indicates that the rAAV-miR5 distribution is primarily contained within the target region. Each group has two AGMs (n=2 / group), and each data point represents the average value per animal (2 punches / SN). [Figure 22C]Figures 22A, 22B, and 22C provide graphical representations of the in vivo distribution analysis of vector genome copy number (CN), reported as CN per microgram of genomic DNA, at individual doses of 3e10vg / SN (Figure 21A), 3e9vg / SN (Figure 21B), and 3e8vg / SN (Figure 21C). CN was determined using primer probes targeting the unique sequence of rabbit globin polyA. The X-axis represents the basal ganglia and adjacent brain structures, and the Y-axis represents DNA in CN / μg. A dose-dependent increase in rAAV-miR5 is observed in the substantia nigra, and relatively low to moderate distributions are found in adjacent anatomically connected brain regions of basal ganglia structures (e.g., subthalamic nucleus and globus pallidus). In vivo distribution analysis of other brain regions of basal ganglia and cortical structures indicates that the rAAV-miR5 distribution is primarily contained within the target region. Each group has two AGMs (n=2 / group), and each data point represents the average value per animal (2 punches / SN).

[0045] [Figure 22D] Figure 22D shows the overall SNCA mRNA knockdown profile in key brain regions by rAAV-miR5. RT-qPCR analysis of SNCA mRNA showed a dose-dependent decrease in SN (>50% at dose 3e10vg / SN) compared to a control group (including diluent-treated animals and a mis11 control group to account for basal fluctuations in endogenous SNCA levels) 2 months after necropsy. Consistent with in vivo distribution analysis and miRNA expression analysis, knockdown of target gene expression was limited to the SN and parts of anatomically linked basal ganglia structures. Each group had two AGMs (n=2 / group), and each data point represents the mean per punch per animal. [Modes for carrying out the invention]

[0046] The present invention is characterized by RNA polynucleotide constructs that target α-synuclein mRNA and include nucleic acid encoding sequences. Examples of RNA polynucleotide constructs include inhibitory RNA polynucleotides containing α-synuclein mRNA targeting sequences, such as pri-miRNA, pre-miRNA, shRNA, miRNA, and optionally modified miRNA.

[0047] In certain embodiments, the inhibitory RNA polynucleotide is an inhibitory RNA duplex. When we refer to an "inhibitory RNA duplex," we mean a double-stranded RNA comprising a guide strand and a passenger strand that target an RNA target region. The passenger strand is sufficiently complementary to the guide strand to hybridize under physiological conditions. The guide strand associated with the RNA-induced silencing complex (RISC) can inhibit target RNA activity. In the case of mRNA targets, for example, it can inhibit protein expression from the target mRNA.

[0048] Inhibitory RNA duplexes such as miRNAs can be produced, for example, by gene expression that produces pri-miRNA, pre-miRNA, or shRNA, and then biosynthesizes them, or they can be produced by chemosynthesis.

[0049] A pri-miRNA scaffold can incorporate a guide sequence and a passenger sequence to produce pri-miRNA. The guide strand can be selected to target different types of RNA sequences or regions, such as the 5'UTR, 3'UTR, or mRNA. Different passenger sequences can be incorporated into the pri-miRNA scaffold for a given guide sequence. The passenger strand can be optimized for a specific scaffold and provide sufficient complementarity to the guide strand for hybridization under physiological conditions.

[0050] Constructs containing α-synuclein mRNA targeting sequences can be used, for example, in or to produce inhibitory RNA, which inhibits α-synuclein expression and / or can treat diseases or disorders of synucleinopathy. Inhibition of α-synuclein expression can be performed, for example, for therapeutic and research purposes. Research purposes include investigating the effects of inhibiting α-synuclein expression in animal models.

[0051] The term "subject" refers to mammals such as humans, apes, gibbons, gorillas, chimpanzees, orangutans, macaques, pets like dogs and cats, poultry, domesticated animals like ducks, horses, cows, goats, sheep, and pigs, and laboratory animals like mice, rats, rabbits, or guinea pigs. The preferred subject is humans.

[0052] In certain embodiments, α-synuclein mRNA targeting inhibitory RNAs are evaluated using transgenic mice expressing human A53T mutant α-synuclein. (See, for example, Menon et al., Brain Communications, Volume 3, Issue 4, 2021, fcab247, hypetext: / / doi.org / 10.1093 / braincomms / fcab247, which is incorporated herein by reference in its entirety.)

[0053] When referring to percentages of “identical,” “identical,” and similar terms, they relate to two sequences that have maximum alignment in a particular region. The provided region is with respect to the indicated reference sequence. For example, a sequence that is “identical” or “identical” to a target sequence can be calculated by determining the number of identical nucleotides in the sequences aligned to provide maximum identity, dividing by the total number of nucleotides in the target sequence, and multiplying by 100. Differences between aligned sequences may include one or more deletions, substitutions, and / or additions.

[0054] When we refer to an indicated percentage of identity or identity with one or more reference sequences, and throughout the specification, any similar expression defining an indicated percentage of identity or identity with one or more reference sequences defines that percentage or percentage range independently for each referenced sequence. For determining identity or identity, RNA and corresponding DNA are considered identical unless otherwise indicated (e.g., referring to the molecules as DNA or RNA). The corresponding RNA of DNA provides uracil instead of thymine and a ribose backbone instead of a deoxyribose backbone.

[0055] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to different forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), unless otherwise specified. When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of providing the sequence in the 5' to 3' direction.

[0056] In certain embodiments, nucleic acids include naturally occurring polynucleotides, synthetic polynucleotides, and intentionally modified or altered polynucleotides. Unless otherwise indicated by the context, nucleic acids can be single-stranded, double-stranded, or triple-stranded, linear or cyclic, and of any length.

[0057] According to certain embodiments, a polynucleotide is a single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule. According to certain embodiments, a dsDNA molecule is a minicircle, nanoplasmid, open-chain linear double-stranded DNA, or closed-end linear double-stranded DNA (CELiD / ceDNA / doggybone DNA). According to certain embodiments, an ssDNA molecule is a closed-circle or open-chain linear DNA.

[0058] A "transgene" refers to a nucleic acid that is intended to be introduced into a cell and is operably linked to a promoter. Transgenes include nucleic acids that encode heterologous polynucleotide sequences and / or heterologous promoters.

[0059] In certain embodiments, the encoded polynucleotide construct is “CpG-depleted” or “CpG-depleted.” In certain embodiments, the “CpG-depleted” or “CpG-depleted” region is the region outside the pri-miRNA or pre-miRNA sequence. “CpG-depleted” or “CpG-depleted” means a nucleotide sequence in which (i) one or more CpG dinucleotides (or motifs) have been removed from the reference nucleic acid sequence, and / or (ii) the percentage of CpG in the polynucleotide referred to is between 0% and 10%. In different embodiments, the CpG percentage is 0–5%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0060] In certain embodiments, the CpG motif is reduced in the 5' and / or 3' untranslated region (UTR), stuffer sequence, promoter, enhancer, polyadenylation signal, 5' and / or 3' ITR, and / or intron.

[0061] The singular forms ("a," "an," and "the") include references to the plural form unless the context clearly indicates otherwise.

[0062] The conjunction term "and / or" between multiple enumerated elements encompasses both individual choices and combined choices. For example, when two elements are joined by "and / or," the first choice refers to the applicability of the first choice without the second choice, the second choice refers to the applicability of the second choice without the first choice, and the third choice refers to the applicability of the first and second choices together. Any one of the choices is understood to be within its scope of meaning and therefore satisfy the requirements of the term "and / or." The simultaneous applicability of two or more choices is also understood to be within the scope of meaning of the term "and / or."

[0063] Unless otherwise indicated by the context in which they are used, the terms "or" and "and" have the same meaning as "and / or" when applied to different alternative members in a list.

[0064] References that are followed by different members or examples after terms such as "including," "for example," "eg," or "such as" are open-ended descriptions in which the listed members or examples are illustrative and can provide or use other members or examples.

[0065] The terms "polypeptide," "protein," and "peptide" can be used interchangeably to refer to amino acid sequences regardless of function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about ten amino acids. The amino acids provided include naturally occurring amino acids and those provided by cellular modifications.

[0066] The terms “comprise,” as used in reference to an element or group of elements, and variations such as “comprises” and “comprising,” are open-ended and do not exclude additional unlisted elements or method steps. Terms such as “including,” “containing,” and “characterized by” are synonymous with including. In the different embodiments and examples described herein, open-ended terms such as “comprising” may be replaced with “consisting” or “consisting essentially of.”

[0067] When the term "consisting of" is used in relation to elements, steps, or components listed in the claimed invention, any elements, steps, or components not explicitly stated in such claims are excluded.

[0068] When the phrase "essentially consists of..." is used, the scope of the claims is limited to specific materials or processes, or to those that do not substantially affect the fundamental and novel features of the claimed invention.

[0069] The term "approximately" refers to a value within 10% (i.e., plus or minus 10%) of the underlying parameter. For example, "approximately 1:10" includes 1.1:10.1 or 0.9:9.9, and "approximately 5 hours" includes 4.5 hours or 5.5 hours. The term "approximately" at the beginning of a set of values ​​modifies each value by 10%. In certain embodiments, the term "approximately" refers to a value within 10% of the underlying parameter.

[0070] Every number or range of numbers includes integers within that range, fractions of those values, or integers within that range, unless the context clearly indicates otherwise. For example, when we say a decrease of 95% or more, we include 95%, 96%, 97%, 98%, 99%, 100%, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., as well as 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. When we say a range of numbers such as "1-4", we include 1, 2, 3, 4, as well as 1.1, 1.2, 1.3, 1.4, etc. As a further example, “1 to 4 weeks” includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0071] Furthermore, when referring to a numerical range such as "0.01 to 10," this includes 0.011, 0.012, 0.013, as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dosage for a target body weight of approximately "0.01 mg / kg to approximately 10 mg / kg" includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

[0072] When we say "greater than" or "less than" an integer, it includes numbers greater than or less than the number being referenced. For example, "greater than 2" includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, and "two or more doses" includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more doses.

[0073] Various references, including papers and patent publications, are cited or mentioned throughout the background art and specification. Each of these references is incorporated herein by reference in its entirety. None of the references are considered prior art with respect to any of the disclosed or claimed inventions. In some cases, to emphasize the incorporation, it is indicated that a particular reference is incorporated herein by reference.

[0074] The definitions provided herein, including those defined in this section and other sections of this application, apply to the entire application.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0076] The detailed description of the invention is separated into various sections and paragraphs, providing examples of various embodiments. These separations should not be considered to separate the content of one paragraph or section or embodiment from the content of another paragraph or section or embodiment. The provided description is broadly applicable and encompasses all possible combinations of sections, paragraphs, and sentences. Any description of an embodiment is for illustrative purposes only and does not imply that the scope of this disclosure, including the claims (unless otherwise stated in the claims), is limited to these examples.

[0077] The present invention is disclosed herein using affirmative language to describe a number of embodiments of the invention in a broad sense. The invention also includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is excluded all or in part. For example, in certain embodiments of the invention, materials and / or method steps are excluded. Thus, with respect to things not included in the invention, embodiments that are not expressly excluded in the invention are nevertheless disclosed herein, even if the invention is not stated herein that the invention does not broadly exclude them.

[0078] I. RNA polynucleotides that target α-synuclein mRNA For example, various α-synuclein mRNA target regions are provided by the reverse complements of any of sequence numbers 129-135 and any of sequence numbers 32-62. RNA polynucleotides that target a particular target region contain a sequence substantially complementary to the target region. A substantially complementary sequence contains a region of at least 10 nucleotides that is at least 70% complementary, at least 80% complementary, at least 90% complementary, or 100% complementary. The degree of complementarity is determined based on sequences aligned for maximum complementarity. Differences between complementary sequences include additions, deletions, and non-complementary bases. Preferably, substantially complementary sequences can hybridize with each other under physiological conditions.

[0079] In certain embodiments, substantially complementary sequences include regions of 10–25 nucleotides, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, which, when aligned to have maximum complementarity, are fully complementary, at least 70% complementary, at least 80% complementary, or at least 90% complementary.

[0080] In certain embodiments, substantially complementary sequences include regions of 10–25 nucleotides, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, which, when aligned to have maximum complementarity, are perfectly complementary except for a difference of 1, 2, or 3 nucleotides.

[0081] In certain embodiments, the constructs containing the α-synuclein mRNA targeting sequence are inhibitory RNA duplexes, pri-miRNAs, pre-miRNAs, or shRNAs. The α-synuclein mRNA targeting sequences present in these constructs provide a guide strand sequence that can hybridize to the α-synuclein mRNA. Such constructs also include a passenger strand sequence substantially complementary to the guide strand. The degree of complementarity of the guide to the passenger strand can vary, for example, considering sufficient complementarity for hybridization and use as a specific scaffold. In certain embodiments, the guide and passenger strands are either perfectly complementary or have a mismatch of 1, 2, 3, or 4 nucleotides from perfect complementarity when aligned for maximum complementarity. Each mismatch may independently be a non-complementary base, addition, and / or deletion.

[0082] In certain embodiments, the RNA polynucleotide includes (1) a targeted sequence containing a region that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the RNA sequences in SEQ ID NOs. 32-62, or (2) a sequence that differs by 0, 1, 2, or 3 nucleotides from any of the RNA sequences in SEQ ID NOs. 32-62, or (4) a sequence that differs by 0, 1, 2, or 3 nucleotides from any of the RNA sequences in SEQ ID NOs. 94, 95, 96, 97, 98, 99, or 100. In certain embodiments, the nucleotide difference is a substitution.

[0083] Table 1 shows various combinations of guide and seed sequences. [Table 1]

[0084] In certain embodiments, the targeted RNA sequence includes: a) A sequence in which sequence number 34 and 0, 1, 2, or 3 nucleotides are different, and the target sequence includes the sequence of sequence number 108, b) A sequence in which sequence 0, 1, 2 or 3 nucleotides are different from sequence number 36, and the target sequence includes the sequence of sequence number 109, c) A sequence in which sequence number 37 and 0, 1, 2, or 3 nucleotides are different, and the target sequence includes the sequence of sequence number 110, d) A sequence in which sequence number 38 is different from sequence number 0, 1, 2 or 3 nucleotides, and the target sequence includes the sequence of sequence number 111, e) A sequence in which sequence number 39 and 0, 1, 2, or 3 nucleotides are different, and the target sequence includes the sequence of sequence number 112, f) Sequences in which 0, 1, 2 or 3 nucleotides are different from sequence number 41, and the target sequence includes the sequence of sequence number 113, and g) A sequence in which 0, 1, 2 or 3 nucleotides differ from sequence number 42, and the targeting sequence includes the sequence of sequence number 114. In the specific embodiments of (a) to (g), any nucleotide difference is a substitution, if present.

[0085] In certain embodiments, the guide sequence includes, consists of, or is essentially composed of, a targeting sequence, and the RNA polynucleotide further includes a passenger sequence substantially complementary to the guide.

[0086] In certain embodiments, the passenger sequence and guide sequence are provided in the combinations shown in Table 2, the guide chain contains, essentially consists of, or comprises the indicated targeting sequence, and the passenger sequence contains, essentially consists of, or comprises the indicated sequence or a sequence different from the indicated sequence by one, two, or three nucleotide substitutions.

[0087] [Table 2]

[0088] In certain embodiments, the passenger-guide strand coding combinations provided in Table 2 are present in pri-miRNA, pre-miRNA, shRNA, or inhibitory RNA duplexes.

[0089] IA inhibitory RNA duplex

[0090] Inhibitory RNA duplexes can inhibit mRNA activity in conjunction with the RNA-induced silencing complex (RISC). The term RISC includes related proteins such as the core algonaut (AGO) protein. While not limited to any specific theory, the guide strand is loaded into the RISC and hybridizes to the target mRNA, with hybridization initiated at a seed region located n-2 to approximately n-8 at the 5' end of the guide strand.

[0091] In certain embodiments, the seed region is either perfectly complementary to its mRNA target or differs from perfect complementarity by only one nucleotide.

[0092] Preferably, the passenger chain is discarded and broken down by RISC. In different embodiments, the guide chain ratio to passenger chain ratio is about 25:1, about 15:1, or about 10:1.

[0093] Depending on the degree of complementarity and the location of the non-complementary region, RISC associated with the guide strand may cause mRNA cleavage or translational repression. (For example, as described in Lam et al., Mol.Ther.Nucleic Acids. (2015) 4(9):e252 and Kobayashi et al., ACS Omega. (2022) 7(2):2398-2410, both of which are incorporated herein by reference in their entirety.)

[0094] In certain embodiments, the guide and passenger strands are independently 18 to 25 nucleotides long and each includes a 3' overhang of 1 to 5 nucleotides. In further embodiments, each overhang is 1, 2, 3, or 4 nucleotides. In further embodiments, each guide and passenger strand is independently 20, 21, 22, or 23 nucleotides, and each overhang is 2 or 3 nucleotides. In further embodiments, each guide and passenger strand is 21, 22, or 23 nucleotides, and each overhang is 2 nucleotides.

[0095] IBmiRNA

[0096] The delivery of inhibitory RNA duplexes to their target is facilitated using precursor constructs such as pri-miRNA, pre-miRNA, and shRNA. These various constructs include stem-loop structures, guide strand sequences, and passenger strand sequences, and utilize the intracellular miRNA biosynthesis mechanism to form inhibitory RNA duplexes. Such constructs can be provided, for example, using transgenes encoding inhibitory RNA.

[0097] The production and processing of naturally occurring miRNAs involve the transcription of pri-miRNA from miRNA genes, as well as the cleavage of the 3' and 5' regions mediated by Drosha-DGCR8, to form pre-miRNAs. The resulting pre-miRNAs contain a stem with a nucleotide overhang at the 3' end and a tip stem-loop. (See, for example, Jin et al., Mol. Cell. 2020 May 7;78(3):423-433, which is incorporated herein in its entirety by reference.)

[0098] A pri-miRNA consists of a 5' flanking sequence, a guide / passenger sequence, a tip loop, a guide / passenger sequence, and a 3' flanking sequence. The guide sequence can be either the 5' or 3' of the loop. If the guide sequence is the 5' of the loop, the passenger sequence is the 3' of the tip loop; if the guide sequence is the 3' of the loop, the passenger sequence is the 5' of the loop.

[0099] The 5' and 3' flanking regions constitute part of the stem region and further include a single-stranded region. The lengths of the flanking regions can vary independently. In certain embodiments, the 5' and 3' pri-miRNA flanking regions are at least 20 nucleotides, at least 50 nucleotides, or at least 100 nucleotides, respectively.

[0100] A pri-miRNA scaffold refers to the pri-miRNA region excluding the guide and passenger sequences. The scaffold can function as a delivery vehicle for the biosynthesis of various inhibitory RNA duplexes, with the guide and passenger sequences embedded within it.

[0101] Scaffolds into which guide and passenger strand sequences can be incorporated may be naturally occurring pri-miRNAs, modifications to naturally occurring miRNAs, or artificially designed to take into account the characteristics of pri-miRNAs. (See, for example, Xie et al., Mol.Ther.(2020)28(2):422-430, Fowler et al., Nucleic Acids Research(2016),44(5):e48, Roden et al.,(2017)Genome Res.27(3):374-384; Jin et al.,(2020)Mol.Cell.7;78(3):423-433, and Fang and Bartel Genes.Mol Cell.(2015)60(1):131-145, each of which is incorporated herein by reference in its entirety.)

[0102] Design considerations for pri-miRNA scaffolds include stem length, loop size, and the presence of specific motifs that can enhance biosynthesis and / or inhibitory RNA. Pri-miRNA stems can contain various structures, including GU or UG wobbles, single base pair mismatches, bulges, and multiple base pair mismatches.

[0103] In certain embodiments, the pri-miRNA scaffold includes a UG motif on the 5' arm at a position -14 or -13 relative to the drosha 5' cleavage site, and / or a CNNC on the 3' arm between positions +14 and +18 relative to the drosha 3' cleavage site.

[0104] In certain embodiments, the pri-miRNA scaffold is the miR-1, miR-26, miR16-1, miR-30, miR-33, miR-101, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold. (U.S. Patent No. 10,457,940, Miniarikova et al.,Mol.Ther.Nucleic Acids(2016)Mar 22;5(3):e297, Fowler et al.,Nucleic Acids Research(2016),44(5):e48,Xie et al.,Molecular Therapy(2020)28:2 422-430, Fang and Bartel Molecular Cell (2015) 60, 131-145, Roden et al. (2017) Genome Res. 27, 374-384, and Calloni and Bonatto, Human Gene Therapy Methods (2015) 25(5): 162-174).

[0105] In certain embodiments, the pri-miRNA contains a mismatched GHG motif in the 3' arm of the stem.

[0106] In certain embodiments, the length of the pri-miRNA stem is 33, 34, 35, 36, 37, 38, or 39 nucleotides.

[0107] In certain embodiments, the length of the pri-miRNA stem is 34, 35, or 36 nucleotides.

[0108] In certain embodiments, the pri-miRNA tip loop is 3 to 23 nucleotides long. In further embodiments, the loop is 10 to 23 nucleotides long. In certain embodiments, the guide and passenger strands do not extend into the loop. In certain embodiments, the guide and / or passenger strands extend into the loop.

[0109] In certain embodiments, the pri-mRNA contains a 5'7-methylguanylate (m7G) cap.

[0110] In certain embodiments, the pri-miRNA contains, consists of, or is essentially derived from, any sequence of sequence 115–121, or any sequence of sequence 0, 1, 2, 3, or 4 nucleotides different from sequence 115–121. The nucleotide differences can be introduced at various positions, taking into account the guidance provided herein, e.g., the characteristics of various pri-miRNA scaffolds, the complementarity of the guide to the target, the complementarity of the passenger strand to the guide strand, and the adjustments to a particular scaffold.

[0111] Pre-miRNA produced by drosate cleavage of pri-miRNA is transported from the nucleus to the cytoplasm, where it is cleaved by a dicer to produce inhibitory RNA duplexes. The term "pre-miRNA" here refers to RNA polynucleotides, including those containing guide and passenger strands as described herein, that can be cleaved by a dicer to produce inhibitory RNA duplexes.

[0112] In certain embodiments, the dicer-inhibiting RNA duplex comprises a guide strand and a passenger strand, each approximately 22 nucleotides long, with each strand containing a 2-nucleotide overhang at its 3' end. (Fang and Bartel (2015) Molecular Cell, 60:131-145).

[0113] In certain embodiments, the pre-miRNA contains, consists of, or is essentially derived from, any of the sequences of SEQ ID NOs. 122–128, or any of the sequences of SEQ ID NOs. 122–128 differs from 0, 1, 2, 3, or 4 nucleotides. The nucleotide differences can be introduced at different positions, such as guide sequences, loop sequences, and / or passenger sequences.

