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WO2025085713A1 - Acide nucléique inhibiteur ciblant l'expression de l'alpha synucléine - Google Patents

Acide nucléique inhibiteur ciblant l'expression de l'alpha synucléine Download PDF

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WO2025085713A1
WO2025085713A1 PCT/US2024/051907 US2024051907W WO2025085713A1 WO 2025085713 A1 WO2025085713 A1 WO 2025085713A1 US 2024051907 W US2024051907 W US 2024051907W WO 2025085713 A1 WO2025085713 A1 WO 2025085713A1
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sequence
seq
nucleic acid
raav
rna
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Pedro Gonzalez-Alegre
Sambuddha Basu
Maria Grazia BIFERI
Vikrant SINGH
Juan Li
Christopher CALI
Francesca CARGNIN
Elizabeth Ramsburg
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Spark Therapeutics Inc
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Spark Therapeutics Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Synucleinopathies are neurodegenerative diseases or disorders characterized by neuronal and/or glial inclusions. Pathologically, synucleinopathies can be divided into two major disease groups: Lewy body diseases or disorders and multiple system atrophy (MSA). Lewy body diseases and disorders are characterized by aggregated a-synuclein, and include Parkinson’s disease, Parkinson’s disease dementia, dementia with Lewy bodies, infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, adult-onset dystonia-parkinsonism, autosomal recessive early-onset parkinsonism, POLG-associated neurodegeneration, Niemann-Pick type Cl, and Krabbe disease. (Koga et al. Molecular Neurodegeneration (2021) 16:83.) [0004] Parkinson’s disease is an age-related progressive neurodegenerative disorder.
  • Parkinson’s disease is characterized by the abnormal accumulation of misfolded a-synuclein protein aggregates in various regions of the brain. Dopaminergic neuronal loss in the substantia nigra is a pathologic hallmark of Parkinson’s disease. (Lee et al. Neuroimmunol.
  • Parkinson symptomatic disease treatments such as dopamine replacement therapy, have been used to treat motor symptoms associated with Parkinson’s disease.
  • motor symptoms can generally be well controlled with symptomatic medications.
  • Parkinson’s disease advances, such treatments becomes less effective and associated with debilitating side effects (e.g., dyskinesia), leading to worsening of functioning and quality of life.
  • side effects e.g., dyskinesia
  • the present invention features polynucleotide constructs comprising sequences targeting a-synuclein mRNA.
  • Polynucleotide constructs include inhibitory RNA polynucleotides comprising a sequence targeting a-synuclein mRNA, such as pri-microRNA (pri-miRNA), pre- microRNA (pre-miRNA), short hairpin RNA (shRNA), microRNA (miRNA) and optionally modified miRNA.
  • Constructs comprising sequences targeting a-synuclein mRNA and/or encoding sequences can be used, for example, in methods for inhibiting a-synuclein expression and/or treating a synucleinopathy disease or disorder.
  • a-synuclein mRNA “targeting” sequence is able to hybridize to a substantially complementary a-synuclein mRNA target sequence.
  • Alpha-synuclein mRNA target sequences described herein include those of any of SEQ ID NOs: 129-135 and the complement of any of SEQ ID NOs: 32-62.
  • RNA polynucleotide indicates the polynucleotide comprises a sequence targeting an mRNA that can inhibit mRNA activity. Inhibition of mRNA activity results in a decrease in protein expression from the targeted mRNA.
  • Inhibitory RNA include, for example, miRNA and miRNA precursors such as pre-miRNA, sh-RNA, and pri-miRNA
  • RNA polynucleotide comprising an RNA sequence targeting a-synuclein mRNA.
  • the RNA polynucleotide comprises a targeting RNA sequence having a sequence identity of at least 90% with the sequence of any of SEQ ID NOs: 32-62.
  • a second aspect of the present invention is directed to an optionally modified inhibitory RNA duplex comprising (a) a guide strand able to hybridize to the target sequence of any of SEQ ID NOs: 129-135; 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 third aspect of the present invention is directed to a polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide comprising an a-synuclein mRNA targeting sequence.
  • a fourth aspect of the present invention is directed to an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide comprising an a-synuclein mRNA targeting sequence and one or more expression control elements operably linked to the encoding nucleic acid sequence.