[0114] In certain embodiments, pri-miRNA is introduced into cells using a transgene containing a sequence encoding the pri-miRNA sequence. The biosynthesis of pri-miRNA results in the production of an inhibitory RNA duplex. The expression cassette containing the transgene can be introduced into cells using a viral or non-viral delivery vehicle.

[0115] In certain embodiments, shRNA is introduced into cells using a transgene containing a sequence encoding the shRNA sequence. The shRNA is structurally and functionally similar to pre-miRNA. The shRNA contains an RNA duplex with substantially complementary arms and loops, which can be transported from the nucleus into the cytoplasm and cleaved by a dicer to form an inhibitory RNA duplex. (Aguiar et al., Transl. Neurodegener. (2017) Nov 27;6:30).

[0116] IC scaffolding

[0117] In certain embodiments, the pri-miRNA scaffold is any of the S155, S155e, S26, S126 or S33 scaffold. The S155 scaffold corresponds to the miR155 scaffold and was used for miR3, miR5, miR6, miR7, miR8, miR10 and miR11 described in the Examples below. The S155e scaffold corresponds to the enhanced miR155 scaffold. (See Fowler et al., Nucleic Acids Research (2016), 44(5):e48). The S33 scaffold corresponds to the scaffold present in miR33. (See Xie et al., Molecular Therapy (2020) 28:2 422-430 2020). The S126 scaffold is an artificial scaffold produced starting from the miR26 scaffold. Various scaffolds can be used to incorporate various guide strands and passenger strands.

[0118] In certain embodiments, the S126 scaffold can be used to incorporate various guide strands and passenger strands, and said scaffold comprises SEQ ID NO: 186: N 01 N 02 N 03 N 04 N 05 N 06 N 07 N 08 N 09 N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 N 19 N 20 N 21 UGUGCAGGUCCCAN 22 N 23 N 24 N 25 N 26 N 27 N 28 N 29 N 30 N 31 N 32 N 33 N 34 N35 N 36 N 37 N 38 N 39 N 40 N 41 N 42 CG (In the formula, N 01 ~N 42 N is a ribonucleotide. 01 is N 42 It is complementary to N 02 is N 41 Not complementary to N 03 ~N 10 is N 33 ~N 40 It is complementary to N 11 is N 32 Not complementary to N 12 ~N 21 is N 22 ~N 31 (Complementary to). In certain embodiments, the passenger chain is N 01 -N 21 Provided by U, where the targeting sequence is N 01 -N 21 It can be inserted into, and the guide chain is N 23 -N 42 Provided by G. The corresponding DNA can be used to encode an RNA sequence.

[0119] In a particular embodiment, the RNA sequence containing SEQ ID NO: 186 has the following structure: [ka]

[0120] In different embodiments, the RNA sequence containing SEQ ID NO: 186 can be diced by a dicer, or by a dross and dicer.

[0121] In a particular embodiment relating to the RNA sequence containing SEQ ID NO: 186, the RNA sequence further comprises a 5' facile region and a 3' facile region, and the polynucleotide comprises the RNA sequence of SEQ ID NO: 187: GUGGCCGN 01 N 02 N 03 N 04 N 05 N 06 N 07 N 08 N 09 N 10 N 11 N 12 N 13 N 14 N 15 N 16 N 17 N 18 N 19 N 20 N 21 UGUGCAGGUCCCAN 22 N 23 N 24 N 25 N 26 N 27 N 28 N 29 N 30 N 31 N 32 N 33 N 34 N 35 N 36 N 37 N 38 N 39 N 40 N 41 N 42 CGGGGACGC (In the formula, N 01 to N 42 are ribonucleotides, N 01 is complementary to N 42 , N 02 is not complementary to N 41 , N 03 to N 10 are complementary to N 33 to N 40 , N 11 is not complementary to N 32 , and N 12 to N 21 are complementary to N 22 to N 31 ). The corresponding DNA can be used to encode the RNA sequence.

[0122] In certain embodiments, an inhibitory RNA polynucleotide targeting α-synuclein is incorporated into an S126 scaffold containing SEQ ID NO: 186 or 187, wherein (1) the guide strand contains the sequence of SEQ ID NO: 95 and the passenger strand contains a sequence selected from the group consisting of SEQ ID NOs: 189-197, or (2) the guide strand contains the sequence of SEQ ID NO: 97 and the passenger strand contains a sequence selected from the group consisting of SEQ ID NOs: 198-206. Tables 3 and 4 show the passenger strand combinations for guide strands of SEQ ID NOs: 95 and SEQ ID NO: 97. Nucleotides shown in bold are located in the bulge formed by non-complementary nucleotides.

[0123] [Table 3]

[0124] [Table 4]

[0125] In certain embodiments, the passenger-guide strand combinations provided in Tables 3 and 4 are present in pri-miRNA, pre-miRNA, shRNA, or inhibitory RNA duplexes. Examples of pre-miRNAs containing the sequence combinations in Table 3 within an S126 scaffold are provided by SEQ ID NOs. 207-215, and examples of pre-miRNAs containing the sequence combinations in Table 4 within an S126 scaffold are provided by SEQ ID NOs. 216-224. Longer examples of pri-miRNAs containing the sequence combinations in Table 3 within an S126 scaffold are provided by the RNA versions (ribose backbone and U instead of T) in SEQ ID NOs. Longer examples of pri-miRNAs containing the sequence combinations in Table 4 within an S126 scaffold are provided by the RNA versions (ribose backbone and U instead of T) in SEQ ID NOs. 252-260.

[0126] ID-modified inhibitory RNA

[0127] An alternative method for providing inhibitory RNA duplexes through transgene expression and biosynthesis is direct administration of the inhibitory RNA duplex. Direct administration can be facilitated using modified RNA. Modified inhibitory RNA duplexes can be produced, for example, by modifying one or more nucleotides of the inhibitory RNA duplex. For example, the inhibitory RNA duplex may be modified to improve pharmacokinetics, enhance activity, suppress innate immune activation, improve targeting, and reduce extratarget toxicity.

[0128] Modifications to inhibitory RNA can be performed at various locations, including the 5' end, 3' end, sugar moiety, phosphate group, and nucleobase. Various types of modifications can be performed to enable or promote Watson-Crick hybridization to the target and inhibitory RNA activity. References describing various modifications and modification patterns include Hu et al., Sig Transduct Target Ther., (2020) 5, 101; Varley and Desauniers (2021) RSC Adv., 11, 2415; Alterman et al., Nature Biotechnology (2019) 37:884-894; International Publication WO2021 / 252649; and U.S. Patent Application Publication 2022 / 125823, each of which is incorporated herein by reference in its entirety.

[0129] In certain embodiments, the inhibitory RNA is a 5' phosphate mimetic (e.g., 5-(E)-vinyl phosphate, 5'-methylene phosphonate, 5'-(R)-methyl phosphate, 5'-(S)-methyl phosphate, 5'(R)-MeOCH3 phosphate, 5'-(S)-methyl-F phosphate, 5-deoxy-5'-morpholino-2'O-methyluridine, and phosphorothioate), or an internal phosphate modification (e.g., Rp phosphorothioate). Oate, Sp phosphorothioate, phosphorodithioate, methoxyphosphonate, phenylethyl phosphate, 2'-5' phosphate bond and amide bond), one or more nucleic acid base modifications (e.g., 5'-nitroindole, pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2'deoxyuridine, N-ethylpiperidine, 7'-EAA triazole-modified adenine) (N-ethylpiperidine 6'-triazole-modified adenine, 6'-phenylpyrrolocytosine, or 2',4'-difluorotoluyl ribonucleotide), sugar modification (e.g., 2'-O-methyl, 2'-O-methoxyethyl, 2'-deoxy-2'-fluoro, 2'-O-(p-oxazol-2-yl)benzyl, (2'S)-2-deoxy-2'-C-methyl, (2'R)-2-deoxy-2'-C-methyl, 2'-arabino- The modifications include one or more modifications selected from lulo, 2'O-benzyl, 2'O-methyl-4-pridin, locked nucleic acids, (s)-cET-BNA, tricyclo-DNA, PMO, unlocked nucleic acids, glycol nucleic acids, althritol, and nucleic acids, as well as conjugation groups (e.g., lipophilic groups, hydrophilic groups, cholesterol, GalNAc, docosahexaenoic acid, or docosahexaenoic acid having a phosphocholine head group).

[0130] In certain embodiments, at least 50%, at least 60%, at least 70%, at least 90%, or 100% of the nucleotides are modified.

[0131] In certain embodiments, the inhibitory RNA duplex is a modified bivalent inhibitory RNA duplex. In further embodiments, the modified bivalent inhibitory RNA duplex is fully chemically stabilized and comprises one or more 2'-OMe, 2'-F, phosphorothioate, phosphodiester, and 5' vinylphosphonate. (See, for example, Alterman et al., Nature Biotechnology (2019) 37:884-894, whose entirety is incorporated herein by reference).

[0132] Modified RNA can be produced using different techniques, such as stepwise synthesis of RNA and / or modification of the produced RNA sequence. The produced RNA sequence itself can be constructed, for example, by stepwise synthesis or using coding nucleic acids.

[0133] II. Nucleic acids encoding RNA polynucleotides containing targeted sequences RNA polynucleotides containing a targeting sequence can be produced from a polynucleotide using a nucleic acid sequence that codes for the RNA polynucleotide. With respect to RNA polynucleotides, the coding nucleic acid sequence provides the same sequence as the corresponding RNA polynucleotide, and if the coding nucleic acid is DNA, the DNA has the nucleic acid base thymine instead of uracil and deoxyribose instead of ribose. RNA polynucleotides are produced from a template strand complementary to the coding strand. The coding strand can, for example, provide a template and / or be used to produce the template strand.

[0134] A polynucleotide containing a nucleic acid sequence encoding an RNA polynucleotide may contain further components such as those that promote the production of RNA polynucleotides, those that promote the delivery of polynucleotides as viral vectors, and / or those that provide further activity. Further activity may be provided, for example, by encoding a protein and / or by encoding a sequence that provides one or more further inhibitory RNAs.

[0135] The sequences providing further inhibitory RNA may be the same or different, and may be directed to the same or different targets. Examples of different configurations include encoding two or more pri-miRNAs of the same sequence, encoding two or more pri-mRNAs having the same guide strand but different scaffolds, and / or encoding two or more pri-miRNAs having different guide strands. Different guide strands may be directed to the same target, e.g., the same mRNA, or to different targets, e.g., different mRNAs. In certain embodiments, the polynucleotide encodes one, two, three, four, or five pri-miRNAs, where each pri-miRNA may be the same or different.

[0136] The same type of coding structure that provides pri-mRNA can be used for polynucleotides that code for shRNA. In certain embodiments, the polynucleotide codes for 1, 2, 3, 4, or 5 shRNAs, each of which may be the same or different.

[0137] The production of RNA polynucleotides containing a targeted sequence is facilitated using an expression cassette. An expression cassette comprises a nucleic acid sequence encoding an RNA polynucleotide and one or more expression regulatory elements operably ligated to the RNA polynucleotide encoding nucleic acid sequence. An expression cassette may, for example, contain nucleic acid sequences encoding the same or different RNA polynucleotides (e.g., different pri-miRNAs or shRNAs), and the different polynucleotides may be operably ligated to the same or different expression elements. For example, the same promoter may be ligated to nucleic acid sequences providing multiple pri-miRNAs, or two or more pri-miRNAs may be operably ligated to different promoters.

[0138] In certain embodiments, a polynucleotide encoding an RNA polynucleotide includes (1) a sequence that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any sequence in sequence numbers 136-142, (2) a sequence that includes at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides in any sequence number 136-142, and / or (3) a sequence that differs from sequence number 136, 137, 138, 139, 140, 141, or 142 by only 0, 1, 2, or 3 nucleotides.

[0139] Table 5 shows various DNAs that encode target sequence and seed combinations. [Table 5]

[0140] In certain embodiments, the coding DNA target sequence includes: a) A sequence in which sequence number 136 differs from sequence number 0, 1, 2, or 3 nucleotides, and the target sequence includes the sequence of sequence number 143, b) A sequence in which sequence number 137 and 0, 1, 2, or 3 nucleotides are different, and the target sequence includes the sequence of sequence number 144, c) A sequence in which sequence number 138 and 0, 1, 2, or 3 nucleotides are different, and the target sequence includes the sequence of sequence number 145, d) A sequence in which sequence number 139 differs from sequence number 0, 1, 2, or 3 nucleotides, and the target sequence includes the sequence of sequence number 146, e) A sequence in which sequence number 140 and 0, 1, 2, or 3 nucleotides are different, and the target sequence includes the sequence of sequence number 147, f) Sequences in which 0, 1, 2 or 3 nucleotides are different from sequence number 141, and the target sequence includes the sequence of sequence number 148, and g) A sequence in which sequence number 142 differs from sequence number 0, 1, 2, or 3 nucleotides, and the target sequence includes the sequence of sequence number 149. In the specific embodiments of (a) to (g), any nucleotide difference is a substitution, if present.

[0141] In certain embodiments, the targeting sequence is a guide chain sequence, and the polynucleotide further comprises a sequence encoding a passenger sequence substantially complementary to the targeting sequence.

[0142] In certain embodiments, the encoding passenger sequence and guide sequence are provided in combinations shown in Tables 6-8, where the encoding guide sequence includes, essentially consists of, or comprises the indicated sequence, and the encoding passenger sequence includes, essentially consists of, or comprises the indicated sequence or a sequence that differs from the indicated sequence by only one, two, or three nucleotides.

[0143] [Table 6]

[0144] [Table 7]

[0145] [Table 8]

[0146] In certain embodiments, the combination of passenger strand and guide strand encoding is as provided in Tables 6, 7, or 8, and the polynucleotide encodes pri-miRNA, pre-miRNA, or shRNA.

[0147] In certain embodiments, a polynucleotide encoding an RNA polynucleotide includes (1) a sequence that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences in SEQ ID NOs. 157-163, (2) a sequence that includes at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides, and / or (3) a sequence that differs from SEQ ID NOs. 157, SEQ ID NOs. 158, SEQ ID NOs. 159, SEQ ID NOs. 160, SEQ ID NOs. 161, SEQ ID NOs. 162, or SEQ ID NOs. 163 by 0, 1, 2, or 3 nucleotides.

[0148] In a particular embodiment, the polynucleotide is (1) a sequence that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any sequence of SEQ ID NOs. 225-260, (2) at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any sequence of SEQ ID NOs. 225-260, and / or (2) sequence of SEQ ID NOs. 225, sequence The sequence contains or codes with 0,1,2 or 3 nucleotides different from any of the following: sequence number 226, sequence number 227, sequence number 228, sequence number 229, sequence number 230, sequence number 231, sequence number 232, sequence number 233, sequence number 234, sequence number 235, sequence number 236, sequence number 237, sequence number 238, sequence number 239, sequence number 240, sequence number 241, and sequence number 242, sequence number 243, sequence number 244, sequence number 245, sequence number 246, sequence number 247, sequence number 248, sequence number 249, sequence number 250, sequence number 251, sequence number 252, sequence number 253, sequence number 254, sequence number 255, sequence number 256, sequence number 257, sequence number 258, sequence number 259, and sequence number 260.

[0149] In certain embodiments, the polynucleotide comprises a sequence encoding a pre-miRNA or pri-miRNA, and the polynucleotide comprises a guide / passenger combination provided in Table 6, (1) any sequence of SEQ ID NOs. 157-163 and at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 1 (2) Contains sequences that are 00% identical and / or contain sequences that differ by 0, 1, 2 or 3 nucleotides from SEQ ID NOs. 157, SEQ ID NOs. 158, SEQ ID NOs. 159, SEQ ID NOs. 160, SEQ ID NOs. 161, SEQ ID NOs. 162 or SEQ ID NOs. 163, and such sequences (including 0% identical or different) are based on the following combinations of DNA encoding guide / passenger / pre-miRNA: SEQ ID NOs. 136 / 150 / 157, SEQ ID NOs. 137 / 151 / 158, SEQ ID NOs. 138 / 152 / 159, SEQ ID NOs. 139 / 153 / 160, SEQ ID NOs. 140 / 154 / 161, SEQ ID NOs. 141 / 155 / 162, and SEQ ID NOs. 142 / 156 / 163.

[0150] In certain embodiments, the polynucleotide comprises a sequence encoding a pre-miRNA or pri-miRNA, and the polynucleotide comprises a guide / passenger combination provided in Table 7, and (1) comprises a sequence that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of sequence numbers 225-233, and / or (2) sequence number Sequence number 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, and SEQ ID NO: 233 contains a sequence that differs by 0, 1, 2, or 3 nucleotides from any of these sequences (including percent identity or difference) based on the following combinations of DNA encoding guide / passenger / pre-miRNA: SEQ ID NO: 137 / 261 / 225, SEQ ID NO: 137 / 262 / 226, SEQ ID NO: 137 / 263 / 227, SEQ ID NO: 137 / 264 / 228, SEQ ID NO: 137 / 265 / 229, SEQ ID NO: 137 / 266 / 230, SEQ ID NO: 137 / 267 / 231, SEQ ID NO: 137 / 268 / 232, and SEQ ID NO: 137 / 269 / 233.

[0151] In certain embodiments, the polynucleotide encodes a pre-miRNA or pri-miRNA polynucleotide, and the polynucleotide comprises a guide / passenger combination provided in Table 8, and (1) comprises a sequence that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences of SEQ ID NOs. 234-242, and / or (2) Sequence number 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, and SEQ ID NO: 242 contains a sequence that differs by 0, 1, 2, or 3 nucleotides from any of these sequences (including percent identity or difference) based on the following combinations of DNA encoding guide / passenger / pre-miRNA: SEQ ID NO: 139 / 270 / 234, SEQ ID NO: 139 / 271 / 235, SEQ ID NO: 139 / 272 / 236, SEQ ID NO: 139 / 273 / 237, SEQ ID NO: 139 / 274 / 238, SEQ ID NO: 139 / 275 / 239, SEQ ID NO: 139 / 276 / 240, SEQ ID NO: 139 / 277 / 241, and SEQ ID NO: 139 / 278 / 242.

[0152] In certain embodiments, the polynucleotide comprises a sequence encoding an inhibitory RNA that targets α-synuclein mRNA, and further comprises a nucleic acid sequence encoding glial cell-derived neurotrophic factor (GDNF). When referring to GDNF, this includes full-length, mature, and variants thereof. GDNF is a potent neurotrophic factor for dopamine neurons. The amino acid sequence of full-length GDNF is provided by SEQ ID NO: 188, where amino acids 1–19 provide a signal sequence. (See, for example, the hypertext transfer protocol / / www.uniprot.org / uniprotkb / P39905 / entry and the hypertext transfer protocol / / www.ncbi.nlm.nih.gov / protein / NP_000505.1). In certain embodiments, the GDNF comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 188 (full-length) or amino acids 20–211 (mature) of SEQ ID NO: 188. In further embodiments, the mature GDNF further comprises heterogeneous signal sequences. Examples of various signal sequences are provided, for example, in International Patent Publication WO2022155665 and International Patent Publication WO2022165027, both of which are incorporated herein by reference in their entirety.

[0153] II.A. Expression Cassette Elements

[0154] An expression cassette comprises a nucleic acid sequence encoding a polynucleotide operably linked to an expression regulatory element. The expression cassette described herein comprises a nucleotide sequence encoding an RNA polynucleotide that targets α-synuclein mRNA, together with one or more regulatory sequences, and may also include additional sequences such as stuffer sequences, as well as additional coding sequences such as additional RNA polynucleotides and / or polypeptide sequences.

[0155] A "regulatory expression element" influences the expression of the sequence to which it is operably linked. Protein regulatory expression elements can, for example, affect transcription, translation, splicing, and message stability. Regulatory expression elements that affect RNA polynucleotide production can, for example, affect transcription levels.

[0156] Regulatory elements are typically located at the 5' ("upstream") or 3' ("downstream") of the transcribed nucleic acid. Regulatory elements can also be located within the protein-coding transcript (e.g., within an intron). Regulatory elements can be located adjacent to or away from the transcribed sequence. One or more regulatory elements may be present. Examples of regulatory elements include promoters, enhancers, introns, polyadenylation signals, Kozak sequences, post-transcriptional regulatory elements, and termination sequences.

[0157] The promoter is the DNA region where transcription begins. Generally, the transcribed nucleic acid is located 3' of the promoter sequence. In certain embodiments, the promoter sequence is ligated to an enhancer. The enhancer is a DNA region that increases promoter transcription. The enhancer may be adjacent to the promoter or distal to it. Typically, the enhancer is located upstream of the promoter, but it can be located downstream or within the promoter sequence.

[0158] Expression regulatory elements, such as promoters and enhancers, can be selected to preferentially drive expression in specific cell or tissue types. Expression regulatory elements are typically active in specific cells, tissues, or organs because they are recognized by transcription-activating proteins or other transcriptional regulators and provide activity in those cell, tissue, or organ types. (e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4) thSee Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al., (2010) Current protocols in molecular biology, John Wiley & Sons, New York.

[0159] The incorporation of tissue-specific regulatory elements into expression constructs provides at least partial tissue tropism for the expression of encoded RNA polynucleotides. When we refer to a promoter or enhancer specific to a particular cell type or tissue, we mean that the promoter or enhancer provides higher levels of expression and / or secretion in the indicated cell type or tissue type. Examples of CNS-specific promoters include NSE (neuron-specific enolase), synapsin or NeuN, platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), methyl-CpG-binding protein 2 (MeCP2), Ca 2 Examples include neuron-specific promoters such as calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), nerve fiber light chain (NFL) or heavy chain (NFH), β-globin minigen nβ2, preproenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters; astrocyte-specific promoters, such as glial fibrillary acidic protein (GFAP) and EAAT2 promoters; oligodendrocyte-specific promoters, such as myelin basic protein (MBP) / myelin-associated glycoprotein and oligodendrocyte transcription factor 2 promoters; and neuron / hypothalamic-specific promoters, such as proopiomelanocortin (POMC) promoters. (For example, U.S. Patent Application Publication No. 2021 / 214749 and Adeno-Associated Virus Vectors (2019), Ed. Castle., 1) st See Edition, Springer, New York, NY, both of which are incorporated herein by reference in their entirety.

[0160] Expression regulatory elements also include ubiquitous or indiscriminate promoters and promoter / enhancers that can drive polynucleotide expression in many different cell types. Such elements include the cytomegalovirus (CMV) pre-early promoter / enhancer sequence, the Roussarcoma virus (RSV) promoter / enhancer sequence, the phosphoglycerate kinase (PKG) promoter, CAG (a complex of the CMV enhancer, chicken β-actin promoter (CBA), and rabbit β-globin intron) (see, e.g., Boshart et al., (1985) Cell, 41:521-530), the SV40 promoter, the dihydrofolate reductase promoter, and the cytoplasmic β-actin promoter.

[0161] Further promoters include superoxide dismutase 1 (SOD1) promoters, U6 promoters, H1, mouse mammary tumor virus LTR promoters, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoters, SFFV promoters, rat insulin promoters, TBG promoters, desmin promoters and similar muscle-specific promoters, synthetic promoters, hybrid promoters, and multi-tissue specific promoters.

[0162] In certain embodiments, the promoter is the EF-1α promoter (e.g., Wang et al., J. Cell Mol. Med. (2017) 21(11):3044-3054, the whole of which is incorporated herein by reference) and / or comprises, or essentially comprises, a sequence identity of at least 95%, at least 97%, at least 99%, or 100% with sequence number 184.

[0163] Expression control elements can also affect expression in a manner that is regulatable by signals or stimuli that increase or decrease expression. Regulatable elements that increase the expression of a transcribed nucleic acid in response to a signal or stimulus are also referred to as "inducible elements" (i.e., induced by a signal). Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present. Specific examples include the zinc-inducible sheep metallothionein (MT) promoter, the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (International Publication No. WO 1998 / 10088), the tetracycline repression system (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline induction system (Gossen et al., Science 268:1766-1769 (1995), and also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)), the RU486 induction system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441 (1997)), and the rapamycin induction system (Magari et al., J. Clin. Invest. 100:2865-2872 (1997), and Rivera et al., Nat. Medicine. 2:1028-1032 (1996)). Other examples of regulatable control elements include those that are regulated by specific physiological states such as temperature, acute phase, or development.

[0164] In certain embodiments, the expression cassette further comprises one or more introns. Various types of introns can be used. Examples of introns that can be used include rabbit β-globin introns containing splice donor / splice acceptor, SV40 introns containing splice donor / splice acceptor, human β-globin intron, intron 2 of the human hemoglobin β gene, hFIX int1 (intron 1 of the human coagulation factor IX gene), CBA-rHHB (a synthetic intron obtained by fusing intron 1 of the chicken β-actin gene with intron 2 of the rabbit hemoglobin β gene), CBA (intron 1 of the chicken β-actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (derived from different regions of the human coagulation factor IX gene, and pLIVE vector (Mirus) Examples include synthetic introns (containing Bio, Madison, Wisconsin), human hemoglobin β-subunit (HBB2) synthetic introns and optimized HBB2, as well as chimeric introns consisting of a 5' splice donor of the human β-globin I intron and the branching and 3' acceptor regions of the intron located between the leader and the main body of the immunoglobulin gene heavy chain variable region. (Buck et al., Int.J.Mol.Sci.(2020),21,4197; Ronzitti et al.Mol.Ther.Methods Clin Dev.(2016)Jul 20;3:16049, and HBB-IG introns provided by pCMVNT® vectors).

[0165] In certain embodiments, nucleic acids encoding RNA polynucleotides, including a targeting sequence, pri-miRNA, or shRNA, are placed within the intron.

[0166] In certain embodiments, the expression cassette includes post-transcriptional regulatory elements. Post-translational regulatory elements such as the Woodchuck post-transcriptional regulatory element (WPRE) and the hepatitis B regulatory element can increase gene expression. (Buck et al., Int.J.Mol.Sci.(2020),21,4197).

[0167] The polyadenylation signal sequence promotes nuclear export, translation and / or mRNA stability, and provides for the formation of a polyA tail that may also be involved in transcription termination. Examples of polyadenylation signal sequences include the SV40 late polyadenylation signal, the bovine growth hormone polyA (bGHpA) signal sequence, synthetic polyA, mouse β-globin pA, rabbit β-globin pA (RbG), and H4 system pA (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197).

[0168] In certain embodiments, the expression cassette comprises a Kozak consensus sequence or a variant thereof. The Kozak consensus sequence plays a role in translation initiation. Kozak consensus sequences and variants are provided, for example, in McClements et al., (2021) Molecular Vision, 27, 233-242.

[0169] In certain embodiments, the expression cassette comprises an upstream promoter and a downstream polyadenylation signal operably linked to a nucleic acid sequence encoding an RNA polynucleotide.

[0170] In certain embodiments, the expression cassette comprises, in 5' to 3' direction, a promoter or promoter / enhancer, an intron, a nucleic acid sequence encoding an RNA polynucleotide, and a polyadenylation signal, which are operably linked to the nucleic acid sequence encoding the RNA polynucleotide.

[0171] In certain embodiments, the expression cassette further comprises a sequence encoding glial cell-derived neurotrophic factor (GDFN). The GDFN-encoding sequence can be operably ligated to a promoter or separate promoter for an RNA polynucleotide coding sequence containing an α-synuclein-targeting sequence. In further embodiments, the expression cassette comprises a nucleic acid encoding GDNF operably ligated to a promoter for an RNA polynucleotide coding sequence containing an α-synuclein-targeting sequence, wherein the GDNF-encoding sequence is upstream of the RNA polynucleotide coding sequence.

[0172] In certain embodiments, the expression cassette further comprises a miRNA target sequence, which in further embodiments is incorporated into the 3'UTR of the expression cassette. The miRNA target sequence is recognized by a miRNA present in a specific cell or tissue that results in the degradation of the mRNA transcript. Incorporating a miRNA target sequence based on the presence of a specific miRNA in a specific cell can be used to reduce expression in a specific cell or tissue type. Multiple tandem repeats of the miRNA target sequence can be used to increase degradation. (Geisle et al., (2016) World Journal of Experimental Medicine 6(2):37-54).

[0173] In certain embodiments, an expression cassette that does not contain a sequence encoding pri-miRNA or shRNA contains 0-5, 0-10, 0-15, 0-50, or 0-100 CpGs, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpGs, and / or 0-5%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% CpGs.

[0174] II.B. Recombinant Viral Vectors Nucleic Acids

[0175] Polynucleotide recombinant viral nucleic acids contain 5' and / or 3' viral elements that provide viral packaging and replication, and may offer further activities such as promoter activity, genomic integration, or episome concatemerization. The 5' and 3' elements are generally located at or near the 5' and 3' ends of recombinant viral nucleic acids and may be naturally occurring sequences or modified versions of naturally occurring sequences. Examples of 5' and 3' elements include adenovirus ITRs, adeno-associated virus ITRs and packaging sequences, and retrovirus 5' and 3' long-terminal repeats (LTRs) and packaging sequences. (Naso et al., (2017) BioDrugs, 31(4), 317-334; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53(2021); and Liu and Seol (2020) BMB Reports; 53(11):565-575).

[0176] The term "recombinant" as a modifier for nucleic acids or vectors indicates a combination of elements not found in nature. For example, recombinant viral vector nucleic acids provide 5' and / or 3' viral elements along with an expression cassette containing one or more elements not naturally associated with the 5' and / or 3' viral elements. Similarly, viral vectors such as rAAV vectors may contain naturally occurring or modified capsids that encapsulate recombinant viral vector nucleic acids.

[0177] Polynucleotides, expression cassettes, and viral vector nucleic acids are compatible with specific viral vectors. For example, they can produce rAAV containing ssDNA or dsDNA, adenovirus vectors containing dsDNA, and retroviral vectors containing ssRNA.

[0178] In certain embodiments, the viral vector nucleic acid outside the pri-miRNA or shRNA coding region may be 0-5, 0-10, 0-15, 0-50, 0-100, or 0-150 CpGs, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 CpGs, and / or containing any of the following: 0% to 10%, 0%, approximately 0.5%, approximately 1.0%, approximately 2.0%, approximately 3.0%, approximately 4.0%, approximately 5.0% CpG, approximately 6%, approximately 7%, approximately 8%, approximately 9%, or approximately 10% CpG.

[0179] III. Viral Vectors In certain embodiments, the gene delivery vehicle is a viral vector. The viral vector comprises a protein capsid that encapsulates recombinant viral nucleic acid and can deliver the nucleic acid to cells or tissues. Depending on the specific vector, the viral vector may further comprise a viral envelope. Examples of viral vectors that can be used include adenovirus vectors, rAAV, retroviral vectors, and herpes simplex vectors.

[0180] Different serotypes exist depending on the type of virus. Different serotypes can result in different activities, such as the potential to induce tropism in cells and tissues or an immune response in the host. The term "serotype" broadly refers to both serologically distinct viruses and serologically undistinguishable viruses that may exist within a given serotype subgroup or variant. Serological distinction can be determined based on the absence of cross-reactivity between antibodies against one capsid and antibodies against another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0181] As more naturally occurring virus isolates are discovered and / or capsid variants are generated, serological differences may or may not be found with any of the currently existing serotypes. Therefore, if a new virus does not exhibit serological differences, it is a subgroup or variant of the corresponding serotype.

[0182] III.A. Adenovirus Vectors

[0183] Adenoviruses are non-enveloped double-stranded DNA viruses. Recombinant adenovirus vectors contain recombinant adenovirus nucleic acid lacking one or more proteins involved in viral replication, and further contain an adenovirus capsid. Recombinant adenovirus vectors may contain varying amounts of adenovirus DNA. The Ad genome is flanked by hairpin-like inverted end repeats (ITRs) that vary in length from 30 to 371 bp at both ends. ITRs function as self-priming structures that promote primase-independent DNA replication. The 5' and 3' inverted repeats do not need to be exact inverted repeats. Packaging of the viral genome requires a packaging signal located on the left arm of the genome. (Liu and Seol (2020) BMB Reports; 53(11): 565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: 53).

[0184] In certain embodiments, the recombinant adenovirus vector is a third-generation vector, also referred to as “gutless” or “helper-dependent.” Gutless vectors can be produced from recombinant adenovirus nucleic acids that lack all, or substantially all, viral sequences except for the ITR and packaging signal. Gutless adenovirus vectors are high-capacity vectors capable of accommodating up to approximately 36 kb of DNA insertion. Preferred recombinant adenovirus nucleic acids are approximately 27 kb to 37 kb. Stuffer sequences can be added to recombinant adenovirus nucleic acids to increase nucleic acid size and capsid incorporation. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences. (Liu and Seol (2020) BMB Reports; 53(11): 565-575; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: 53; and Sandig et al., PNAS (2000) 97(3): 1002-1007, both of which are incorporated herein by reference in their entirety).

[0185] In certain embodiments, recombinant adenovirus vectors may be produced based on rare human or chimpanzee serotypes. The use of chimpanzee and rare human serotypes may help reduce the host immune response to recombinant adenovirus vectors due to pre-existing immunity. (Guo et al., (2018) Human vaccines & immunootherapeutics, 14(7):1679-1685 and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53).

[0186] Adenoviral vectors can be produced, for example, by using appropriate helper viruses or plasmids and cell lines to supply the viral proteins required for vector production in trans. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53).

[0187] III.B. AAV Vectors

[0188] Recombinant adeno-associated virus (also referred to herein as "rAAV") vectors are based on adeno-associated virus. Adeno-associated virus is a single-stranded DNA virus that contains a 4.7 kb genome flanked by 145 nt ITRs at both ends of the genome. ITR activity is important for self-priming and packaging, and may also provide additional activities such as promoter activity. The rAAV 5' and 3' ITRs may differ in size, and the 5' and 3' inverted repeats need not be exact inverted repeats.

[0189] rAAV vectors comprise an AAV recombinant nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks sequences encoding one or more AAV proteins involved in viral replication. In certain embodiments, the rAAV vector comprises AAV 5' and / or 3' ITRs together with a DNA insert. In certain embodiments, the rAAV nucleic acid comprises a 5' ITR and / or a 3' ITR independently selected from the 5' and 3' ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74 and AAV3B ITRs. In a further embodiment, the 5' and 3' ITRs are present, and both ITRs are derived from the same serotype genome.

[0190] In a further embodiment, the 5'ITR includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 182, and the 3'ITR independently includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 183.

[0191] In certain embodiments, the rAAV nucleic acid is at least about 2.5 kb. If necessary, a stuffer sequence may be used to increase the rAAV nucleic acid size and packaging efficiency. In different embodiments, the rAAV nucleic acid with stuffer is 4–5.2 kb, 3.0–5.5 kb, 4.0–5.0 kb, 4.3–4.8 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, or about 4.7 kb. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences.

[0192] In certain embodiments, rAAV is a self-complementary adeno-associated virus vector (scAAV) or a short hairpin adeno-associated virus vector (shAAV). scAAV and shAAV provide double-stranded recombinant adeno-associated virus nucleic acid that can be incorporated into an AAV capsid. scAAV and shAAV contain inverse dimer repeats that provide intramolecular double-stranded DNA. Since scAAV can be produced by mutating the ITR terminal dissociation site, the repeat cannot nick the terminal degradation site. shAAV can utilize a short hairpin to produce dsAAV. Being double-stranded DNA, scAAV and shAAV offer an advantage in avoiding the DNA synthesis steps required for single-stranded rAAV nucleic acid upon entry into cells. A potential disadvantage of scAAV and shAAV is that the size of the DNA insert that can be incorporated is reduced to about half compared to single-stranded rAAV nucleic acid. (U.S. Patent No. 10,457,940, Xie et al., Mol Ther. (2017) Jun 7;25(6):1363-1374, and McCarty Mol. Ther. (2008), 16(10):1648-1656, each of which is incorporated herein by reference in its entirety).

[0193] Naturally occurring AAV capsids contain viral proteins VP1, VP2, and VP3 in a ratio of approximately 1:1:10. AAV vectors can be produced when all three viral proteins are based on a specific serotype, or when one, two, or all three viral proteins are based on different serotypes.

[0194] Recombinant AAV capsids and nucleic acids may be based on the same serotype (or subgroup or variant) or may be different from each other. In certain embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITR) as the capsid protein it encapsulates.

[0195] In different embodiments, the rAAV capsid is at least 80% and at least 8% of VP1 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10, or VP1, VP2, or VP3 of SEQ ID NO: 164 or SEQ ID NO: 167. The proteins contain sequences that are 5%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9%, or 100% identical.

[0196] In certain embodiments, the AAV capsid is Each includes VP1, VP2, and VP3 having at least 80%, at least 90%, at least 95%, or 100% amino acid sequence identity with any of the following: VP1, VP2, or VP3 of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10, or VP1 of SEQ ID NO: 164 or SEQ ID NO: 167, as well as their variants (e.g., capsid variants such as amino acid insertions, additions, substitutions, and deletions). (See, for example, U.S. Patent Applications No. 9,909,142 and No. 9,840,719 disclosing RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6, U.S. Patent Application Publication No. 2013 / 0059732 and U.S. Patent No. 9,169,299 disclosing LK01, LK02 and LK03; and U.S. Patent No. 11,110,153, the entirety of which is incorporated herein by reference.)

[0197] In a particular embodiment, the capsid includes VP1 having the sequence of sequence number 164, VP2 having the sequence of sequence number 165, and VP3 having the sequence of sequence number 166.

[0198] In certain embodiments, AAV capsids can cross the blood-brain barrier and provide CNS expression. Examples of such AAV capsids and designs of AAV capsids capable of providing CNS expression are provided in Chen et al., (2021) J. Control. Release 333, 129-138 (e.g., AAV9, AAV-PHP·B, AAV-PHP.eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10) U.S. Patent No. 9,585,971, and Goertsen et al., (2022) Nat. Neurosci. 25, 106-115 (2022).

[0199] The AAV genome contains two major genes: rep and cap. Transcription from the rep gene is initiated by two different promoters, producing non-structural proteins called Rep78, Rep68, Rep52, and Rep40. The rep proteins function in genome replication and / or inclusion. The cap gene encodes structural proteins (VP1, VP2, VP3) that constitute the capsid, non-structural assembly activating proteins (APP) that perform functions related to capsid assembly, and membrane-associated accessory proteins that may be involved in the production stages of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther. 31(9-10):499-511, incorporated herein by reference in its entirety).

[0200] AAV requires helper virus function to complete its replication cycle. Helper virus function can be supplied by different viruses in tolerant cell lines. Tolerant cell lines are cell lines that can support viral replication. Examples of AAV helper viruses include adenoviruses, HSV-1, HPV-16, and HboV1, which can be used in combination with tolerant primate cells, for example, and baculoviruses, which can be used in combination with tolerant insect cells, for example, sf9. (Maurer and Weitzman Hum. Gene Ther. (2020) 31(9-10):499-511, and Meier et al., (2020) Viruses 19;12(6):662, both of which are incorporated herein in their entirety by reference).

[0201] Recombinant AAV can be produced, for example, by supplying the viral proteins required for vector production in trans using a suitable helper virus or plasmid and cell line. In certain embodiments, rAAV is produced using an rAAV vector genome plasmid. The plasmid contains that portion of the rAAV nucleic acid that is ultimately packaged or capsidized to form a viral (e.g., rAAV) vector. The "plasmid backbone" contains elements essential for proliferation and recombinant virus production. Except for possible 3'ITR and / or 5'ITR cloning remnants, the plasmid backbone itself is not packaged or capsidized into viral particles.

[0202] A vector genome plasmid may contain regions such as the origin of replication and selection markers. Further possible sites include the cloning site.

[0203] Recombinant AAVs can be produced from a variety of cell lines, including HeLa, A549, BHK, Vero, and HEK293, or their derivatives. In certain embodiments, HEK293 cells are used (American Type Culture Collection Accession Number ATCC CRL1573). Other host cell lines suitable for rAAV vector production are described, for example, in Robert et al., (2017) Biotechnol. J. (2017) 12(3), 1600193, and International Application No. PCT / US2017 / 024951 (these disclosures are incorporated herein in their entirety).

[0204] Recombinant AAVs can be cultured under a variety of different conditions suitable for providing cell proliferation and gene expression. References describing rAAV production include Clement and Grieger (2016) Mol.Ther.Methods Clin.Dev.16;3:16002, Robert et al., (2017) Biotechnol.J.12(3),1600193, and Adeno-Associated Virus Vectors (2019), Ed.Castle.,1 st Edition, Springer, New York, NY are cited, and each of these is incorporated herein by reference in its entirety.

[0205] In certain embodiments, AAV helper function is introduced into host cells by transfecting them with an AAV helper construct before or simultaneously with transfection of an AAV expression vector. Host cells possessing AAV helper function may be called “helper cells” or “packaging helper cells.” AAV helper constructs may be used to complement the deficient AAV function required for productive AAV transduction by providing at least transient expression of AAV rep and / or cap genes. AAV helper constructs often lack AAV ITR and are unable to replicate or package. These constructs may take the form of plasmids, phages, transposons, cosmids, viruses, or virions, for example. Many AAV helper constructs have been described, for example, the commonly used plasmids pAAV / Ad and pIM29+45, which encode both rep and cap expression products. Several other vectors encoding rep and / or cap expression products are known. Recombinant AAVs can be produced, for example, as described in U.S. Patent No. 9,408,904, and International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414, which are incorporated in whole herein.

[0206] In certain embodiments, the rAAV vector is produced by rAAV-producing cells containing rAAV helper virus activity. The genome of the rAAV-producing cells includes rAAV nucleic acid, rep genes, and cap genes.

[0207] In certain embodiments, the rAAV vector is produced by culturing rAAV-tolerant cells containing an AAV genome plasmid, the rAAV-tolerant cells further comprising rep and cap genes provided as part of the cell genome and / or by one or more separate plasmids, and helper virus activity provided as part of the cell genome and / or by one or more separate plasmids. In further embodiments, (a) the rAAV-tolerant cell line is a packaging cell, and the genome of the packaging cell comprises a cap gene and a rep gene, (b) the rep gene, cap gene and helper activity are provided from the same plasmid, or (c) the rep gene and cap gene are provided by a rep / cap plasmid and helper activity is provided by a helper plasmid.

[0208] In certain embodiments, including the use of HSV helper functions, the helper functions are provided by genes encoding at least UL5, UL8, UL52, and ICP8.

[0209] In certain embodiments, including the use of adenovirus helper functions, the helper functions are provided by genes encoding at least E1A, E1B19K, E1B55K, E2A, E4orf6, and VA RNA. In certain embodiments, E1, E2A, and VR RNA functions are provided by helper plasmids, and additional helper functions are provided by the host strain.

[0210] In certain embodiments, rAAV vectors are obtained by producing rAAV using the methods described herein and purifying the rAAV. Purification of rAAV can be carried out using techniques such as gradient-based purification, column-based purification, and combined methods. (See, for example, Ayuso et al., Curr Gene Ther. (2010) 10(6):423-36, which is incorporated herein by reference in its entirety.)

[0211] III.C. Retrovirus Vector

[0212] Retroviruses are enveloped single-stranded RNA viruses containing 5' and 3' LTRs and a packaging signal sequence located just outside the LTR. Different types of retroviral vectors may contain varying amounts of viral genome. In certain embodiments, the retroviral vector is an HIV-based lentiviral vector that retains all cis-acting sequences necessary for viral RNA packaging, reverse transcription, and proviral DNA incorporation, while removing all HIV protein-coding genes. Lentiviral vectors have a packaging capacity of up to approximately 9 kb. If necessary, stuffer sequences may be used to increase rAAV nucleic acid size and packaging efficiency. Lentiviral vectors can be produced by trans-supplying the viral proteins required for vector production using appropriate plasmids and cell lines. (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53).

[0213] III.D. Additional Parvovirus Vectors

[0214] In certain embodiments, the vector is based on a parvovirus other than AAV, such as erythroparvovirus, protoparvovirus, or tetraparvovirus. Members of erythroparvovirus (e.g., human parvovirus B19) typically have a genome size of about 5.6 kb, and therefore, recombinant vectors based on erythroparvovirus are expected to accommodate a larger recombinant payload than AAV vectors. Protoparvoviruses and tetraparvoviruses have a genome size of about 5.3 kb, and therefore, recombinant vectors based on these viruses are also expected to accommodate a larger recombinant payload than AAV vectors.

[0215] Examples of erythroparvovirus capsids are disclosed in International Publication Nos. 2023 / 220043, 2023 / 220035, and 2023 / 220040, all of which are incorporated herein by reference in their entirety.

[0216] Examples of protoparvovirus and tetraparvovirus capsids are disclosed in International Publication No. 2022 / 140683, which is incorporated in its entirety herein by reference.

[0217] IV. Nonviral vectors In certain embodiments, the gene delivery vehicle is a non-viral vector. Preferred non-viral vectors are nanoparticles. Various types of nanoparticles can be used, including lipid nanoparticles (LNPs), polymer nanoparticles, lipid polymer nanoparticles (LPNPs), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. (See, for example, Riley and Vermerris Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019), 24, 3744; Bochicchio et al., (2021), 13, 198; Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) NanoImpact 20, 100261; Neshat et al. (2020) Current Opin. Biotechnol. 66:1-10; Ouranidis et al., (2022) Biomedicines, 10, 50; and Qin et al., Signal Transduct Target Ther. (2022) May 21; 7(1):166, each of which is incorporated herein by reference in its entirety.)