  • a fifth aspect of the present invention is directed to recombinant viral vector nucleic acid comprising (a) an expression cassette comprising a nucleic acid sequence encoding an RNA polynucleotide comprising an a-synuclein mRNA targeting sequence and one or more expression control elements operably linked to the encoding nucleic acid sequence and (b) 5’ and/or 3’ viral elements providing for viral packaging and/or replication.
  • a sixth aspect of the present invention is directed to a delivery vehicle comprising a viral or a non-viral vector and (a) an RNA polynucleotide comprising a sequence targeting a- synuclein mRNA; (b) an optionally modified inhibitory RNA duplex targeting a-synuclein mRNA; or (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding for an RNA polynucleotide comprising an a-synuclein mRNA targeting sequence.
  • a seventh aspect of the present invention is directed to a pharmaceutical compositions comprising (a) an RNA polynucleotide comprising a sequence targeting a-synuclein mRNA, (b) an optionally modified inhibitory RNA duplex targeting a-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding for an a-synuclein mRNA targeting sequence or (d) a delivery vehicle comprising (a), (b) or (c); and a pharmaceutically acceptable carrier.
  • An eight aspect of the present invention is directed to a method of reducing a-synuclein expression and/or treating a synucleinopathy disease or disorder in a subject comprising administration of (a) an RNA polynucleotide comprising a sequence targeting a-synuclein mRNA, (b) an optionally modified inhibitory RNA duplex targeting a-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding for an a-synuclein mRNA targeting sequence or (d) a delivery vehicle comprising (a), (b) or (c) and a pharmaceutically acceptable carrier.
  • a ninth aspect of the present invention is directed to a method of treating a disease or disorder associated with low dopamine comprising administration of (a) an RNA polynucleotide comprising a sequence targeting a-synuclein mRNA, (b) an optionally modified inhibitory RNA duplex targeting a-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding for an a-synuclein mRNA targeting sequence or (d) a delivery vehicle comprising (a), (b) or (c) and a pharmaceutically acceptable carrier.
  • Additional aspects of the present invention include (a) an RNA polynucleotide comprising a sequence targeting a-synuclein mRNA, (b) an optionally modified inhibitory RNA duplex targeting a-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding for an a-synuclein mRNA targeting sequence or (d) a delivery vehicle comprising (a), (b) or (c) and a pharmaceutically acceptable carrier, for use in medicine, reducing a-synuclein, or treating a synucleinopathy disease or disorder; and use of (a), (b), (c), or (d) in preparation of a medicament (for example, for use in medicine, reducing a-synuclein or treating a synucleinopathy disease or disorder).
  • FIG. 1 illustrates the ability of different small interfering RNA (siRNA) to reduce expression of the human gene encoding for a-synuclein (SCNA) in HEK293 cells.
  • siRNA were provided at concentrations of 1 nM, 10 nM, or 100 nM.
  • GAPDH refers to glyceraldehyde 3- phosphate dehydrogenase.
  • FIG. 2A, FIG. 2B and FIG. 2C illustrate percent survival of HEK293 cells administered different amount of siRNA.
  • FIG. 2 A shows results using 1 nM.
  • FIG. 2B show results using 10 nM.
  • FIG. 2C show results using 100 nM.
  • GAPDH refers to glyceraldehyde 3 -phosphate dehydrogenase.
  • FIG. 3 illustrates the ability of different siRNAs to inhibit expression of an a-synuclein- GFP fusion protein in HEK293 cells.
  • Plotted activity was green fluorescent protein (GFP) /Vinculin normalized to naive control.
  • the provided controls were siGLO, a scrambled control (non-targeting control with red fluorescence); mi smatch 11 (non-targeting control); and naive conditions.
  • FIG. 4A and FIG. 4B illustrate the impact of different siRNA targeting SNCA expression, on related genes in HEK293 cells.
  • FIG. 4A illustrates expression of SNCB (gene encoding P-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM.
  • FIG. 4B illustrates expression of SNCG (gene encoding y-synuclein) at siRNA concentrations of 1 nM, 10 nM, or 100 nM.
  • GAPDH refers to glyceraldehyde 3 -phosphate dehydrogenase.
  • FIG. 5 illustrates the ability of different siRNA to inhibit expression in SH-SY5Y cells, at different concentrations.
  • FIG. 6 is a graph showing differential expression (DE) of genes resulting from different siRNAs.