[0218] If desired, nanoparticles can target certain cell types using, for example, targeted ligands that recognize target cell receptors. Examples of targeted ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies and proteins / peptides (e.g., RGD, epidermal growth factor, and low-density lipoprotein), and peptides. (e.g., Teo et al., Advanced Drug Delivery Reviews (2016), 98, 41).

[0219] Nanoparticles can be used to deliver inhibitory RNA or coding polynucleotide constructs to cells. In different embodiments, nanoparticles can deliver further therapeutic compounds, and one or more additional compounds are provided in different nanoparticles. When referring to compounds, this includes small molecules and large molecules (e.g., therapeutic proteins and antibodies).

[0220] The production of various nanoparticles and their incorporation of nucleic acids and other compounds is well known in the art. Examples of publications demonstrating the incorporation of nucleic acids into specific nanoparticles such as LPNPs and LNPs include Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct; 68: 1-11 (each of these in whole is incorporated herein by reference). Factors that may affect the incorporation of small molecules into nanoparticles include the presence of hydrophobic and ionizable moieties. (See, for example, Nii and Ishii, International Journal of Pharmaceutics (2005) 298, 198; and Chen et al., Journal of Controlled Release (2018) 286, 46).

[0221] IV.A. Lipid-based delivery system Lipid-based delivery systems involve the use of lipids as components. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.

[0222] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles with nanoscale dimensions that are useful for delivering nucleic acid molecules. In different embodiments, the nanoparticles are approximately 10 nm to 1000 nm, approximately 50 nm to 500 nm, or approximately 50 nm to 200 nm.

[0223] DNA is negatively charged. Therefore, it may be beneficial for LNPs to contain cationic lipids, such as aminolipids. Exemplary aminolipids are described in U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122 and 7,745,651, as well as U.S. Patent Publication No. These are described in U.S. Patent Publication Nos. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125, all of which are incorporated herein by reference in their entirety. In certain embodiments, the LNP includes an aminolipid described in U.S. Patent No. 9,512,073, which is incorporated herein by reference in its entirety.

[0224] The terms “cationic lipid” and “aminolipid” are used interchangeably herein to include lipids and salts thereof having one, two, three or more fatty acid or fatty alkyl chains and pH titrable amino groups (e.g., alkylamino or dialkylamino groups). Cationic lipids are typically protonated (i.e., positively charged) at pH lower than the pKa of the cationic lipid and substantially neutral at pH higher than the pKa. Cationic lipids can be titrable cationic lipids. In certain embodiments, cationic lipids include a protonable tertiary amine (e.g., pH titrable) group, a C18 alkyl chain in which each alkyl chain may independently have one or more double bonds, one or more triple bonds, and an ether bond, ester bond, or ketal bond between the head group and the alkyl chain.

[0225] Cationic lipids include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (Dlin-K-C2-DMA, also known as Dlin-C2K-DMA, XTC2, and C2K). Examples include 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (Dlin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (Dlin-M-C3-DMA, also known as MC3), salts of these, and mixtures thereof. Other cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (Dlin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (Dlin-K-C4-DMA), Dlen-C2K-DMA, γ-Dlen-C2K-DMA, and (Dlin-MP-DMA) (also known as 1-B11).

[0226] Further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (Dlin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (Dlin-K-MPZ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), 1,2-dilinoleoxy-3-(dimethylamino)acetoxypropane (Dlin-DAC), and 1,2-dilinoleoxy-3-morpholinopropane. (Dlin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (Dlin-S-DMA), 1-Linoleoyl-2-Linoleyloxy-3-aminopropane-dimethylaminopropane (Dlin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (Dlin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DlinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleylxo-3-(2-N,N-dimethylamino)ethoxypropane (Dlin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleyloxy) N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyroxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DM RIE), 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9'-12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy) Examples include -3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS), and disubstituted spermine (D2S), or mixtures thereof.

[0227] Several commercially available preparations of cationic lipids, such as lipofectin® (including DOTMA and DOPE available from GIBCO / BRL) and lipofectamine® (including DOSPA and DOPE available from GIBCO / BRL), can be used.

[0228] Further ionizable lipids that can be used include C12-200, 306Oi10, MC3, cKK-E12, bCKK-E12, Lipid5, Lipid9, ATX-002, ATX-003, and Merck-32. U.S. Patent Application Publication 2017 / 0367988 describes Merck-32.

[0229] In further embodiments, cationic lipids may be present in amounts ranging from approximately 10% to approximately 85% of LNP by molar ratio, or from approximately 50% to approximately 75% of LNP by molar ratio.

[0230] LNPs may contain neutral lipids. These neutral lipids may include lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Examples of such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by considerations including particle size and stability. In certain embodiments, the neutral lipid component may be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0231] Lipids having acyl chain groups with various chain lengths and degrees of saturation are available, or can be isolated or synthesized. In certain embodiments, lipids containing saturated fatty acids having carbon chain lengths in the range of C14 to C22 can be used. In certain embodiments, lipids having mono- or diunsaturated fatty acids having carbon chain lengths in the range of C14 to C22 can be used. Furthermore, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or phosphatidylcholine. Neutral lipids may also consist of phospholipids having other head groups such as sphingomyelin, dihydrosphingomyelin, or serine and inositol.

[0232] In further embodiments, by providing neutral lipids, the neutral lipids may be present in amounts ranging from about 0.1% to about 99% by weight of LNP, or from about 5% to about 15% by weight of LNP, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0233] LNPs may contain further components such as sterols and polyethylene glycol. Sterols can impart fluidity to LNPs. As used herein, “sterols” refers to naturally occurring sterols of plant (plant sterols) or animal (animal sterols) origin, as well as synthetic sterols that exist unnaturally, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. Suitable sterols include those conventionally used in the field of liposome, lipid vesicle, or lipid particle preparation, most commonly cholesterol. Examples of plant sterols include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication 2011 / 0177156. In different embodiments providing sterols, the sterols are present in amounts ranging from about 1% (by weight) to about 80% (by weight) of the LNP, or from about 10% (by weight) to about 25% (by weight) of the LNP.

[0234] Polyethylene glycol (PEG) is a water-soluble polymer of ethylene PEG repeating units having terminal hydroxyl groups. PEG is classified by molecular weight; for example, PEG2000 has an average molecular weight of approximately 2,000 daltons, and PEG5000 has an average molecular weight of approximately 5,000 daltons. Commercially available PEGs from Sigma Chemical Co. and other companies include monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol torecylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM).

[0235] In certain embodiments relating to PEG, PEG has an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In further embodiments, PEG is substituted with methyl groups at the terminal hydroxyl positions. In further embodiments, PEG has an average molecular weight of about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or about 2,000 daltons, or about 750 daltons.

[0236] Examples of PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAAs) described in U.S. Patents 8,936,942 and 7,803,397. PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have various "anchor" lipid moieties for fixing the PEG moiety to the surface of lipid vesicles. Suitable examples of PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates described in U.S. Patent 5,820,873 (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. In certain embodiments, the PEG-modified lipids may be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, the amount of PEG may be approximately 0.1% (by weight) to approximately 50% (by weight) of LNP, or approximately 5% (by weight) to approximately 15% (by weight) of LNP.

[0237] In further embodiments relating to LNP size, the LNPs have a size range of approximately 10 nm to 500 nm, or approximately 50 nm to approximately 200 nm, or 75 nm to approximately 125 nm before encapsulating the nucleic acid.

[0238] In certain embodiments relating to LNPs, the LNPs are described in Billingsley et al., Nano Lett. 2020, 20, 1578, International Publication No. 2021 / 077066, or U.S. Patent Application No. 2022 / 396556 (each of which is incorporated herein by reference in whole). These publications describe LNPs containing lipid-immobilized PEG, cholesterol, phospholipids, and ionizable lipids. In certain embodiments, the LNPs comprise a C14-4 polyamine core and / or have a particle size of about 70 nm. C14-4 has the following structure: [ka]

[0239] In certain embodiments, the LNP comprises a cationic lipid or lipopeptide as described in U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374, or International Publication No. 2021 / 077066 (each in whole being incorporated herein by reference). In certain embodiments, the LNP comprises a cationic lipid, a cholesterol-based lipid, and / or one or more PEG-modified lipids. In certain embodiments, the LNP comprises cKK-E12 (Dong et al., PNAS (2014) 111(11), 3955): [ka]

[0240] In certain embodiments, the LNP includes a modified form of cKK-E12, referred to herein as "bCKK-E12", having the following structure: [ka]

[0241] In certain embodiments, the LNP comprises lipids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described by Sabnis et al., Molecular Therapy 2018, 26:6, 1509-1519 (which is incorporated herein by reference in its entirety). In certain embodiments, the LNP comprises lipids 5, 8, 9, 10, or 11 as described by Sabnis et al.

[0242] Lipid 5 by Sabnis et al. has the following structure: [ka]

[0243] Lipid 9 by Sabnis et al. has the following structure: [ka]

[0244] Further lipids that may be utilized include: Roces et al., Pharmaceutics, 2020, 12, 1095; Jayaraman et al, Angew. Chem. Int. Ed., 2012, 51, 8529-8533; Maier et al., www.moleculartherapy.org, 2013, Vol.21, No.8, 1570-1578; Liu et al., Adv. Mater. 2019, 31, 1902575, e.g., BAMEA-O16B; Cheng et al., Adv. Mater., 2018, 30, 1805308, e.g., 5A2-SC8; Hajj and Ball, Small, 2019 15, 1805097, e.g., 306Oi10; Du et al., US Patent Application Publication Examples include those described in No.20160376224; and Tanaka et al., Adv.Funct.Mater.,2020,30,1910575, each of which is incorporated herein by reference in its entirety.

[0245] In further embodiments, the nanoparticles are LNPs. In further embodiments, the LPNs contain, essentially consist of, or consist of, the following components in mol%: (1) about 20% to about 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipids, about 0.1% to about 10% of one or more PEG-conjugated lipids, and 0% to about 70% of cholesterol; (2) about 20% to about 50% of one or more cationic lipids, about 5% to about 20% of one or more phospholipids, about 0.1% to about 5% of one or more PEG-conjugated lipids, and about 20% to about 60% of cholesterol. In further embodiments, the phospholipids are neutral lipids, and the phospholipids are DOPE or DSPC.

[0246] In further embodiments, LNP contains, essentially consists of, or comprises, the following components in mol%: (1) about 35% cKK-E12, about 2.5% C14-PEG2000, about 46.5% cholesterol and about 16% DOPE; (2) about 35% bCKK-E12, about 2.5% C14-PEG2000, about 46.5% cholesterol and about 16% DOPE; (3) about 50% lipid 9 and C14-PE (4) Approximately 1.5% G2000, 38.5% cholesterol, and 10% DSPC, (5) Approximately 50% lipid 5, 1.5% C14-PEG2000, 38.5% cholesterol, and 10% DSPC, (6) Approximately 50% ionizable lipids, 10% DSPC, 37.5% cholesterol, and 2.5% stabilizer (PEG-Lipid), or (6) GenVoy-ILM® LNP (Precision NanoSystems).

[0247] IV.B. Polymer-based nanoparticles

[0248] Polymer-based delivery systems can be fabricated from a variety of natural and synthetic materials. DNA and other compounds can be encapsulated in the polymer matrix of polymer nanoparticles or adsorbed or conjugated onto the surface of the nanoparticles. Examples of polymers commonly used for nucleic acid delivery include poly(lactic acid-coglycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI) and PEI derivatives, chitosan, dendrimers, polyanhydrides, polycaprolactone, polymethacrylate, poly-L-lysine, pullulan, dextran, hyaluronic acid, and poly-β-aminoesters. (Thomas et al., (2019) Molecules 24, 3744).

[0249] Polymer nanoparticles can have different sizes in the ranges of approximately 1 nm to approximately 1000 nm, approximately 10 nm to approximately 500 nm, approximately 50 nm to approximately 200 nm, approximately 100 nm to approximately 150 nm, and approximately 150 nm or less.

[0250] IV.C. Lipid Polymer Nanoparticles

[0251] Lipid polymer nanoparticles are hybrid nanoparticles that provide both lipid and polymer components, and can therefore be considered LNPs or LPNPs. The composition of LPNPs can provide an outer polymer and an inner lipid or an outer lipid and an inner polymer. The presence of two different types of materials facilitates the design of nanoparticles to provide delayed release of components. Different lipid and polymer components can be selected considering the material to be delivered. (See, for example, Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct; 68: 1-11).

[0252] IV.D. Protein and peptide-based nanoparticles

[0253] Protein and peptide-based systems can utilize a variety of different proteins and peptides. Examples of proteins that can be used include gelatin and elastin. Peptide-based systems can utilize, for example, CPP.

[0254] CPPs are short peptides (6-30 amino acid residues) that are potentially capable of intracellular penetration for the delivery of therapeutic molecules. The majority of CPPs consist mainly of arginine and lysine residues, which make them cationic and hydrophilic, but CPPs may also be amphiphilic, anionic, or hydrophobic. CPPs may be derived from natural biomolecules (e.g., HIV-1 Tat protein) or obtained by synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996-2006). Examples of CPPs include cationic CPPs (highly positively charged) such as Tat peptide, penetratin, protamine, poly-L-lysine, and polyarginine; amphiphilic CPPs (chimeric or fusion peptides constructed from different sources containing both positively and negatively charged amino acid sequences), such as transportan, VT5, bactenesin-7 (Bac7), proline-rich peptide (PPR), SAP(VRLPPP)3, TP10, pep-1, and MPG; membrane-directed CPPs (exhibiting both hydrophobic and amphiphilic properties simultaneously, containing both large and small aromatic residues), such as H625, SPIONs-PEG-CPP, and NP; and hydrophobic CPPs (containing only nonpolar motifs or residues), such as SG3, PFVYLI, pep-7, and fibroblast growth factor.

[0255] Protein and peptide nanoparticles can be supplied in various sizes, for example, in the ranges of approximately 1 nm to 1000 nm, approximately 10 nm to 500 nm, approximately 50 nm to 200 nm, approximately 100 nm to 150 nm, or less than approximately 150 nm.

[0256] IV.E. Peptide Cage Nanoparticles

[0257] Peptide cage delivery systems can be produced from proteinaceous materials that can assemble into cage-like structures forming a restricted internal environment. Peptide cages may include a proteinaceous shell that self-assembles to form a protein cage (e.g., a structure with an internal cavity that is naturally accessible to a solvent, or can be made so by altering the solvent concentration, pH, or equilibrium ratio). The monomers of the protein cage may be in naturally occurring forms or mutant forms involving amino acid substitutions, insertions, and deletions (e.g., fragments).

[0258] Different types of protein "shells" can be assembled to load different types of materials. Protein cages can be produced using viral coat proteins (e.g., from the Cowpea Chlorotic Mottle Virus protein coat) as well as non-viral proteins (e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Application Publication No. 20040028694, and U.S. Patent Application Publication No. 20090035389 (each of which is incorporated herein by reference in their identity)).

[0259] Examples of protein cages derived from non-viral proteins include apoferritins such as ferritin and 12 and 24-subunit ferritins from eukaryotes or prokaryotes, heat shock proteins (HSPs) such as the class of 24-subunit heat shock proteins that form an internal core space, small HSPs from Methanococcus jannaschii, dodecameric Dsp HSPs from E. coli, and MrgA proteins.

[0260] Protein cages can have various core sizes, such as in the range of approximately 1 nm to 1000 nm, approximately 10 nm to 500 nm, approximately 50 nm to 200 nm, approximately 100 nm to 150 nm, or less than approximately 150 nm.

[0261] IVF exosome

[0262] Exosomes are small biological membrane vesicles. Exosomes have been used to deliver a variety of cargoes, including small molecules, peptides, proteins, and nucleic acids. Exosomes are generally in size from approximately 30 nm to 100 nm and can be taken up by cells to deliver their cargo (e.g., expression cassettes containing nucleic acids encoding RNA polynucleotides, including α-synuclein-targeting sequences). Cargo can either associate with the exosome surface structure or be encapsulated within the exosome bilayer.

[0263] Exosomes can be modified in various ways to facilitate cargo delivery and cell targeting. Modifications to facilitate cargo delivery include structures for associating with cargo, such as protein scaffolds and polymers. Modifications for cell targeting include modifications to targeting ligands and surface charge. Publications describing the production, modification, and use of exosomes for the delivery of various cargoes include Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) NanoImpact 20, 100261; and Dooley et al., (2021) Molecular Therapy 29(5), 1729 (each of these in its entirety is incorporated herein by reference).

[0264] V. Pharmaceutical Compositions Pharmaceutical compositions can be used to facilitate the storage and / or delivery of drugs administered to a subject. In certain embodiments, a pharmaceutical composition comprises (a) an RNA polynucleotide containing an α-synuclein mRNA targeting sequence, (b) an optionally modified inhibitory RNA duplex that targets α-synuclein mRNA, (c) a polynucleotide, expression cassette or recombinant viral nucleic acid containing a sequence encoding an α-synuclein mRNA targeting sequence, or (d) a delivery vehicle containing (a), (b) or (c), and a pharmaceutically acceptable carrier.

[0265] The term "pharmaceutically acceptable" indicates that the component does not produce substantially undesirable biological effects in the amounts used. A pharmaceutically acceptable carrier may contain a variety of components, such as salts, sugars, buffers, solvents, preservatives, proteins, and one or more pharmaceutically acceptable excipients, such as surfactants. A particular excipient may have two or more functions. Examples of pharmaceutically acceptable excipients and carriers that can be used in viral vectors are provided, for example, in International Publication 2021 / 071835 and International Publication 2024 / 138129 (these in their entirety are incorporated herein by reference).

[0266] Pharmaceutical compositions can be formulated to suit a specific route of administration or delivery. Suitable compositions for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions, which are typically sterile and may be isotonic with the intended recipient's blood. Exemplary examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal oils, vegetable oils, and synthetic oils. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0267] In one embodiment, the pharmaceutical composition includes a formulation that can be injected into a subject. Examples of injectable formulation components include isotonic sterile saline, salts (e.g., monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, and mixtures of such salts), buffered saline, sugars (e.g., dextrose), and water for injection. The pharmaceutical composition also includes a dry composition, such as a lyophilized composition, which, when combined with sterile water or saline, allows for the formation of a solution suitable for administration.

[0268] Furthermore, the suspension can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain a suitable stabilizer or agent to increase the solubility of the compound, facilitating the preparation of a concentrated solution.

[0269] "Effective dose" or "sufficient dose" refers to the amount that provides the indicated or desired effect. The effective dose may be administered as a single or multiple dose, alone, or in combination with one or more other compositions (e.g., additional therapeutic agents or immunosuppressants), treatments, protocols, or therapeutic regimens, to provide a long-term or short-term response.

[0270] A pharmaceutical composition containing a transgene encoding an RNA polynucleotide that targets α-synuclein mRNA can be delivered to a subject to enable the production of the encoded polynucleotide. Delivery may be in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains sufficient genetic material to enable the recipient to produce a therapeutically effective dose in the subject.

[0271] The "therapeutic effective dose" refers to the amount that elicits a desired or demonstrated biological or medical response in a subject. The therapeutic effective dose can be determined based on observed symptoms and / or by the use of biomarkers associated with a particular disease or disorder. The selection of a specific effective dose can be optimized by considering various factors, including the disease being treated or prevented, the symptoms involved, the target disease or disorder, safety and efficacy in animal models, patient weight, and the patient's immune status. The optimal dose used in a formulation also depends on the route of administration and the severity of the disease or disorder, and can be evaluated according to the patient's circumstances. The effective dose can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

[0272] In certain embodiments, the pharmaceutical composition comprising an rAAV vector comprises an empty AAV capsid. In certain embodiments, in a pharmaceutical composition comprising an rAAV vector and an empty AAV capsid, the ratio of empty AAV capsid to rAAV vector is within or between the ranges of approximately 100:1 to 50:1, approximately 50:1 to 25:1, approximately 25:1 to 10:1, approximately 10:1 to 1:1, approximately 1:1 to 1:10, approximately 1:10 to 1:25, approximately 1:25 to 1:50, or approximately 1:50 to 1:100. In certain embodiments, the ratio of empty AAV capsid to rAAV vector is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0273] Further guidance and examples of pharmaceutical compositions and delivery systems can be found, for example, in Remington: The Science and Practice of Pharmacy (2020) 23rd ed., University of the Science in Philadelphia, published by Elsevier, and The Merck Index (2013) 15. thed., Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa., and Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th Provided to ed. Lippincott Williams & Wilkins, Baltimore, MD.

[0274] VI. Administration and Treatment RNA polynucleotides containing inhibitory sequences that target α-synuclein can be used in methods for reducing α-synuclein expression in a subject and / or for treating a disease or disorder of synucleinopathy. Such methods may include, for example, the administration of (a) RNA polynucleotides containing an α-synuclein mRNA targeting sequence, (b) optionally modified inhibitory RNA duplexes that target α-synuclein mRNA, (c) polynucleotides, expression cassettes or recombinant viral nucleic acids containing a sequence encoding an α-synuclein mRNA targeting sequence, or (d) a delivery vehicle containing (a), (b) or (c) and a pharmaceutically acceptable carrier.

[0275] A treatment can be administered to a target, preferably a human target, for preventive treatment to reduce the likelihood or severity of a disease or disorder, and / or to treat a diagnosed disease or disorder. Treating a diagnosed disease or disorder includes improvement of one or more symptoms and / or delay of the onset of one or more symptoms. In certain embodiments, a particular therapeutic agent, route of administration, and / or pharmaceutical composition are selected with consideration to the particular disease or disorder being treated.

[0276] Subjects with a specific disease or disorder, or at high risk of developing a specific disease or disorder, can be identified, for example, based on symptoms, α-synuclein, biomarkers, and / or genetic markers. Treatment can be administered, for example, to subjects at high risk of developing a synucleinopathy. Examples of markers include SNCA A53T and SNCA A30P substitutions. (Er et al., Biochem Soc Trans. 2023 Feb 27;51(1):245-257, the entire work is incorporated herein by reference).

[0277] In certain embodiments, the diagnosis involves an amplification assay of α-synuclein seeds. (Siderowf et al., Lancet Neurol. 2023 May;22(5):407-417, the entire article is incorporated herein by reference.)

[0278] In certain embodiments, the treatment is for Lewy body disease or disorder. In further embodiments, Lewy body disease or disorder is characterized by aggregated α-synuclein and includes Parkinson's disease, Parkinson's dementia, Lewy body dementia, infant axonal dystrophy, atypical axonal dystrophy, adult-onset dystonia-parkinsonism, autosomal recessive early-onset parkinsonism, POLG-associated neurodegeneration, Niemann-Pick type C1, and Krabbe disease.