  • FIG. 7 illustrates the ability of different pre-miRNA to reduce normalized GFP signal (as a measure of SCNA expression) compared to pCC41. The pre-miRNA are shown with a miR designation. Statistical analysis was carried out using 2-way ANOVA with Dunnett’s multiple comparison analysis.
  • FIG. 8A and FIG. 8B illustrate the ability of different rAAV vectors comprising nucleic acid encoding for pre-miRNA to inhibit a-synuclein expression and a-synuclein aggregation at two different multiplicity of infections.
  • FIG. 8A illustrates normalized human a-synuclein to AAV-a-synuclein (AAV-a-syn).
  • FIG. 8B illustrates normalized pS129 intensity to AAV-a- synuclein. pS129 intensity is a measure of a-synuclein aggregation.
  • Statistical analysis was carried out using 2-way ANOVA against AAV-aSyn.
  • FIGs. 9A-9D illustrates the ability of different rAAV vectors comprising nucleic acid encoding for pre-miRNA to inhibit a-synuclein expression.
  • FIG. 9A illustrates fold change relative to human a-synuclein + misl 1.
  • FIG. 9B illustrates inhibition of human a-synuclein production.
  • FIG. 9C illustrates reduction in mutant a-synuclein (A53T).
  • FIG. 9D illustrates the effect on mouse a-synuclein expression.
  • Statistical analysis was carried out using 1-way ANOVA with Dunnett’s multiple comparison.
  • FIGs. 10A-10D illustrate miRNA biodistribution and inhibition of murine a-synuclein.
  • FIG. 10A illustrates biodistribution in the substantia nigra (SN) and striatum (Str).
  • FIG. 10B illustrates murine SNCA (mSCNA) expression in the SN and Str.
  • FIG. 10C illustrates mSCNA expression in the substantia nigra.
  • FIG. 10D illustrates mSCNA expression in the striatum.
  • FIG. 11 illustrates guide and passenger strand levels from rAAV vectors.
  • FIG. 12 illustrates the effect of different doses on aSyn treated mice body weight.
  • Mice were administered a 7.5e9 vg/SN dose of human aSyn (AAVl/2-A53TaSyn) to induce a- synuclein mediate neuronal loss.
  • Treatment animals received 5e7, 5e8, or 5e9 vector genomes (vg) doses of rAAV-miR5.
  • Controls were administered diluent, or misl 1 (missense construct) at 5e9 vg + aSyn.
  • Statistics Ordinary 1-way ANOVA with Tukey’s post-hoc analysis.
  • FIGs. 13 A and 13B illustrate rAVV-miR5 distribution in the substantia nigra.
  • Treatment animals received 5e7, 5e8, or 5e8 vector genomes (vg) doses of rAAV-miR5.
  • Controls were administered diluent, or misl 1 (missense construct) at 5e9 vg + aSyn.
  • Vector presence in the substantia nigra was assessed using specific primers designed based on the BgH and RbG polyA sequences.
  • the AAVl/2-A53TaSyn vector has a BgH polyA; rAAV-miR5 and rAAV-CAG- misl 1 have a RbG PolyA.
  • FIG. 13A illustrates measurement of BgH polyA.
  • FIG. 13B illustrates measurement of RbG polyA.
  • FIG. 14 illustrates the ability of rAAV-miR5 to knock down aSyn, as measured by qRT- PCR.
  • Treatment animals received 5e7, 5e8, or 5e8 vector genomes (vg) doses of rAAV-miR5.
  • Controls were administered diluent, or misl 1 (missense construct) at 5e9 vg + aSyn.
  • Statistics were done by Ordinary 1-way Anova with Tukey’s multiple comparison test. **p ⁇ 0.01, *p ⁇ 0.05
  • FIG. 15 illustrates the ability of rAAV-miR5 to knock down aSyn in the cortex, as measured by JESS assay.
  • Treatment animals received 5e7, 5e8, or 5e8 vector genomes (vg) doses of rAAV-miR5. Controls were administered diluent, or misl 1 (missense construct) at 5e9 vg + aSyn. Ordinary 1-way ANOVA Tukey’s multiple comparison. ***p ⁇ 0.001. Comparison done with negative control + AST a-Syn.
  • FIG. 16 illustrates the ability of rAAV-miR5 to inhibit dopamine loss caused by A53T a- Syn.
  • Treatment animals received 5e7, 5e8, or 5e8 vector genomes (vg) doses of rAAV-miR5.