[0279] Specific embodiments relate to the treatment of Parkinson's disease, Parkinsonian dementia, or Lewy body dementia. Symptoms of Parkinson's disease include resting tremor, bradykinesia, gait disturbance, dysphonia, decreased voice volume, muscular dystrophy, instability, and postural deformities. Parkinson's disease may also be accompanied by non-motor symptoms such as REM sleep behavior disorder (RBD), olfactory disturbance, depression, and autonomic dysfunction. Early symptoms include pain, rigidity or numbness of the limbs, bradykinesia, tremor, and decreased facial expression. Later symptoms include motor variability, dyskinesia, gait disturbances, and falls. The initial diagnosis may be based on the evaluation of the patient's medical history and clinical features on examination. Responsiveness to dopamine may also be used over time in the diagnosis of Parkinson's disease. Most Parkinson's disease patients eventually develop dementia. (Koga et al. Molecular Neurodegeneration (2021) 16:83, and Lee et al. Neuroimmunol Neuroinflammation 2021;8:222-44, both of which are incorporated herein in their entirety).

[0280] Symptoms of Lewy body dementia include dementia, fluctuating cognition, visual hallucinations, rapid eye movements, sleep behavior disturbances, and parkinsonism. (Koga et al. Molecular Neurodegeneration (2021) 16:83).

[0281] In certain embodiments, diagnosis, patient monitoring, and / or clinical assessment include the use of questionnaires, scales, and / or assessment tools used by clinicians in the treatment and clinical outcome assessment of movement disorders such as Parkinson's disease. Such clinical outcome assessments can be found, for example, on the International Parkinson and Movement Disorder website (http: / / www.movementdisorders.org / MDS / MDS-Clinical-Outcome-Assessment.htm), which is incorporated by reference.

[0282] In certain embodiments, the progression of Parkinson's disease is assessed using the Movement Disorder Society of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS). (Goetz et al, 2023 Mov Disord 2023;38(2):342-7, and Goetz et al, Mov Disord 2008;23(15):2129-70, both of which are incorporated herein by reference in their entirety).

[0283] In certain embodiments, the progression of Parkinson's disease can be assessed using imaging biomarkers. In further embodiments, imaging biomarkers and techniques for monitoring the pathogenesis and progression of Parkinson's disease include SV2A and VMAT2 PET to investigate changes in dopamine terminal integrity in the caudate nucleus and putamen, structural MRI to examine volume loss in the substantia nigra, and quantitative sensitivity mapping to assess structural changes based on iron accumulation in the substantia nigra, and biofluidic biomarkers to track disease progression, such as total α-Syn, total tau and phosphorylated tau, dopa decarboxylase, and CSF levels of 3,4-dihydroxyphenylacetic acid, where α-Syn protein knockdown can be measured in neuron-derived exosomes isolated from plasma.

[0284] In an additional embodiment for disease progression, the Roche Parkinson's Disease Mobile Application (DHT) can be used, which includes a series of performance outcome ("PerfO") assessments designed to measure the progression of major motor function in individuals with early-stage Parkinson's disease. (Essioux et al., Digital health technologies can reduce sample size and enable shorter proof of concept clinical trial in Parkinson's disease. Presented at the 17th AD / PD 2023 International Conference on Alzheimer's and Parkinson's Diseases and Related Neurological Disorders, March 28–April 1, 2023, Gothenburg, Sweden). DHT provides a measure of the impairment underlying core motor symptoms of Parkinson's disease, including bradykinesia of the upper and lower body, gait impairment, postural instability, tremor, and speech and voice difficulties. These can be derived from the seven-motor DHT PerfO test, which includes hand rotation, dexterity, U-turn, resting tremor, postural tremor, speech, and voice.

[0285] Specific embodiments relate to the treatment of MSA. MSA is characterized by a variable combination of autonomic dysfunction, levodopa (L-DOPA) unresponsive parkinsonism, cerebellar ataxia, pyramidal tract signs, and non-motor symptoms. Non-motor symptoms may include RBD, sleep-disordered breathing, dysphagia, severe dysphonia, and dysarthria. (Koga et al. Molecular Neurodegeneration (2021) 16:83).

[0286] Administration may be by various routes, such as subcutaneous, epidermal, intradermal, intrathecal, intraorbital, intramucosal, intranasal, intraperitoneal, intravenous, intrapleural, intra-arterial, intracavitary, oral, portal vein, intramuscular, intraparenchymal, intracisional, or intraventricular. In certain embodiments, administration to the patient is by infusion in a pharmaceutical carrier.

[0287] In certain embodiments, administration is direct administration to the CNS, for example, intraparenchymal, intracisional, or intraventricular.

[0288] In certain embodiments, the route of administration and / or the vector targets the caudate nucleus, putamen, and / or substantia nigra. In further embodiments, the caudate nucleus, putamen, and substantia nigra are targeted to treat MSA, or the substantia nigra is targeted to treat Parkinson's disease.

[0289] In certain embodiments, administration is, for example, direct administration to the CNS by intraparenchymal, intracisional, or intraventricular administration.

[0290] In certain embodiments, CNS administration is performed by direct administration to the brain using a needle or catheter. (For example, International Publication No. 2021 / 108809, Cohen-Pferrer et al., Pediatric Neurology 67(2017)23-35, and U.S. Patent No. 10369329, each of which is incorporated herein by reference in its entirety).

[0291] In certain embodiments, CNS administration is performed by convection-enhanced delivery. Convection-enhanced delivery includes surgical exposure of the brain, direct placement of a catheter into the target area, and subsequent injection of the therapeutic agent. (U.S. Patent Application Publication No. 2022 / 010001, and Devinski et al. (2009) Expert Rev Neurother. 9(10):1519-27, both of which are incorporated herein by reference in their entirety).

[0292] In certain embodiments, intraparenchymal administration is performed by bilateral injection using convection-enhanced delivery via IP injection into the caudate nucleus and putamen, or substantia nigra. In further embodiments, administration uses bilateral stereotactic injection. In even further embodiments, administration is performed under MRI guidance, and rAAV is provided in a formulation containing a contrast agent. Various infusion devices are available, such as the ClearPoint® SmartFlow cannula and the ClearPoint® Neuro Navigation System, for example, with convection-enhanced delivery (CED) under MRI guidance.

[0293] In certain embodiments, the presence of inhibitory nucleic acids encoded by the transgene is measured in cerebrospinal fluid (CSF) after IP injection. The presence of inhibitory nucleic acids in CSF can be monitored at different time points, e.g., baseline, as well as at 3, 6, 12, 18, and 24 months. In further embodiments, a vector encoding miR5 is administered, and the presence of miR5 is measured in cerebrospinal fluid (CSF). Measurement of inhibitory nucleic acids in CSF can be used, for example, as a sustained-release marker.

[0294] Examples of CNS delivery devices, systems, and technologies include those described in U.S. Patent No. 8,128,600, U.S. Patent Publication No. 2020 / 0324089, U.S. Patent No. 1,1129643, U.S. Patent No. 1,1154377, U.S. Patent Publication No. 2021 / 0343397, U.S. Patent Publication No. 2021 / 0282866, U.S. Patent No. 9,572,928, U.S. Patent No. 8,337,458, U.S. Patent No. 1,0722,265, and U.S. Patent Publication No. 2021 / 214749, each of which is incorporated herein by reference in its entirety.

[0295] CNS administration can also be carried out using techniques that facilitate transport across the blood-brain barrier, such as disruption of the blood-brain barrier and the use of blood-brain barrier carriers. (Chen et al., (2021) Journal of Controlled Release 333, 129-138 and Bellettato and Scrapa, Italian Journal of Pediatrics (2018) 44(Suppl 2):131, Haumann et al., (2020) CNS Drugs 34, 1121-1131, and Camalleri et al., J. Clin. Neurophysiol. (2020) Mar; 37(2): 104-117, all of which are incorporated herein by reference in their entirety). Techniques that facilitate crossing the blood-brain barrier can be utilized in viral and non-viral delivery vehicles.

[0296] In certain embodiments, CNS delivery is achieved using techniques and / or drugs that facilitate cross-brain barrier passage.

[0297] In certain embodiments, CNS delivery is achieved by using techniques that bypass the blood-brain barrier.

[0298] In certain embodiments, CNS delivery is achieved by direct administration to the brain.

[0299] Techniques to facilitate blood-brain barrier (BlBC) crossing include disrupting the BlBC, using BlBC carriers, and using vectors capable of crossing the BlBC. Common techniques for facilitating BlBC crossing can be utilized with both viral and nonviral delivery vehicles. Furthermore, certain delivery vehicles, such as specific AAV serotypes, can facilitate BlBC crossing. (Chen et al., (2021) Journal of Controlled Release 333, 129-138; Bellettato and Scrapa, Italian Journal of Pediatrics (2018) 44(Suppl 2):131; Haumann et al., (2020) CNS Drugs 34, 1121-1131; Camalleri et al., J. Clin. Neurophysiol. (2020) Mar; 37(2):104-117); and Fischell and Fishman, Front. Neurosci., 23 September 2021, volume 15, Article 747726, doi:10.3389 / fnins.2021.747726; each of these is incorporated herein by reference in its entirety).

[0300] In certain embodiments, AAV capsids that facilitate CNS entry are used. Examples of such capsids are provided in Chen et al., (2021) Journal of Controlled Release 333, 129-138 (e.g., AAV9, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent No. 9,585,971, U.S. Patent Application Publication No. 2021 / 214749 and U.S. Patent Application Publication No. 2021 / 107947, U.S. Patent No. 11,518,787, and International Publication No. 2019 / 158619.

[0301] In certain embodiments, the AAV capsid includes an insert that targets CNS cells, such as those located in the caudate nucleus and / or putamen and / or substantia nigra and / or ventral tegmental area. Examples of targeting sequences are provided in U.S. Patent Application Publication 2021 / 214749 and International Publication 2019 / 158619, both of which are incorporated herein by reference in their entirety. Targeting peptides can be inserted at different AAV capsid locations. In certain embodiments, the AAV capsid insertion sites are located outside the lipase domain of VP1 and outside the aggregate activation protein (AAP).

[0302] In certain embodiments, the AAV capsid includes a targeted peptide insert containing an amino acid sequence described in U.S. Patent Application Publication 2021 / 214749 or International Publication 2019 / 158619. In further embodiments, the capsid is an AAV2 capsid containing a peptide insert containing SEQ ID NOs. 279, 280, 281, or 282, wherein the insert is centered at amino acid residue 587 of AAV2 VP1, or the insert is located between N587 and R588 of the AAV2 VP1 capsid protein.

[0303] In certain embodiments, CNS delivery involves the use of focused ultrasound and microbubbles. Focused ultrasound combined with microbubbles can transiently disrupt the blood-brain barrier to facilitate the entry of therapeutic agents, including transgene delivery vehicles. Focused ultrasound can accumulate energy in selected regions of human anatomical structures, and its use can be assisted by magnetic resonance imaging (MRI) guidance. Various types of microbubbles can be used in conjunction with focused ultrasound to transiently disrupt the blood-brain barrier. (Cammalleri et al., J Clin Neurophysiol. (2020) Mar;37(2):104-117; Noroozian et al., Methods Mol Biol. (2019) 1950,177-197, U.S. Patent No. 10,322,178, International Publication No. 2019 / 113538, each of which is incorporated herein by reference in whole).

[0304] Microbubbles are typically administered intravenously. In certain embodiments, the microbubbles are one of the following: Definity® (Lantheus Medical Imaging, Northvillerica, Massachusetts, USA, containing perflutren lipid microspheres (average diameter 1.1–3.3 μm, maximum diameter 20 μm)), SonoVue® (Bracco Imaging, Milan, Italy, suspension of phospholipid microspheres containing sulfur hexafluoride gas (average diameter approximately 2.5 μm, more than 90% of bubbles less than 8 μm)), and Optison® (GE Healthcare, Princeton, New Jersey, USA, sterile, nonpyrogenic suspension of human serum albumin microspheres containing perflutren (average diameter 3.0–4.5 μm, maximum diameter 32 μm)). (See Camalleri et al., J Clin Neurophysiol. (2020) Mar;37(2):104–117, which is incorporated herein in its entirety by reference).

[0305] In certain embodiments, microbubbles are produced and administered as described in U.S. Patent No. 10,322,178, which is incorporated herein in its entirety by reference.

[0306] In certain embodiments, the rAAV vector is selected or manipulated to facilitate use in focused ultrasound delivery. (Kofed et al., Journal of Controlled Release 351(2022)667-680; Kofed et al., Molecular Therapy: Methods & Clinical Development Vol.27 December 2022,167-195; U.S. Patent Application Publication No. 2023 / 0047753; International Publication No. 2023 / 0044161, each of which is incorporated herein by reference in its entirety).

[0307] In certain embodiments, the rAAV vector comprises an acoustically targeted peptide described in U.S. Patent Application Publication No. 2023 / 0047753 and / or International Publication No. 2023 / 004416.

[0308] In certain embodiments, intranasal administration is used to achieve CNS vector delivery. Intranasal delivery bypasses the blood-brain barrier and reduces systemic exposure. In further embodiments, intranasal delivery includes the use of focused ultrasound and microbubbles. The use of focused ultrasound and microbubbles in combination with intranasal delivery has been shown to facilitate the penetration of therapeutic agents already in the perivascular space across the blood-brain barrier. (The entire work is incorporated herein by reference, Ye et al., The Lancet (2022) Oct: Vol 84: 1-14).

[0309] In certain embodiments providing CNS expression, the polynucleotide expressing the RNA polynucleotide includes a PGK promoter, a CBh promoter, and / or an EF1-α promoter.

[0310] The optimal dose may vary depending on various factors, including the specific treatment and the desired endpoint. The amount, number, frequency, or duration of the dose may be proportionally increased or decreased in consideration of any adverse side effects, complications, or other risk factors for the treatment or treatment, as well as the condition of the subject.

[0311] A "unit dosage form" refers to a physically distinct unit containing a predetermined effective amount of the active ingredient in combination with a pharmaceutically acceptable carrier. Unit dosage forms can be provided, for example, in ampoules and vials that may contain a pharmaceutically acceptable carrier or a composition in a lyophilized or lyophilized state. In the case of a lyophilized or lyophilized state, a sterile liquid carrier may be added before administration. Individual unit dosage forms can be included in multi-dose kits or containers.

[0312] An "effective dose" is one that achieves the desired or indicated effect. For example, an effective dose in treatment reduces one or more adverse symptoms, reduces the likelihood of one or more symptoms associated with a disease or disorder, or reduces the progression of a disease or disorder. A preferred effective dose for treatment is one that is effective in reducing several or all adverse symptoms.

[0313] Since α-Syn is a naturally occurring protein, a certain amount of this protein may be important or useful for specific functions. The biological function of α-Syn is not well characterized, but α-Syn null animals in which the SNCA gene is experimentally deleted develop normally and remain healthy throughout their lives (Chandra et al., Proc Natl Acad Sci USA 2004;101(41):14966-71), although slight learning deficits have been reported in one strain of α-Syn-deficient mice (Kokhan et al., Behav Brain Res 2012;231(1):226-30). Population-based studies have shown that individuals identified with deletion of a single SNCA allele lack the features of parkinsonism or dementia, negating a significant loss-of-function effect of α-Syn (Blauwendraat et al., UK Biobank. Neurobiol Dis 2021;148:105182, and Burre et al., Mov Disord. 2024 Jun 30. doi:10.1002 / mds.29897).

[0314] Non-clinical studies in rodents and NHPs suggest that reductions in SNCA mRNA (up to 90%) and α-Syn protein (up to 70%) were well tolerated for 12 months and did not cause dopaminergic neurodegeneration or dysfunction in ascending dopaminergic projections (Zharikov et al., Neurobiol Dis 2019;125:146-53, and Zharikov et al., J Clin Invest 2015;125(7):2721-35). In NHPs, repeated intrathecal delivery of antisense oligonucleotides (ASOs) resulted in up to 70% knockdown of SNCA transcripts and α-Syn protein in the brain, with no adverse effects up to 90 days (Cole et al., JCI Insight 2021;6(5):e135633). Immunotherapy aimed at reducing α-Syn levels in the brain has shown a safety profile that supports further development (Jankovic et al., JAMA Neurol 2018;75(10):1206-14, and Pagano N Engl J Med 2022;387(5):421-32), indicating that a reduction in α-Syn may be acceptable in adults.

[0315] In certain embodiments, a pharmaceutical composition comprising a viral or nonviral vector is administered to a subject in a dose suitable for reducing α-synuclein or α-synuclein expression. In different embodiments, α-synuclein is reduced by at least 15%, at least 20%, at least 50%, at least 60%, or at least 70% in a transduced brain region (e.g., substantia nigra) compared to baseline levels, and / or α-synuclein or α-synuclein expression is reduced by at least 20%, at least 50%, at least 60%, at least 70%, or about 30% to about 70%, or about 30% to about 60%, or about 25% to about 70%.

[0316] In different embodiments, appropriate doses are approximately 0.01 mg / kg to 10 mg / kg of vector per kg of body weight of the subject, approximately 0.01 mg / kg to 0.1 mg / kg of vector per kg of body weight of the subject, approximately 0.1 mg / kg to 1.0 mg / kg of vector per kg of body weight of the subject, or approximately 1.0 mg / kg to 10 mg / kg of vector per kg of body weight of the subject.

[0317] Generally, the dose of rAAV vectors is at least 1 × 10¹⁶ per kilogram of body weight of the subject. 8 The vector genome (vg / kg) is greater than or equal to 1 × 10⁻¹⁶. 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , or 1 × 10 14 The therapeutic effect is achieved using the number of vector genomes per kilogram of the subject's body weight (vg / kg). In different embodiments, the rAAV dose is approximately 5 × 10⁶. 11 rAAVvg / kg or approximately 5 × 10 11 More than rAAVvg / kg, approximately 1×10 12 rAAVvg / kg or approximately 1 × 10⁻⁶ 12 More than rAAVvg / kg, approximately 2×10 12 rAAVvg / kg or approximately 2 × 10⁻⁶ 12 More than rAAVvg / kg, approximately 3×10 12 rAAVvg / kg or approximately 3 × 10⁻⁶ 12 More than rAAVvg / kg, approximately 4×10 12 rAAVvg / kg or approximately 4 × 10 12 More than rAAVvg / kg, approximately 5×10 12 rAAVvg / kg or approximately 5 × 10 12 More than rAAVvg / kg, approximately 1×10 13 rAAVvg / kg or approximately 1 × 10⁻⁶ 13 More than rAAVvg / kg, approximately 2×10 13 rAAVvg / kg or approximately 2 × 10⁻⁶ 13 More than rAAVvg / kg, approximately 3×10 13 rAAVvg / kg or approximately 3 × 10⁻⁶ 13More than rAAVvg / kg, approximately 4×10 13 rAAVvg / kg or approximately 4 × 10 13 More than rAAVvg / kg, approximately 5×10 13 rAAVvg / kg or approximately 5 × 10 13 More than rAAVvg / kg, approximately 6×10 13 rAAVvg / kg or approximately 6 × 10 13 rAAVvg / kg is greater than rAAVvg / kg.

[0318] An example of a dose range for rAAVvg / kg is a dose range of approximately 5 × 10 11 ~about 6×10 13 rAAVvg / kg, dose range approximately 5 × 10 11 ~Approx. 5.5×10 11 rAAVvg / kg, dose range approximately 5.5 × 10⁻⁶ 11 ~about 6×10 11 rAAVvg / kg, dose range approximately 6 × 10 11 ~Approx. 6.5×10 11 rAAVvg / kg, dose range approximately 6.5 × 10⁻⁶ 11 ~Approx. 7×10 11 rAAVvg / kg, dose range approximately 7 × 10 11 ~Approx. 7.5×10 11 rAAVvg / kg, dose range approximately 7.5 × 10⁻⁶ 11 ~Approx. 8×10 11 rAAVvg / kg, dose range approximately 8 × 10 11 ~Approx. 8.5×10 11 rAAVvg / kg, dose range approximately 8.5 × 10⁻⁶ 11 ~Approx. 9×10 11 rAAVvg / kg, dose range approximately 9 × 10 11 ~Approx. 9.5×10 11 rAAVvg / kg, dose range approximately 9.5 × 10⁻⁶ 11 ~Approx. 1×10 12 rAAVvg / kg, dose range approximately 1 × 10⁻⁶ 12 ~Approx. 1.5×10 12 rAAVvg / kg, dose range approximately 1.5 × 10⁻⁶ 12 ~about 2×10 12 rAAVvg / kg, dose range approximately 2 × 10⁻⁶ 12 ~Approx. 2.5×10 12rAAVvg / kg, dose range approximately 2.5 × 10⁻⁶ 12 ~Approx. 3×10 12 rAAVvg / kg, dose range approximately 3 × 10 12 ~Approx. 3.5×10 12 rAAVvg / kg, dose range approximately 3.5 × 10⁻⁶ 12 ~Approx. 4×10 12 rAAVvg / kg, dose range approximately 4 × 10 12 ~Approx. 4.5×10 12 rAAVvg / kg, dose range approximately 4.5 × 10⁻⁶ 12 ~Approx. 5×10 12 rAAVvg / kg, dose range approximately 5 × 10 12 ~Approx. 5.5×10 12 rAAVvg / kg, dose range approximately 5.5 × 10⁻⁶ 12 ~about 6×10 12 rAAVvg / kg, dose range approximately 6 × 10 12 ~Approx. 6.5×10 12 rAAVvg / kg, dose range approximately 6.5 × 10⁻⁶ 12 ~Approx. 7×10 12 rAAVvg / kg, dose range approximately 7 × 10 12 ~Approx. 7.5×10 12 rAAVvg / kg, dose range approximately 7.5 × 10⁻⁶ 12 ~Approx. 8×10 12 rAAVvg / kg, dose range approximately 8 × 10 12 ~Approx. 8.5×10 12 rAAVvg / kg, dose range approximately 8.5 × 10⁻⁶ 12 ~Approx. 9×10 12 rAAVvg / kg, dose range approximately 9 × 10 12 ~Approx. 9.5×10 12 rAAVvg / kg, dose range approximately 9.5 × 10⁻⁶ 12 ~Approx. 1×10 13 rAAVvg / kg, dose range approximately 1 × 10⁻⁶ 13 ~Approx. 1.5×10 13 rAAVvg / kg, dose range approximately 1.5 × 10⁻⁶ 13 ~about 2×10 13 rAAVvg / kg, dose range approximately 2 × 10⁻⁶ 13 ~Approx. 2.5×10 13 rAAVvg / kg, dose range approximately 2.5 × 10⁻⁶ 13 ~Approx. 3×1013 rAAVvg / kg, dose range approximately 3 × 10 13 ~Approx. 3.5×10 13 rAAVvg / kg, dose range approximately 3.5 × 10⁻⁶ 13 ~Approx. 4×10 13 rAAVvg / kg, dose range approximately 4 × 10 13 ~Approx. 4.5×10 13 rAAVvg / kg, dose range approximately 4.5 × 10⁻⁶ 13 ~Approx. 5×10 13 rAAVvg / kg, dose range approximately 5 × 10 13 ~Approx. 5.5×10 13 rAAVvg / kg, dose range approximately 5.5 × 10⁻⁶ 13 ~about 6×10 13 rAAVvg / kg, dose range approximately 6 × 10 13 ~Approx. 1×10 14 rAAVvg / kg is one example.

[0319] In a particular embodiment, rAAVvg / kg is approximately 5 x 10 11 vg / kg, approx. 6x10 11 vg / kg, approx. 7x10 11 vg / kg, approx. 8x10 11 vg / kg, approx. 9x10 11 vg / kg, approx. 1x10 12 vg / kg, approx. 2x10 12 vg / kg, approx. 3x10 12 vg / kg, approx. 4x10 12 vg / kg, approx. 5x10 12 vg / kg, approx. 6x10 12 vg / kg, approx. 7x10 12 vg / kg, approx. 8x10 12 vg / kg, approx. 9x10 12 vg / kg, approx. 1x10 13 vg / kg, approx. 2x10 13 vg / kg, approx. 3x10 13 vg / kg, approx. 4x10 13 vg / kg, approx. 5×10 13 vg / kg, or approximately 6 × 10⁻⁶ 13 It is administered at a dose of vg / kg.

[0320] In certain embodiments, the doses and dose ranges of other viral vectors are as provided herein with respect to rAAV. For example, in certain embodiments, the doses and dose ranges of recombinant adenovirus vectors, recombinant retrovirus vectors (e.g., lentiviruses), and recombinant herpes simplex virus vectors are the same as those exemplified above with respect to rAAV.

[0321] In certain embodiments, the provided vector genome dose is based on a target region such as the caudate nucleus, putamen and / or substantia nigra (SN). In further embodiments, the provided dose is 3e8vg / targeted brain region (TBR) ~ 3e11vg / TBR, about 3e8vg / TBR, about 3e9vg / TBR, about 3e10vg / TBR, or about 3e11vg / TBR, or 3e8vg / SN ~ 3e11vg / SN, about 3e8vg / SN, about 3e9vg / SN, about 3e10vg / SN, or about 3e11vg / SN.

[0322] In certain embodiments, the polynucleotide constructs, viral vectors, and nonviral vectors described herein are administered in combination with additional compounds or therapies for specific diseases of impairment, and / or in combination with compounds that reduce the immune response produced against the polynucleotide, delivery vehicle, and / or the produced protein. The additional compounds or therapies may be provided in different modalities, such as being administered separately, and may be administered or carried out before, substantially simultaneously with, or after the administration of the polynucleotide constructs, viral vectors, and nonviral vectors described herein.

[0323] In certain embodiments, the administration of polynucleotide constructs, viral vectors, and nonviral vectors described herein is carried out in combination with one or more further treatments for synucleinopathy-related diseases or disorders. Examples of therapeutic agents for Parkinson's disease include dopamine agonists (e.g., levodopa), monoamine oxidase inhibitors and antagonists, and glial cell line-derived neurotrophic factor (GDNF). (Lee et al. Neuroimmunol Neuroinflammation 2021;8:222-44, the entire work is incorporated herein by reference).

[0324] In certain embodiments, the treatment may include (a) dopamine decarboxylase inhibitors / dopamine precursors (e.g., carbidopa-levodopa), catechol-o-methyltransferase inhibitors that inhibit the breakdown of levodopa (e.g., entacapone, tolcapone, or opicapone), dopamine agonists (e.g., pramipexole, ropinirole, apomorphine, or rotigotine), monoaminooxidase B inhibitors that inhibit the breakdown of dopamine (e.g., selegiline, rasagiline, or safinamide), mixed-mechanism inhibitors including N-methyl-D-aspartate antagonists (e.g., amantadine and The treatment is carried out in combination with (b) the patient having received or is receiving treatment with (a), one or more additional compounds used to treat Parkinson's disease, selected from memantine, an adenosine 2A antagonist (e.g., istradefylline), and / or anticholinergics (e.g., trihexyphenidyl and benztropine) (see, for example, the hypertext transfer protocol / / www.apdaparkinson.org / what-is-parkinsons / treatment-medication / medication), and / or (b) the patient having received or is receiving treatment with (a). In a further embodiment, the patient has received or is receiving treatment with (a) and has a reduced response to treatment including (a).

[0325] In certain embodiments, treatment is carried out in combination with (a) one or more additional compounds used to treat Lewy body dementia, selected from cholinesterase inhibitors (e.g., rivastigmine, donepezil, and galantamine), N-methyl-D-aspartate antagonists (e.g., memantine and amantadine), dopamine decarboxylase inhibitors / dopamine precursors (e.g., carbidopa-levodopa), and antipsychotics (e.g., clozapine and ketiapine), and / or (b) the patient has received or is receiving treatment with (a). In further embodiments, the patient has received or is receiving treatment with (a) and has a reduced response to treatment including (a).

[0326] In certain embodiments, multiple system atrophy is treated in combination with one or more additional compounds or other treatments for specific symptoms. Examples of such additional compounds and treatments include blood pressure-raising agents (e.g., fludrocortisone, midododrine, pyridostigmine, and droxidopa), agents that alleviate Parkinson's disease-like symptoms (e.g., levodopa and carbidopa), and drugs that treat erectile dysfunction (e.g., sildenafil). (See, for example, the hypertext transfer protocol incorporated herein by reference: / / www.mayoclinic.org / diseases-conditions / multiple-system-atrophy / diagnosis-treatment / drc-20356157).

[0327] In certain embodiments, an RNA polynucleotide containing an inhibitory sequence targeting α-synuclein is used in combination with GDNF. In further embodiments, GDNF or a nucleic acid encoding GDNF is administered. The nucleic acid encoding GDNF can be provided on the same or a different polynucleotide as the polynucleotide encoding the RNA inhibitory sequence targeting α-synuclein.

[0328] In certain embodiments, the administration of polynucleotide constructs, viral vectors, and nonviral vectors described herein is carried out in combination with immunosuppressants or regimens. Such agents and regimens may be used, as needed, to achieve immune tolerance or to mitigate the immune response to the delivered polynucleotide or delivery vehicle. Examples of immunosuppressants and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particles (SVP)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles)), ImmTOR-IL® (ImmTOR containing a Treg-selective IL-2 agonist), B-cell depletion, immunoadsorption, and plasma ferresis.

[0329] In certain embodiments, the polynucleotide construct, viral vector, or nonviral vector is administered in combination with one or more immunosuppressants, which are administered before, substantially concurrently with, or after the administration of the polynucleotide construct, viral vector, or nonviral vector. In certain embodiments, one or more immunosuppressants are administered concurrently with the polynucleotide construct, viral vector, or nonviral vector. In certain embodiments, one or more immunosuppressants are administered 1–12, 12–24, or 24–48 hours, or 2–4, 4–6, 6–8, 8–10, 10–14, 14–20, 20–25, 25–30, 30–50 days, or more than 50 days before the administration of the polynucleotide construct, viral vector, or nonviral vector. In certain embodiments, one or more immunosuppressants are administered 1–12, 12–24, or 24–48 hours, or 2–4, 4–6, 6–8, 8–10, 10–14, 14–20, 20–25, 25–30, 30–50 days, or more than 50 days after administration of a polynucleotide construct, viral vector, or nonviral vector.

[0330] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid, such as a corticosteroid. In certain embodiments, immunosuppressants include prednisone, prednisolone, calcineurin inhibitors (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g., CellCept®, Myfortic®), CD52 inhibitors (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, beratacept), anti-CD3 mAb, anti-LFA-1 mAb (e.g., ephalizumab), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epratuzumab), anti-CD20 mAb (e.g., rituximab, olelizumab, ofatumumab, bertuzumab), proteasome inhibitors (e.g., bortezomib), TACI-Ig (e.g., atacicept), and anti-C5 mAbs (e.g., eculizumab), mycophenolate, azathioprine, sirolimus everolimus, TNFR-Ig, anti-TNF mAbs, tofacitinib, anti-IL-2R (e.g., basiliximab), anti-IL-17 mAbs (e.g., secukinumab), anti-IL-6 mAbs (e.g., anti-IL-6 antibody silkumab, anti-IL-6 receptor antibody tocilizumab (Actemra®)), IL-10 inhibitors, TGF-β inhibitors, B-cell targeted antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particles (SVP®)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles), intravenous gamma globulin (IVIG), omalizumab, methotrexate, tyrosine kinase inhibitors (e.g., ibrutinib), cyclophosphamide, fingolimod, B-cell activator (BAFF) inhibitors (e.g., anti-BAFF mAbs, e.g., belimumab), proliferation-inducing ligand inhibitors (APRIL), anti-IL-1β These include mAbs (e.g., canakinumab (Haris®)), C3a inhibitors, Tregitope (see, for example, U.S. Patent No. 10,213,496), or combinations and / or derivatives thereof.

[0331] Strategies to reduce (overcome), avoid, or mitigate the effects of humoral immunity on viral vectors (e.g., rAAV vectors) include administering high vector doses, using empty AAV capsids as decoys to adsorb anti-AAV antibodies, administering immunosuppressants to reduce, decrease, inhibit, prevent, or eradicate the humoral immune response to rAAV, altering the rAAV capsid serotype, or manipulating rAAV capsids to make them less susceptible to neutralizing antibodies, using plasma exchange cycles to adsorb anti-AAV immunoglobulins and thereby reduce anti-AAV antibody titers, and using delivery techniques such as balloon catheters followed by saline flushing. Such strategies are described, for example, in Mingozzi et al., (2013) Blood, 122:23-36. Similar techniques and strategies can be used for other types of viral vectors.

[0332] An empty capsid used as a decoy probe, provided in the same or a separate formulation as the rAAV vector, can be provided in different ratios to the viral vector. In certain embodiments, the decoy probe is provided simultaneously with the rAAV. The amount of empty capsid administered can be calibrated based on the amount (titer) of antibody produced in a particular subject. In certain embodiments, the ratio of empty AAV capsid to rAAV vector is within or between approximately 100:1 to 50:1, approximately 50:1 to 25:1, approximately 25:1 to 10:1, approximately 10:1 to 1:1, approximately 1:1 to 1:10, approximately 1:10 to 1:25, approximately 1:25 to 1:50, or approximately 1:50 to 1:100. In certain embodiments, the ratio of the empty AAV capsid to the rAAV vector administered is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Preferably, the serotype of the empty capsid is the same as the rAAV serotype.

[0333] In certain embodiments, viral vehicles are delivered using methods that bypass the bloodstream and viral antibodies. Examples of such techniques include delivery to the liver via the hepatic artery and delivery to the liver by endoscopic retrograde cholangiopancreatography (ERCP). Other duct systems, such as the submandibular gland duct, can also be used as entry points for delivering viral vectors to subjects that express or have pre-existing anti-antibodies against the viral vector.

[0334] Further strategies to reduce humoral immunity against rAAV (which can be applied to other viral vectors) include methods for removing, depleting, capturing, and / or inactivating AAV antibodies, commonly known as apheresis, or more specifically, plasmapheresis involving blood products. Apheresis, or plasmapheresis, is the process of circulating human target plasma ex vivo (outside the body) through a device that modifies the plasma by adding, removing, and / or replacing components before returning it to the patient. Using plasmapheresis, human immunoglobulins (e.g., IgG, IgE, IgA, IgD) can be removed from blood products (e.g., plasma). This procedure can be used to deplete, capture, inactivate, reduce, or remove immunoglobulins (antibodies) that bind to AAV, thereby lowering the titer of the target AAV antibody that may contribute to rAAV neutralization. One example involves using a device consisting of an AAV capsid affinity matrix column, through which a blood product (e.g., plasma) is passed to the AAV capsid affinity matrix, resulting in the binding of different isotypes of AAV antibodies. (For example, Bertin et al., 2020, Sci. Rep. 10, 864, the whole of which is incorporated herein by reference).

[0335] In certain embodiments, polynucleotide constructs, viral vectors, and non-viral vectors can be used in combination with agents such as anti-FcRn antibodies that block, inhibit, or reduce the interaction between IgG and neonatal Fc receptors (FcRn) to reduce IgG recycling and enhance in vivo IgG clearance, and / or agents that conjugate to recombinant viral vectors or to nucleic acids or polypeptides, proteins or peptides encoded by polynucleotides capsid-formed by recombinant viral vectors, or agents that reduce circulating antibodies that conjugate to polynucleotides. In certain embodiments, antibody binding to viral vectors is reduced or inhibited by agents that reduce the interaction of IgG with FcRn, proteases, or glycosidases.

[0336] In certain embodiments, the polynucleotide constructs, viral vectors, and non-viral vectors described herein may be used in combination with endopeptidases (e.g., IdeS derived from Streptococcus pyogenes) or modified variants thereof, or with endoglycosidases (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof. Such therapies may be performed, for example, to reduce or eliminate neutralizing antibodies against gene delivery vehicles (e.g., viral vector capsids) and enable the treatment of patients previously deemed ineligible for gene therapy or patients developing antibodies resulting from gene therapy. Such strategies are described, for example, in Leborgne et al., (2020) Nat. Med., 26:1096-1101.

[0337] VII. Kit The present invention includes a kit comprising a packaging material and one or more components thereof. The kit typically includes a label or package insert containing a description or instructions for use of the components therein for in vitro, in vivo, or ex vivo use. The kit may include an aggregate of such components, such as a polynucleotide construct, a viral vector, or a non-viral vector, and optionally a second active substance such as another compound, drug, or composition.

[0338] A kit refers to a physical structure that contains one or more components. Packaging materials can be made from materials commonly used for such purposes, such as paper, corrugated fiber, glass, plastic, foil, ampoules, vials, and tubes, which can keep the components sterile.

[0339] The label or insert may include clinical pharmacology of the active ingredient, including identification information, dosage, mechanism of action, pharmacokinetics, and pharmacodynamics of one or more of its components. The label or insert may include information identifying the manufacturer, lot number, place and date of manufacture, and expiration date. The label or insert may include information about diseases in which the kit components can be used. The label or insert may include instructions for clinicians or subjects to use one or more of the kit components in a method, use, or treatment protocol or treatment regimen. The instructions may include dosage, frequency, or duration for implementing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein, and instructions.

[0340] Labels or inserts may include information about one or more benefits that the components may offer, such as preventive or therapeutic benefits. Labels or inserts may also include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which the use of a particular composition is inappropriate. If a subject is taking, plans to take, or is currently taking, one or more other medications that may be incompatible with this product, or is receiving, plans to receive, or is currently receiving another treatment protocol or regimen that is incompatible with this product, adverse side effects or complications may occur, and the instructions may include information about such interactions.

[0341] Labels or inserts include "printed material," such as paper or cardboard, which is separate from or fixed to the components, kit, or packaging material, or attached to ampoules, tubes, or vials containing the kit components. Labels or inserts may further include computer-readable media such as barcoded printed labels, discs, optical discs such as CDs or DVD-ROMs / RAMs, DVDs, MP3s, magnetic tapes, or electrical storage media such as RAM and ROMs, or hybrids thereof such as magnetic / optical storage media, FLASH media, or memory cards.

[0342] VIII. Additional aspects and embodiments Examples of additional aspects, embodiments, and combinations thereof include: 1. RNA polynucleotides comprising a targeted RNA sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences of SEQ ID NOs. 36, 38, 32-35, 37, or 39-62, and / or differing by one or two nucleotides from any of the sequences of SEQ ID NOs. 36, 38, 32-35, 37, or 39-62. 2. The RNA polynucleotide described in 1, wherein the targeted RNA sequence includes a sequence selected from the group consisting of the following: a) A sequence in which sequence number 34 is different from sequence number 0, 1, 2 or 3 nucleotides, and the target sequence includes the sequence of sequence number 108, b) A sequence in which 0, 1, 2 or 3 nucleotides are different from sequence number 36, and the target sequence includes the sequence of sequence number 109, c) A sequence in which 0, 1, 2 or 3 nucleotides are different from sequence number 37, and the targeting sequence includes the sequence of sequence number 110, d) A sequence in which sequence number 38 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 111, e) A sequence in which sequence number 39 is different from sequence number 0, 1, 2 or 3 nucleotides, and the target sequence includes the sequence of sequence number 112, f) A sequence in which the sequence of sequence number 41 is different from the sequence of 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 113, and g) A sequence in which the sequence of sequence number 42 is different from sequence number 0, 1, 2, or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 114. 3. The RNA polynucleotide described in 1, wherein the targeted RNA sequence includes the sequence of SEQ ID NO: 36 or SEQ ID NO: 38. 4. The RNA polynucleotide described in 3, wherein the targeting RNA sequence includes the sequence of SEQ ID NO: 95 or SEQ ID NO: 97. 5. The RNA polynucleotide according to any one of items 1 to 4, wherein the RNA polynucleotide further comprises a second RNA sequence, the second RNA sequence being substantially complementary to the targeting RNA sequence. 6. The RNA polynucleotide according to 5, wherein the RNA polynucleotide is a pre-miRNA, and the pre-miRNA comprises a pre-miRNA scaffold, an integrated guide sequence, and an integrated passenger sequence, the guide sequence comprising a targeting sequence, and the passenger sequence comprising a second sequence. 7. The RNA polynucleotide according to 6, comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of sequence numbers 122-128 and 207-224. 8. The RNA polynucleotide described in 1, comprising the sequence of SEQ ID NO: 123 or 125. 9. The RNA polynucleotide according to 5, wherein the RNA polynucleotide is shRNA, and the shRNA comprises an shRNA scaffold, an integrated guide sequence, and an integrated passenger sequence, the guide sequence comprising a targeting sequence, and the passenger sequence comprising a second sequence. 10. The RNA polynucleotide according to 5, wherein the RNA polynucleotide is a pri-miRNA, and the pri-miRNA comprises a pri-miRNA scaffold, an integrated guide sequence, and an integrated passenger sequence, the guide sequence comprising a targeting sequence, and the passenger sequence comprising a second sequence. 11. RNA polynucleotides as described in 10, wherein the scaffold is miR-1, miR-26, miR16-1, miR-30, miR-33, miR-101, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold. 12. The RNA polynucleotide according to 10, wherein the scaffold is S155e scaffold, S26 scaffold, S126 scaffold, S33 scaffold or S155 scaffold. In further embodiments, the scaffold includes SEQ ID NO: 186 or 187. 13. The RNA polynucleotide according to 10, comprising an RNA version of any of SEQ ID NOs. 115-121, 207-224, or 243-260 (with a ribose backbone and U instead of T) having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In further embodiments, the RNA polynucleotide comprises a passenger / guide combination provided in Tables 2, 3, or 4. 14. The RNA polynucleotide described in 11, comprising the sequence of SEQ ID NO: 116 or SEQ ID NO: 118. 15. An optionally modified inhibitory RNA comprising (a) a guide strand capable of hybridizing with any of the target sequences of Sequence ID No. 129 to 135, and (b) a substantially complementary passenger sequence, wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide as described in any one of sections 16.1 to 14. 17. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide as described in any one of items 1 to 14, operably linked to one or more expression regulatory elements. 18. An expression cassette according to 17, comprising an upstream promoter and a downstream polyadenylation signal operably ligated to a nucleic acid sequence encoding an RNA polynucleotide. The expression cassette according to 18, comprising a promoter or promoter / enhancer, a nucleic acid sequence encoding an RNA polynucleotide, and a polyadenylation signal from 19.5' to 3', wherein the promoter or promoter / enhancer and the polyadenylation signal are operably ligated to the nucleic acid sequence encoding an RNA polynucleotide. 20. An expression cassette according to 18 or 19, wherein the promoter provides CNS expression. 21. An expression cassette according to 18 or 19, wherein the promoter is the EF-1α promoter. 22. An expression cassette described in any one of items 17-21, wherein the expression cassette is DNA. 23. An expression cassette according to 22, comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of sequence numbers 168 to 174. 24. An expression cassette according to 23, comprising the sequence of sequence number 169 or 171. 25. An expression cassette according to any one of items 17 to 24, further comprising a nucleic acid sequence encoding glial cell-derived neurotrophic factor (GDFN) operably linked to a promoter or to a second promoter. A recombinant viral vector nucleic acid comprising an expression cassette described in any one of sections 26.17-25 and 5' and 3' viral elements that provide packaging and / or replication of the virus. 27. The recombinant viral vector nucleic acid according to 26, wherein the recombinant viral vector nucleic acid is recombinant DNA and comprises an adeno-associated virus (AAV) reverse repeat sequence (ITR) adjacent to the 5' end of the recombinant viral vector nucleic acid and an AAV ITR adjacent to the 3' end of the recombinant viral vector nucleic acid. 28. Recombinant viral vector nucleic acid as described in 27, wherein the 5'ITR and 3'ITR are selected from the 5'ITR and 3'ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, or AAV3B. 29. The recombinant viral vector nucleic acid described in 26, wherein the recombinant viral vector nucleic acid is self-complementary. The recombinant viral vector nucleic acid according to 27, wherein the 30.5'ITR contains a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 182, and the 3'ITR contains a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 183. 31. Recombinant viral vector nucleic acid according to 27 or 30, comprising a sequence having at least 90%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences of sequence numbers 175 to 181. 32. Recombinant viral vector nucleic acid according to 27, comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 176 or 178. 33. A delivery vehicle comprising a viral vector or a non-viral vector and an RNA polynucleotide as described in any one of items 1 to 14, an inhibitory RNA as described in item 15, a polynucleotide as described in item 16, an expression cassette as described in any one of items 17 to 25, or a recombinant viral vector nucleic acid as described in any one of items 26 to 32. 34. The delivery vehicle described in 33, wherein the delivery vehicle is a viral vector. 35. The delivery vehicle according to 34, wherein the viral vector is recombinant AAV, recombinant lentiviral vector, or recombinant adenovirus vector. 36. The delivery vehicle according to 35, wherein the viral vector is a recombinant AAV comprising a recombinant viral vector nucleic acid and a capsid as described in any one of items 26 to 32. 37. The delivery vehicle according to 36, wherein the recombinant AAV vector comprises a capsid containing VP1, VP2, or VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10, and VP1, VP2, or VP3 having at least 90% amino acid sequence identity, or VP1 of SEQ ID NO: 164 or SEQ ID NO: 167. 38. A delivery vehicle according to 36, wherein the capsid is the capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10, or the capsid contains VP1 of Sequence ID No. 164 or Sequence ID No. 167. 39. The delivery vehicle according to 36, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166. 40. A delivery vehicle according to any one of claims 36 to 39, wherein the capsid is an AAV2 capsid containing the targeting sequence of sequence number 279, 280, 281, or 282, which is inserted between the positions corresponding to N587 and R588 of VP1. 41. A delivery vehicle according to 39 or 40, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequences of SEQ ID NOs. 137 and 151. 42. The delivery vehicle according to 41, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of Sequence ID No. 158. 43. The delivery vehicle according to 41, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 169. 44. The delivery vehicle according to 41, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 176. 45. A delivery vehicle according to any one of paragraphs 41 to 44, wherein the capsid includes VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166. 46. ​​A delivery vehicle according to any one of claims 41 to 44, wherein the capsid is an AAV2 capsid containing the targeted sequence of sequence number 279 or 282, which is inserted between the positions corresponding to N587 and R588 of VP1. 47. The delivery vehicle according to 33, wherein the delivery vehicle is a nanoparticle selected from the group consisting of lipid nanoparticles (LNPs), polymer nanoparticles, lipid polymer nanoparticles (LPNPs), protein or peptide nanoparticles, DNA dendrimers or DNA nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. 48. The delivery vehicle according to 47, wherein the delivery vehicle is an LNP or LPNP. 49. A recombinant adeno-associated virus vector comprising a capsidized rAAV nucleic acid in a capsid, wherein the rAAV nucleic acid comprises (a) the 5'ITR of SEQ ID NO: 182, (b) the EF1a promoter of SEQ ID NO: 184, (c) a pri-miRNA coding sequence comprising the sequence of SEQ ID NO: 158, (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 185, and (e) the 3'ITR of SEQ ID NO: 183, with (c) operably ligated to (b) and (d). 50. A recombinant AAV vector according to 49, comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of sequence number 169, or the sequence of sequence number 169. 51. A recombinant AAV vector according to 49 or 50, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166. A pharmaceutical composition comprising an RNA polynucleotide as described in any one of items 52.1 to 14, an inhibitory RNA as described in item 15, a polynucleotide as described in item 16, an expression cassette as described in any one of items 17 to 25, a recombinant viral vector nucleic acid as described in any one of items 26 to 32, a delivery vehicle as described in any one of items 33 to 48, an rAAV vector as described in any one of items 49 to 51, and a pharmaceutically acceptable carrier. 53. A method for reducing or inhibiting α-synuclein aggregation in cells or a subject, comprising administering to cells or a subject an RNA polynucleotide described in any one of items 1 to 14, an inhibitory RNA described in item 15, a polynucleotide described in item 16, an expression cassette described in any one of items 17 to 25, a recombinant viral vector nucleic acid described in any one of items 26 to 32, a delivery vehicle described in any one of items 33 to 48, an rAAV vector described in any one of items 49 to 51, or a pharmaceutical composition described in item 52. 54. A method for treating a subject with a synucleinopathy, comprising administering to the subject an RNA polynucleotide described in any one of items 1 to 14, an inhibitory RNA described in 15, a polynucleotide described in 16, an expression cassette described in any one of items 17 to 25, a recombinant viral vector nucleic acid described in any one of items 26 to 32, a delivery vehicle described in any one of items 33 to 48, an rAAV vector described in any one of items 49 to 51, or a pharmaceutical composition described in 52. 55. The method according to 54, wherein the synucleinopathy is either Lewy body disease or multiple system atrophy. 56. The method according to 54, wherein the synucleinopathy is one of the following: infantile axonal dystrophy, atypical axonal dystrophy, adult-onset dystonia-parkinsonism, autosomal recessive early-onset parkinsonism, POLG-associated neurodegeneration, Niemann-Pick type C1, or Krabbe disease. 57. The method described in 54, wherein the synucleinopathy is a disease or disorder that is Parkinson's disease. 58. A method for treating a disease or disorder related to a decrease in dopamine concentration, comprising administering to a subject an RNA polynucleotide described in any one of items 1 to 14, an inhibitory RNA described in 15, a polynucleotide described in 16, an expression cassette described in any one of items 17 to 25, a recombinant viral vector nucleic acid described in any one of items 26 to 32, a delivery vehicle described in any one of items 33 to 48, an rAAV vector described in any one of items 49 to 51, or a pharmaceutical composition described in 52. 59. The method according to any one of items 53 to 58, wherein the administration includes direct intraparenchymal administration, intracisional administration, or intraventricular administration. 60. The method described in any one of items 53-59, wherein the initial dose is administered outside the CNS. 61. The method described in any one of items 53-60, wherein the subject is a human. 62. The method according to any one of items 53-61, which reduces α-synuclein to approximately 25% to approximately 70%, 30% to 65%, or 35% to 60% of baseline levels in a transduced brain region (e.g., the substantia nigra). The method according to any one of claims 53 to 62, wherein a recombinant AAV from any of claims 63.36 to 46 and 59 to 51 is provided in doses of 3e8vg / SN to 3e11vg / SN, about 3e8vg / SN, about 3e9vg / SN, about 3e10vg / SN, or about 3e11vg / SN. An AAV vector genome plasmid containing recombinant viral nucleic acid as described in any one of sections 64.26-64.32. 65. The AAV genome plasmid described in 64, lacking the rep and cap genes. 66. A method for producing an rAAV vector, comprising the step of culturing an rAAV packaging cell line containing rAAV helper virus activity, wherein the genome of the producing cell contains the nucleic acid, rep gene and cap gene described in any one of items 26 to 32, and an rAAV vector is produced. A method for producing an rAAV vector, comprising the step of culturing rAAV-tolerant cells containing the rAAV genome plasmid described in 67.64, wherein the rAAV-tolerant cells further comprise (a) rep genes and cap genes provided as part of the cell genome and / or by one or more separate plasmids, and (b) helper virus activity provided by the cell genome and / or by one or more separate plasmids. 68. The method according to 67, wherein the rAAV-tolerant cells are packaging cells, and the genome of the packaging cells contains a cap gene and a rep gene. 69. The method according to 67, wherein (a) the rep gene, cap gene and helper activity are provided in a single plasmid, or (b) the rep gene and cap gene are provided in a rep / cap plasmid and the helper activity is provided by a helper plasmid. 70. A method for obtaining an rAAV vector, comprising (a) a step of producing an rAAV vector using the method described in any one of items 66 to 69, and (b) a step of purifying the rAAV vector.

[0343] IX. Array Table 9 provides various nucleic acid and amino acid sequences. In various embodiments, a polynucleotide comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the nucleic acid sequences provided in Table 9, and a polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the amino acid sequences provided in Table 9.

[0344] [Table 9] JPEG2026536535000016.jpg215149JPEG2026536535000017.jpg215149JPEG2026536535000018.j pg204149JPEG2026536535000019.jpg159149JPEG2026536535000020.jpg153149JPEG2026536535 000021.jpg149149JPEG2026536535000022.jpg149149JPEG2026536535000023.jpg149149JPEG20 26536535000024.jpg149149JPEG2026536535000025.jpg149149JPEG2026536535000026.jpg14914 9JPEG2026536535000027.jpg165149JPEG2026536535000028.jpg161149JPEG2026536535000029. jpg161149JPEG2026536535000030.jpg161149JPEG2026536535000031.jpg161149JPEG2026536535 000032.jpg162149JPEG2026536535000033.jpg161149JPEG2026536535000034.jpg175149JPEG20 26536535000035.jpg213149JPEG2026536535000036.jpg208149JPEG2026536535000037.jpg23149

[0345] Table 10 provides sequence numbers for various miR constructs, including guide RNA, passenger, seed region, pri-miRNA, and pre-miRNA, and provides the encoding / corresponding DNA (except for pre-miRNA). In various embodiments, the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences provided in Table 10.

[0346] [Table 10] [Examples]

[0347] Examples illustrating different features of the present invention and methods for carrying it out are provided below. The provided examples do not limit the claimed invention.

[0348] Example 1: Decreased siRNA expression of SNCA in HEK293 cells

[0349] The effects of different siRNAs on SNCA expression and cell survival were evaluated in HEK293T cells. Lipofectamine® RNAiMAX (0.3 μL) containing 1 nM, 10 nM, or 100 nM siRNAs was administered in approximately 1.5 × 10⁶ units. 4 Cells with the specified cell density were transfected. SNCA expression was determined 72 hours after transfection by measuring α-synuclein mRNA using PCR. Cell viability was measured using Presto blue staining. Various constructs are summarized in Table 11. The siRNA sequence contained two 5' deoxythymidines.

[0350] [Table 11]

[0351] The effects of siRNA on SNCA expression are shown in Figure 1 and Table 12. Figure 1 shows the ability of different constructs to reduce SNCA expression, represented as RQ. For each siRNA, the center concentration is 10 nM, the concentration to the left is 1 nM, and the concentration to the right is 100 nM.

[0352] Table 12 shows the siRNA specifications and provides inhibition data for several constructs at 100 mM.

[0353] [Table 12]

[0354] Figures 2A, 2B, and 2C show survival percentages at different siRNA concentrations. Figure 2A shows survival with 1 nM siRNA, Figure 2B shows survival with 10 nM siRNA, and Figure 2C shows survival with 100 nM siRNA.

[0355] Example 2: Decreased siRNA expression of SNCA in HEK293 cells

[0356] The effects of different siRNAs on SNCA expression and cell survival were evaluated in cell lines overexpressing SNCA fused to green fluorescent protein (GFP). A decrease in GFP indicates a decrease in SNCA expression.

[0357] Cells were transfected with 100 nM siRNAs 1-25 (see Table 11). Fluorescence from GFP was measured 72 hours after transduction (n=2). Scrambled controls, commercially available siGLO (a non-targeted control with red fluorescence), mismatch 11 (a non-targeted control), and naive conditions were used as controls. Figure 3 shows the ability of different siRNAs to reduce SNCA expression.

[0358] Example 3: siRNA inhibition of SNCB and SNCG expression

[0359] The effects of various constructs targeting SNCA expression on SNCB and SNCG expression were evaluated using the method described in Example 1, except for the detection of SNCB and SNCG transcripts. SNCB encodes β-synuclein, and SNCG encodes γ-synuclein. β-synuclein and γ-synuclein have a similar structure to α-synuclein.

[0360] Figures 4A and 4B show the effects of siRNA on the off-target genes SNCB and SNCG 72 hours after transfection with 1 nM (left value), 10 nM (center value), and 100 nM (right value) siRNAs. The siRNA constructs are summarized in Table 11. Figure 4A shows the expression of SNCB with 1 nM, 10 nM, or 100 nM siRNAs. Figure 4B shows the expression of SNCG with 1 nM, 10 nM, or 100 nM siRNAs.

[0361] Example 4: siRNA inhibition of SNCA expression in SH-SY5Y

[0362] The effects of different siRNAs on SNCA expression and cell survival were evaluated in SH-SY5Y cells (neuroblastoma cells). The siRNA constructs are summarized in Table 11. As described in Example 1, cells were transduced with 10 nM or 100 nM siRNAs, and expression was evaluated by measuring SNCA mRNA transcripts using qPCR. Expression was measured 72 hours after transduction.

[0363] Figure 5 shows the ability of different siRNAs to inhibit expression in SH-SY5Y cells. siRNA-treated cells were compared to siGLO controls. This data supports the ability of the tested siRNAs to inhibit SNCA expression in SH-SY5Y cells.

[0364] Example 5: Differential gene expression analysis

[0365] Figure 6 shows the results of differential gene expression analysis, which measures the ability of siRNA targeting SNCA expression to reduce or increase the expression of non-target genes. Generally, preferred inhibitory nucleic acids have less impact on the expression of non-target genes.

[0366] Example 6: Inhibition of SNCA by Pri-miRNA

[0367] Pri-miRNA sequences were designed by modifying selected siRNA sequences based on their inhibitory activity, effects on cell viability, and off-target effects. The siRNA guide strand was modified by replacing the 3' terminal dT with a nucleotide complementary to the target and embedded in an S155 scaffold. The siRNA passenger strand was modified by replacing the 3' terminal dT with a nucleotide complementary to the guide strand and removing two internal nucleotides for embedding in an S155 scaffold.

[0368] Nucleic acids encoding different pri-miRNAs were inserted into a plasmid containing a reporter gene (mCherry driven by the CMV promoter), and the pri-miRNA coding sequence was placed under the control of the CBH promoter. The resulting plasmid contained CBH-miRNA-CMV-mCherry.

[0369] A plasmid encoding a human α-synuclein-GFP fusion protein was inserted into HEK293 cells. GFP signaling provided a measure of SCNA expression, while mCherry(Red)-positive cells indicated cells transfected with pri-miRNA-expressing nucleic acids. Flow cytometry assays (CytoFLEX) were performed. Provided controls were naive (no transfection) and mock (transfected with reagent only, no plasmid).

[0370] Table 13 provides nomenclature for pri-miRNAs, summarizes the DNA encoding the guide DNA sequence, the passenger DNA sequence, and the pri-miRNA encoding DNA, and provides a reference to the parental siRNA. The parental siRNA is shown as siRNA with modified guide and passenger sequences embedded in an S155 scaffold.

[0371] [Table 13]

[0372] Figure 7 shows the ability of different miRs to inhibit SCNA expression. The miR-treated groups showed a significant reduction in normalized GFP signaling (as a measure of SCNA expression).

[0373] Example 7: In vitro inhibition of α-synuclein aggregation and expression

[0374] The ability of viral delivery nucleic acids containing nucleic acids encoding different miRNAs to inhibit α-synuclein aggregation and expression was tested in primary rat neurons. The miRNAs are summarized in Table 14. Recombinant AAV vectors were produced by incorporating rAAV nucleic acids into a capsid containing VP1 (SEQ ID NO: 164), VP2 (SEQ ID NO: 165), and VP3 (SEQ ID NO: 166). rAAV production was performed using triple transfection.

[0375] Primary rat cortical neurons were seeded in 96-well plates at a rate of 5e4 cells / well. On day 6, the cells were (1) introduced with AAV1-CMV-A53Tα-Syn-WPRE (a recombinant viral vector encoding α-synuclein with an A53T substitution) at an infection factor (MOI) of 10,000, and with rAAV encoding miR at MOIs of 10,000 and 100,000; (2) on day 10, pre-formed human α-synuclein fibrils (PFFs) were added at a concentration of 1 μg / mL; and (3) on day 20, the cells were fixed and permeabilized and stained with antibodies against α-synuclein and pS129.

[0376] The test constructs are summarized in Table 14. Pri-miRNA was operably ligated to the EF1 promoter (SEQ ID NO: 184). The control construct was rAAV-CAG-EGFP containing a CAG promoter ligated to EGFP; rAAV-EF1-mis11 containing a missense miRNA sequence; and rAAV1 / 2-3XmiR-GFP (AAV1 / 2-CAG-Human-SNCA-3xmiR / GFP-WPRE-BGH-polyA) encoding three copies of miRNA that targets α-synuclein encoding mRNA. (AAV1 / 2-CAG-Human-SNCA-3xmiR / GFP-WPRE-BGH-polyA was obtained from Charles River).

[0377] [Table 14]

[0378] Figure 8A shows the percentage of normalized α-synuclein intensity relative to the α-synuclein control. Different miRs significantly inhibited α-synuclein expression.

[0379] Figure 8B shows the percentage of pS129 intensity as a measure of aggregation. Aggregate formation was determined by measuring the fluorescence intensity of pS129 staining and acquiring images using an Opera Phenix 20X air objective lens. Different miRs significantly inhibited α-synuclein aggregation.

[0380] Example 8: In vivo inhibition in a simultaneous transduction model

[0381] The ability of rAAV vectors containing viral nucleic acids encoding miR5 or miR7 to express and inhibit α-synuclein aggregation was evaluated in C57BL6 mice. Inclusion of rAAV nucleic acids encoding miR5 or miR7 was performed as described in Example 7. The experimental design is shown in Table 15.

[0382] [Table 15] IPa refers to intraparenchymal administration to either both hemispheres (two values) or one hemisphere.

[0383] Inhibition of SCNA expression was evaluated using qPCR, which measures mRNA transcripts encoding α-synuclein. Figure 9A shows the change in α-synuclein compared to the rAAV1 / 2-A53TαSyn+rAAV-mis11(mis11) control. Recombinant AAVs encoding miR5 and miR7 significantly reduced SCNA expression at doses of 5e9 and 1e10vg / hemisphere at 7 weeks post-injection in the mouse striatum. AAV1 / 2-3XmiRNA was included as a positive control for knockdown.

[0384] Inhibition of α-synuclein aggregation in the prefrontal cortex was evaluated using the α-synuclein-specific antibody syn204 at a dose of 1 e10 vg / hemispheric dose. Proteins were analyzed by JESS using antibodies targeting human α-Syn. The results are shown in Figure 9B. Both miR5 and miR7 reduced α-synuclein protein.

[0385] The average count of miRNA reads obtained by small RNA sequencing indicates that miR5 and miR7 are successfully expressed from the rAAV vector in the striatum of WT mice when co-introduced with AAV-A53TαSyn (Figure 9C).

[0386] Figure 9D shows the effects of viral vectors on endogenous mouse SNCA (mSCNA). Overexpression of human A53Tα-synuclein did not affect endogenous α-synuclein expression. miR7 has a one-nucleotide mismatch (non-complementary at position 6) with mouse α-synuclein mRNA, while miR5 has a two-nucleotide mismatch (non-complementary at positions 7 and 10) with mouse α-synuclein mRNA. miR7 reduced α-synuclein in the striatum, but miR5 did not have a significant effect.

[0387] Example 9: Inhibition of endogenous mSNCA expression in substantia nigra

[0388] The ability of rAAV vectors to inhibit endogenous mSCNA expression in the substantia nigra was evaluated in mice. The research plan is summarized in Table 16.

[0389] [Table 16] SN refers to injection into the substantia nigra.

[0390] The in vivo distribution and its effect on mSCNA expression are shown in Figures 10A-10D. Figure 10A shows the in vivo distribution in the substantia nigra (SN) and striatum (Str). Recombinant AAV-miR5 and rAAV-miR7 vectors injected into the substantia nigra were detected with low VCGN in the striatum, suggesting anterograde movement of the viral vectors.

[0391] Figure 10B shows mSCNA expression in the substantia nigra (SN) and striatum (Str). Mouse endogenous α-synuclein mRNA levels in the striatum were significantly higher than in the substantia nigra.

[0392] Figure 10C shows mSCNA expression in the substantia nigra (SN). Mouse endogenous α-synuclein mRNA in the substantia nigra was significantly reduced by rAAV-miR7 (1 mismatch), but not by rAAV-miR5 (2 mismatches).

[0393] Figure 10D shows mSCNA expression in the striatum (Str). Mouse endogenous α-synuclein mRNA in the striatum was significantly reduced by both rAAV-miR7 (1 mismatch) and rAAV-miR5 (2 mismatches).

[0394] Example 11: miRNA processing

[0395] Figure 11 shows guide and passenger processing of miR5 and miR7. Next-generation sequencing (NGS) was performed on small RNAs to evaluate miRNA processing after intrasubliminal (IP) delivery of miRNA rAAV vectors to the mouse brain striatum. Briefly, C57BL / 6 mice were co-injected into the mouse brain striatum at two doses: rAAV1 / 2-A53TαSyn (expressing a human SNCA with the A53T mutation), rAAV-mis11 (encoding a missense miRNA), rAAV-miR5, or rAAV-miR7. After 7 weeks, tissue was collected and processed for NGS sequencing of small RNAs. The amounts of guide RNA and passenger RNA generated from two different rAAV-miRNA vectors at two different doses indicate a preferred guide-to-passenger ratio (in the case of a guide-dominant species) that promotes specific degradation of target mRNA (Figure 11).

[0396] Example 12: 10-week rAAV-miR5 study

[0397] The effects of an rAAV vector containing miR5-encoding viral nucleic acid, delivered bilaterally to the substantia nigra of C57BL6 mice, were evaluated 10 weeks after administration. Various parameters, including body weight, in vivo distribution, α-synuclein knockdown, dopamine levels, and dopamine turnover, were measured. Furthermore, the effects on miRNA processing and endogenous miRNA mechanisms were assessed.

[0398] Recombinant AAV vectors were produced by incorporating rAAV nucleic acids into a capsid containing VP1 (SEQ ID NO: 164), VP2 (SEQ ID NO: 165), and VP3 (SEQ ID NO: 166). rAAV production was performed using triple transfection.

[0399] Mice were administered a fixed amount of human αSyn (AAV1 / 2-A53TαSyn) to induce a decrease in dopamine, similar to that seen in synucleinopathy, including Parkinson's disease. In addition, the treated animals were administered rAAV-miR5 in doses of 5e7, 5e8, or 5e9 vector genome (vg). The control group was administered a diluent or 5e9vg of mis11 (missense construct) + αSyn.

[0400] Figure 12 shows the statistical significance of endpoint body weight. Endpoint body weight analysis showed a significant reduction in body weight in the human α-Syn (AAV1 / 2-A53Tα-Syn) and control miRNA (rAAV-CAG-mis11) treatment groups compared to diluent-treated animals. Compared to the control, 5e7vg of rAAV-miR5 showed a reduction in body weight. No statistically significant reduction in body weight was observed in the rAAV-miR5 treatment groups at high and medium doses (5e9 and 5e8vg).

[0401] The distribution of rAVV-miR5 in the substantia nigra is shown in Figures 13A and 13B. The presence of the vector was evaluated using specific primers designed based on BgH and RbG polyA sequences. The AAV1 / 2-A53TαSyn vector possesses BgH polyA; rAAV-miR5 and rAAV-CAG-mis11 possess RbG polyA. Figure 13A shows the measurement of BgH polyA. Figure 13B shows the measurement of RbG polyA.

[0402] Figure 14 shows the ability of rAAV-miR5 to knock down α-Syn, as measured by qRT-PCR. Brain tissue from the substantia nigra was analyzed by qRT-PCR using α-Syn primers. No signal was observed in the diluent injection group, indicating specificity for human α-Syn. Statistically significant knockdown of human α-Syn was observed in the rAAV-miR5 treatment groups at doses of 5e9 (highest dose) and 5e8 (medium dose) vg compared to rAAV-CAG-mis11 (control miRNA), but not at 5e7 (lowest dose). Statistical analysis was performed using Tukey's multiple comparison test with standard one-way Anova.

[0403] Figure 15 shows the ability of rAAV-miR5 to knock down α-Syn in the cortex, as measured by the JESS assay. The first diluent lacks human α-Syn, and the second diluent contains α-Syn. Statistically significant knockdown of human α-Syn was observed in the rAAV-miR5 treatment group at all doses: 5e9vg (highest dose), 5e8vg (medium dose), and 5e7vg (lowest dose) compared to rAAV-CAG-mis11.

[0404] In Parkinson's disease, dopaminergic neurons in the substantia nigra project into the striatum, where they release the neurotransmitter dopamine. Dopamine levels and turnover were measured to evaluate whether rAAV-miR5 treatment inhibited dopamine loss.

[0405] Figure 16 shows the ability of rAAV-miR5 to inhibit dopamine loss induced by A53Tα-Syn. Compared to the dilution group, administration of rAAV encoding human A53Tα-Syn (AAV1 / 2-A53TαSyn) and the negative control miRNA (rAAV-CAG-mis11) resulted in significantly greater dopamine loss in the striatum. Statistically significant inhibition of dopamine loss in the striatum was observed in all miRNA treatment groups (rAAV-miR5, 5e9vg, 5e8vg, and 5e7vg) compared to the AAV1 / 2-A53TαSyn treatment group and the negative control miRNA (rAAV-CAG-mis11) treatment group.

[0406] In Parkinson's disease, the ratio of dopamine downstream metabolites (DOPAC and HVA) to dopamine (DOPAC + HVA) indicates stable dopamine turnover and healthy dopamine neurons. Figure 17 shows the ability of rAAV-miR5 to protect against impaired dopamine turnover. Impaired dopamine turnover was observed during treatment with AAV1 / 2-A53TαSyn and the negative control miRNA (rAAV-CAG-mis11). Protection against impaired dopamine turnover in the rAAV-miR5 treatment group was observed at medium and low doses, but not at high doses. This suggests that the dopamine mechanism is impaired due to total viral load at the highest dose, despite good α-Syn knockdown and protection against dopamine loss.

[0407] Figure 18 shows the evaluation of microRNA processing by small RNA sequencing. Small RNA sequencing after rAAV-miR5 shows that the miRNA guide was expressed better than the passenger strand.

[0408] Figure 19 summarizes the results of small RNA sequencing. The endogenous miRNA mechanism was not disrupted by rAAV-miR5 overexpression. Even at the maximum dose of 5e9vg, the expressed exogenous mature miRNAs accounted for only 1% of the total endogenous miRNA pool. The medium and minimum doses of 5e8 and 5e7vg accounted for less than 0.1% of the total endogenous miRNA pool. The dilution treatment group was a negative control group that did not have miRNAs.

[0409] Example 13: SNCA-OVX mouse study

[0410] SNCA-OVX mice were administered rAAV-miR5 at 5e8vg / SN and 5e9vg / SN. SNCA-OVX mice are genetically modified, with both copies of the mouse SNCA gene deleted; they contain a BAC construct that provides one copy of the human SNCA gene under the control of its endogenous promoter, providing approximately 1.6–1.9 times normal levels of α-Syn expression. (Janezic et al., Proc Natl Acad Sci USA 2013;110(42):E4016-25). Human SNCA mRNA levels were measured by RT-qPCR.

[0411] Significant variability in the abundance of SNCA mRNA in the substantia nigra was observed between untreated males and females, and even among same-sex members. Furthermore, male SNCA-OVX mice had significantly higher body weight compared to females, which resulted in less precise targeting of the substantia nigra and a lower distribution of rAAV-miR5 in males compared to females in the same group. While the variability made it difficult to interpret rAAV-miR5-administered studies, a significant 45% decrease was observed in SNCA mRNA at 5e9vg / SN per hemisphere of rAAV-miR5 in females at 6 weeks (Figure 20).

[0412] Example 14: Dose-setting study in non-human primates (NHPs)

[0413] A two-month NHP dose-finding study was conducted to evaluate the bilateral targeting, pharmacology, and preliminary safety of rAAV-miR5 in the substantia nigra of Chlorocephalus sabaeus (African green monkey, AGM, n=2 / group, 3 doses). Recombinant AAV vectors were produced by incorporating rAAV nucleic acids into a capsid containing VP1 (SEQ ID NO: 164), VP2 (SEQ ID NO: 165), and VP3 (SEQ ID NO: 166). rAAV production was performed using triple transfection. Potential histopathology was assessed by classical hematoxylin and eosin (H&E) staining.

[0414] The primary objective of this study was to evaluate the intracerebral distribution of gene therapy AAV vectors after delivery via MRI-guided convection-enhanced bilateral intraparenchymal (IP) injection into the substantia nigra pars compacta of male Chlorocebus sabaeus, followed by an 8-week observation period. Secondary analyses of this study focused on evaluating the expression levels of target genes in the brain regions in question, including the substantia nigra and its surrounding tissues, as well as their projection pathways.

[0415] Each NHP was administered a bilateral dose of either the vehicle, a control miRNA vector (missense 11 expressed under the EF-1α promoter), or rAAV-miR5. The experimental design is shown in Table 17.

[0416] [Table 17] *15 μL was administered to the left hemisphere of animal C1303.

[0417] On day 60±3, all NHPs were euthanized, their brains were removed, placed in a pre-cooled brain matrix, sliced ​​into 4mm sections, and divided into left and right hemispheres for processing. The left hemisphere was fixed for histology, and the tissue from the right hemisphere was dissected or perforated and processed for in vivo distribution analysis.

[0418] MRI images using gadolinium contrast showed that the substantia nigra was successfully targeted in the test animals. Table 18 shows the estimated coverage of the substantia nigra pars compacta in each test animal. The values ​​were calculated from post-injection images and are estimates of the anatomical target and injection range. Due to the limited resolution of the 1.5T MRI scanner, it was not possible to distinguish between the substantia nigra pars compacta and the reticular region. The values ​​shown in Table 18 correspond to the coverage of the entire anatomical structure. Given that the compacta is located dorsal to the reticular region, the overall coverage of the compacta is expected to be higher than the estimates.

[0419] [Table 18]

[0420] Dose-dependent distribution and expression of rAAV-miR5 were observed, corresponding to a decrease in SNCA mRNA over a two-month period (the longest time point tested) following bilateral injection of rAAV-miR5 into the substantia nigra of NHPs (Figures 21A, 21B, and 21C). Overall SNCA mRNA data also indicated that the rAAV-miR5-mediated decrease in SNCA mRNA was observed primarily in the substantia nigra (>40%). Quantification of cytoplasmic α-syn protein by immunohistochemical intensity staining in TH-positive DA neurons of SNs showed a dose-dependent rAAV-miR5-mediated decrease in cytoplasmic α-syn protein (Figure 21D).

[0421] The distribution of rAAV-miR5 administered at doses of 3e10vg / SN (Figure 22A), 3e9vg / SN (Figure 22B), and 3e8vg / SN (Figure 22C) was primarily contained in the substantia nigra, with limited spread to the connected basal ganglia structures of the subthalamic nucleus and globus pallidus. Correspondingly, SNCA mRNA levels showed a dose-dependent decrease, mainly in the SN and several adjacent basal ganglia regions (Figure 22D). RT-qPCR analysis of SNCA mRNA, performed using validated primer-probe combinations, demonstrated a dose-dependent decrease in SNCA mRNA in the substantia nigra (>50% at dose 3e10vg / SN) when compared to a control group (including diluent-treated animals and a mis11 control group to account for basal fluctuations in endogenous SNCA levels) 2 months after necropsy. Very minimal spread to the spinal cord region was observed, and no vector spread to any peripheral tissues outside the CNS was observed. The expression of the transgene product miR5 followed the distribution pattern of rAAV-miR5, showing significant presence only in the substantia nigra and limited expression only in adjacent basal ganglia structures.

[0422] Overall, the data indicate that the in vivo distribution of the vector genome and the transgene product miR5 were dose-dependent and enriched in the substantia nigra. rAAV-miR5 did not significantly extend beyond the injected and anatomically connected brain regions.

[0423] Neurosurgical treatment was well tolerated, no adverse effects were reported during the life-span of the study, and only minimal histopathological changes along the needle track were observed in all NHPs. No rAAV-miR5-related microscopic findings were observed. All microscopic findings in the study were incidental or related to the injection procedure. Cannula entry points and insertion routes consistent with the injection procedure were macroscopically visible in the brains of all NHPs and primarily included parenchymal loss and vacuolation, pigmented macrophages, gliosis, and axonal degeneration. The incidence and severity of these findings were not associated with rAAV-miR5 treatment.

[0424] The NHP study demonstrated that rAAV-miR5 induced a stable, dose-dependent reduction in both SNCA mRNA and α-Syn protein when administered via neurosurgical delivery to the NHP brain. In the NHP, the surgical administration procedure, similar to that used in human patients, was well-tolerated and resulted in good coverage of the target brain region with minimal histopathological changes.

[0425] The expression of the transgenic product (miR5) is The distribution pattern was consistent with that of rAAV-miR5, with significant expression observed only in the substantia nigra (SN), and limited expression in the adjacent basal ganglia structures. The reduction of SNCA mRNA by rAAV-miR5 was mainly observed in the SN (over 40%), with a reduction within the target range of 25% to 70%. Overall, the data suggest that the in vivo distribution of the vector genome and transgenic product was dose-dependent and concentrated in the SN. rAAV-miR5 did not significantly diffuse outside the injection site or anatomically connected brain regions.

[0426] Example 15: 4-month NHP dose trial

[0427] Considering the target 25% α-syn reduction - 70% and previous preclinical data, a 4-month dose-range study using cynomolgus monkeys, ranging from 3e10vg to 3e11vg per hemisphere, is planned to provide additional biodistribution, pharmacological, and safety data. The study design is shown in Table 19.

[0428] [Table 19]

[0429] The dosage is planned to be a single injection into the substantia nigra via direct convection-enhanced IP infusion using ClearPoint Neuro navigation software, with a ClearPoint Orchestra® neurosurgical frame paired with a ClearPoint SmartFlow Neuro Ventricular cannula. MRI (3T Siemens Skyra) is used for imaging.

[0430] A variety of parameters and endpoints can be evaluated, including some, some, or all of the following: observational findings, body weight, physical examination, neurological FOB, ophthalmological examination, blood pressure, electrocardiogram, clinicopathological evaluation, organ weights, and macroscopic and microscopic examinations; tissue samples taken at autopsy (multiple regions of the brain, DRG [cervical, thoracic, lumbar], adrenal glands, liver, lungs, heart, kidneys, spleen, testes, ovaries), as well as blood and CSF taken at specific points in time for biodistribution and / or biomarker analysis. Tissues for microscopic examination may include brain regions, spinal cord including nerve roots (cervical, thoracic, and lumbar), dorsal root ganglia ("DRG") (cervical, thoracic, and lumbar), sciatic nerve, bones, eyes, liver, spleen, heart, lungs, kidneys, scaffolding muscles, stomach, testes, epididymis, ovaries, uterus, and lymph nodes (cervical and submandibular).

[0431] By examining rAAV-miR5 genomic DNA, miRNA transgenes, and cynomolgus monkey SNCA mRNA levels in the central nervous system (CNS), the extent and magnitude of the in vivo distribution of SNCA mRNA reduction (pharmacodynamics) at each dose level can be determined. α-Syn protein levels can be measured using multiple methods, including enhanced chemiluminescence and immunohistochemical detection.

[0432] Example 16: Human Dose

[0433] Dosage data from non-human primates can be used as a starting point for extrapolating to doses targeting the substantia nigra to achieve a 25-75% reduction in α-syn levels in the human brain. NHP doses can be scaled to humans, for example, based on the volume difference between substantia nigra volume and substantia nigra volume determined from adult cynomolgus monkeys with Parkinson's disease at the same disease stage as the target patient population (modified Hoehn & Yahr stages 1-2.5). Substantia nigra MRI volumetric data are available from the Parkinson's Progression Markers Initiative database and related published reports (Langley et al., medRxiv [Preprint] 2023.08:19.23294281).

[0434] While the present invention has been described and illustrated with reference to its specific embodiments, those skilled in the art will understand that various adaptations, changes, modifications, substitutions, deletions, or additions to the procedures and protocols can be made without departing from the spirit and scope of the invention.

Claims

1. An RNA polynucleotide comprising a targeted RNA sequence having at least 90% sequence identity with any of the sequences of sequence numbers 36, 38, 32-35, 37, or 39-62.

2. The RNA polynucleotide according to claim 1, wherein the targeted RNA sequence includes a sequence selected from the group consisting of the following: a) A sequence in which sequence number 34 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 108, b) A sequence in which sequence number 36 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 109, c) A sequence in which sequence number 37 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 110, d) A sequence in which sequence number 38 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 111, e) A sequence in which sequence number 39 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 112, f) A sequence in which sequence number 41 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 113, and g) A sequence in which the sequence of sequence number 42 is different from sequence number 0, 1, 2 or 3 nucleotides, and the targeting sequence includes the sequence of sequence number 114.

3. The RNA polynucleotide according to claim 1, wherein the targeted RNA sequence includes the sequence of SEQ ID NO: 36 or SEQ ID NO:

38.

4. The RNA polynucleotide according to claim 3, wherein the targeted RNA sequence includes the sequence of SEQ ID NO: 95 or SEQ ID NO:

97.

5. The RNA polynucleotide according to any one of claims 1 to 4, wherein the RNA polynucleotide further comprises a second RNA sequence, the second RNA sequence being substantially complementary to the targeted RNA sequence.

6. The RNA polynucleotide according to claim 5, wherein the RNA polynucleotide is a pre-miRNA, and the pre-miRNA comprises a pre-miRNA scaffold, an incorporated guide sequence, and an incorporated passenger sequence, the guide sequence comprises the targeting sequence, and the passenger sequence comprises the second sequence.

7. The RNA polynucleotide according to claim 6, comprising a sequence having at least 90% sequence identity with any of sequence numbers 122-128 and 207-224.

8. The RNA polynucleotide according to claim 1, comprising the sequence of sequence number 123 or 125.

9. The RNA polynucleotide according to claim 5, wherein the RNA polynucleotide is shRNA, and the shRNA comprises an shRNA scaffold, an incorporated guide sequence, and an incorporated passenger sequence, the guide sequence comprises the targeting sequence, and the passenger sequence comprises the second sequence.

10. The RNA polynucleotide according to claim 5, wherein the RNA polynucleotide is a primi-miRNA, and the primi-miRNA comprises a primi-miRNA scaffold, an incorporated guide sequence, and an incorporated passenger sequence, the guide sequence comprises the targeting sequence, and the passenger sequence comprises the second sequence.

11. The RNA polynucleotide according to claim 10, wherein the scaffold is a scaffold of miR-1, miR-26, miR16-1, miR-30, miR-33, miR-101, miR-64, miR-122, miR-125, miR-135, miR-155, reinforced miR-155 (eSIBR), or miR-451.

12. The RNA polynucleotide according to claim 10, wherein the scaffold is an S155e scaffold, an S26 scaffold, an S126 scaffold, an S33 scaffold, or an S155 scaffold.

13. The RNA polynucleotide according to claim 10, wherein the RNA polynucleotide includes a sequence having at least 90% sequence identity with any RNA version of SEQ ID NOs. 115-121, 207-224, or 243-260.

14. The RNA polynucleotide according to claim 1, comprising the sequence of SEQ ID NO: 116 or SEQ ID NO:

118.

15. An optionally modified inhibitory RNA comprising (a) a guide strand capable of hybridizing with any of the target sequences of Sequence ID No. 129 to 135, and (b) a substantially complementary passenger sequence, wherein one or more nucleotides of the guide strand and the passenger strand are optionally modified RNA.

16. A polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1 to 14.

17. An expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide according to any one of claims 1 to 14, operably linked to one or more expression control elements.

18. The expression cassette according to claim 17, comprising an upstream promoter and a downstream polyadenylation signal operably linked to the nucleic acid sequence encoding the RNA polynucleotide.

19. The expression cassette according to claim 18, comprising a promoter or promoter / enhancer, the nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal from 5' to 3', wherein the promoter or promoter / enhancer and the polyadenylation signal are operably linked to the nucleic acid sequence encoding the RNA polynucleotide.

20. The expression cassette according to claim 18 or 19, wherein the promoter provides CNS expression.

21. The expression cassette according to claim 18 or 19, wherein the promoter is an EF-1α promoter.

22. The expression cassette according to any one of claims 17 to 21, wherein the expression cassette is DNA.

23. The expression cassette according to claim 22, wherein the expression cassette includes a sequence having at least 95% sequence identity with any one of sequence numbers 168 to 174.

24. The expression cassette according to claim 23, wherein the expression cassette includes the sequence of sequence number 169 or 171.

25. The expression cassette according to any one of claims 17 to 24, further comprising a nucleic acid sequence encoding glial cell-derived neurotrophic factor (GDFN) operably coupled to the promoter or to a second promoter.

26. Recombinant viral vector nucleic acid comprising an expression cassette according to any one of claims 17 to 25 and 5' and 3' viral elements for packaging and / or replicating the virus.

27. The recombinant viral vector nucleic acid according to claim 26, wherein the recombinant viral vector nucleic acid is recombinant DNA and comprises an adeno-associated virus (AAV) reverse repeat sequence (ITR) adjacent to the 5' end of the recombinant viral vector nucleic acid and an AAV ITR adjacent to the 3' end of the recombinant viral vector nucleic acid.

28. Recombinant viral vector nucleic acid according to claim 27, wherein the 5'ITR and 3'ITR are selected from the 5'ITR and 3'ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh. 10, AAVrh. 74, or AAV3B.

29. The recombinant viral vector nucleic acid according to claim 27, wherein the recombinant viral vector nucleic acid is self-complementary.

30. The recombinant viral vector nucleic acid according to claim 27, wherein the 5'ITR includes a sequence having at least 95% sequence identity with sequence number 182, and the 3'ITR includes a sequence having at least 95% sequence identity with sequence number 183.

31. Recombinant viral vector nucleic acid according to claim 27 or 30, comprising a sequence having at least 90% sequence identity with any of sequence numbers 175 to 181.

32. The recombinant viral vector nucleic acid according to claim 27, comprising a sequence having at least 95% sequence identity with sequence number 176 or 178.

33. A delivery vehicle comprising a viral vector or a non-viral vector, and an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, or a recombinant viral vector nucleic acid according to any one of claims 26 to 32.

34. The delivery vehicle according to claim 33, wherein the delivery vehicle is the viral vector.

35. The delivery vehicle according to claim 34, wherein the viral vector is recombinant AAV, recombinant lentiviral vector, or recombinant adenovirus vector.

36. The delivery vehicle according to claim 35, wherein the viral vector is a recombinant AAV comprising the recombinant viral vector nucleic acid and capsid described in any one of claims 26 to 32.

37. The delivery vehicle according to claim 36, wherein the recombinant AAV vector comprises a capsid containing VP1, VP2, or VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh. 74, AAV3B, AAV-2i8, AAVrh. 10, AAVrh. 8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh. 10, and VP1, VP2, or VP3 having at least 90% sequence identity, or VP1 of SEQ ID NO: 164 or SEQ ID NO:

167.

38. The delivery vehicle according to claim 36, wherein the capsid is the capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh. 74, AAV3B, AAV-2i8, AAVrh. 10, AAVrh. 8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh. 10, or the capsid includes VP1 of sequence number 164 or sequence number 167.

39. The delivery vehicle according to claim 36, wherein the capsid includes VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO:

166.

40. The delivery vehicle according to claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequences of sequence numbers 137 and 151.

41. The delivery vehicle according to claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of sequence number 158.

42. The delivery vehicle according to claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:

169.

43. The delivery vehicle according to claim 39, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:

176.

44. The delivery vehicle according to claim 33, wherein the delivery vehicle is a nanoparticle selected from the group consisting of lipid nanoparticles (LNPs), polymer nanoparticles, lipid polymer nanoparticles (LPNPs), protein or peptide nanoparticles, DNA dendrimers or DNA nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes.

45. The delivery vehicle according to claim 44, wherein the delivery vehicle is an LNP or an LPNP.

46. A recombinant adeno-associated vector comprising a capsidized rAAV nucleic acid in a capsid, wherein the rAAV nucleic acid comprises (a) the 5' ITR of SEQ ID NO: 182, (b) the EF1a promoter of SEQ ID NO: 184, (c) a prim-miRNA coding sequence comprising the sequence of SEQ ID NO: 158, (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 185, and (e) the 3' ITR of SEQ ID NO: 183, wherein (c) is operably ligated to (b) and (d).

47. The recombinant AAV vector according to claim 46, comprising a sequence at least 95% identical to sequence number 169.

48. The recombinant AAV vector according to claim 46 or 47, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO:

166.

49. A pharmaceutical composition comprising an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery vehicle according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, and a pharmaceutically acceptable carrier.

50. A method for reducing or inhibiting α-synuclein aggregation in cells or a subject, comprising administering to the cells or subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery vehicle according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 49.

51. A method for treating a subject with a synucleinopathy, comprising administering to the subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery vehicle according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 49.

52. The method according to claim 51, wherein the synucleinopathy disease or disorder is either Lewy body disease or multiple system atrophy.

53. The method according to claim 51, wherein the synucleinopathy disease or disorder is any of infantile axonal dystrophy, atypical axonal dystrophy, adult-onset dystonia-parkinsonism, autosomal recessive early-onset parkinsonism, POLG-associated neurodegeneration, Niemann-Pick type C1, or Krabbe disease.

54. The method according to claim 51, wherein the synucleinopathy disease or disorder is Parkinson's disease.

55. A method for treating a disease or disorder related to a decrease in dopamine concentration, comprising administering to a subject an RNA polynucleotide according to any one of claims 1 to 14, an inhibitory RNA according to claim 15, a polynucleotide according to claim 16, an expression cassette according to any one of claims 17 to 25, a recombinant viral vector nucleic acid according to any one of claims 26 to 32, a delivery vehicle according to any one of claims 33 to 45, an rAAV vector according to any one of claims 46 to 48, or a pharmaceutical composition according to claim 46.

56. The method according to any one of claims 50 to 55, wherein the administration includes direct intraparenchymal administration, intracisional administration, or intraventricular administration.

57. The method according to any one of claims 50 to 56, wherein the initial dose is administered outside the CNS.

58. The method according to any one of claims 50 to 57, wherein the subject is a human.

59. The method according to any one of claims 50 to 58, wherein α-synuclein is reduced to about 25% to about 70% of the baseline level in the transduced brain region.

60. The method according to claim 59, wherein the transfected brain region is the substantia nigra.

61. AAV vector genome plasmid comprising recombinant viral nucleic acid according to any one of claims 26 to 32.

62. The AAV genome plasmid according to claim 61, wherein the plasmid lacks the rep and cap genes.

63. A method for producing an rAAV vector, comprising the step of culturing an rAAV packaging cell line containing rAAV helper virus activity, wherein the genome of the producing cell contains the nucleic acid, rep gene, and cap gene described in any one of claims 26 to 32, and the rAAV vector is produced.

64. A method for producing an rAAV vector, comprising the step of culturing rAAV-tolerant cells comprising the rAAV genome plasmid described in claim 62, wherein the rAAV-tolerant cells further comprise (a) rep genes and cap genes provided as part of the cell genome and / or by one or more separate plasmids, and (b) helper virus activity provided by the cell genome and / or by one or more separate plasmids.

65. The method according to claim 64, wherein the rAAV-tolerant cells are packaging cells, and the genome of the packaging cells comprises a cap gene and a rep gene.

66. The method according to claim 64, wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided in a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

67. A method for obtaining an rAAV vector, comprising: (a) producing an rAAV vector using the method described in any one of claims 63 to 66; and (b) purifying the rAAV vector.

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