  • Controls were administered diluent, or misl 1 (missense construct) at 5e9 vg + aSyn. Data represent an absolute number. Ordinary 1-way ANOVA Tukey’s post hoc test performed on log scale values of the data points. Comparison done with negative control + AST a-Syn. ****p ⁇ 0.0001, ***p ⁇ 0.001, *p ⁇ 0.05.
  • FIG. 17 illustrates the ability of rAAV-miR5 to inhibit dopamine loss caused by A53T a- Syn.
  • Treatment animals received doses of rAAV-miR5 vector genomes (vg) at doses of 5e7, 5e8, or 5e9.
  • Controls were administered diluent, or misl 1 (missense construct) at 5e9 vg + aSyn.
  • FIG. 18 illustrates microRNA processing determined by small RNA sequencing after rAAV-miR5 administration.
  • G/P refers to guide/passenger ratio.
  • FIG. 19 summarizes results of small RNA-sequencing. Endogenous miRNA machinery was not perturbed by rAAV-miR5 overexpression. Treatment animals received doses of rAAV- miR5 vector genomes (vg) at doses of 5e7, 5e8, or 5e9. At the highest dose, 5e9 vg, exogenous matured miRNA was 1% of the total endogenous miRNA pool. The mid and low doses 5e8 and 5e7 vg, resulted in exogenous miRNA of ⁇ 0.1% of the total endogenous miRNA pool. Diluent treated group, the negative control group, had no miRNA.
  • FIG. 20 illustrates the ability of rAAV-miR5 to significantly reduce human SNCA mRNA levels at a dose of 5e9 vg/SN, as measured by RT-qPCR of human SNCA mRNA in the SN compared to diluent injected group in SNCA-OVX female mice.
  • Statistical comparisons between groups were assessed by 1-way ANOVA with Tukey’s multiple comparisons test. Data are plotted as individual (symbols) and group means ⁇ SEM (bars).
  • FIGs. 21 A and 21B illustrate the biodistribution of vector genomes and expression of miR5, respectively, in the substantia nigra of African green monkeys (“AGM”) two months after the administration of rAAV-miR5.
  • the X axis depicts three tested doses of rAAV-miR5 (3e8 vg, 3e9 vg, and 3el0 vg per substantia nigra) and the Y axis depicts copy number (CN) of vector genomes/pg of DNA (FIG. 21 A) or CN of miR5 miRNA/pg of RNA (FIG. 2 IB).
  • miRNA expression analysis showed a dose-dependent distribution of rAAV-miR5 in the substantia nigra.
  • FIG. 21C illustrates the relative expression of SNCA mRNA by dosage group of AGM.
  • the X axis depicts the two control groups (diluent and misl 1) as well as the three doses of rAAV-miR5 that were administered (3e8 vg, 3e9 vg, and 3el0 vg per substantia nigra).
  • the Y axis depicts fold change in relation to the diluent group.
  • n 2 AGM.
  • Misl 1 refers to a nontargeting miRNA expressed under a synthetic EF- la promoter.
  • FIG. 2 ID shows immunohistochemistry (IHC) analysis of cytoplasmic a-synuclein protein (also referred to herein as a-Syn) intensity in tyrosine hydroxylase (TH)-positive dopaminergic (DA) neurons of the substantia nigra, indicating rAAV-miR5-mediated dosedependent reduction of cytoplasmic a-Syn protein.
  • a-Syn cytoplasmic a-synuclein protein
  • TH tyrosine hydroxylase
  • DA dopaminergic
  • the X axis depicts Group 1 (diluent), Group 2 (misl 1), Group 3 (rAAV-miR5 administered at 3e8 vg per substantia nigra), Group 4 (rAAV-miR5 administered at 3e9 vg per substantia nigra), and Group 5 (rAAV-miR5 administered at 3el0 vg per substantia nigra).
  • the Y axis depicts a-Syn intensity. Each data point represents mean values per monkey where 2 tissue punches were collected for analysis. Error bars represent SEM.
  • FIGs. 22A, 22B, and 22C provide graphical representations of biodistribution analysis of vector genome copy number (CN), reported as CN per microgram of genomic DNA, at individual doses of 3el0 vg/SN (FIG. 21 A), 3e9 vg/SN (FIG. 21B), and 3e8 vg/SN (FIG. 21C).
  • CN was determined using a primer probe targeting a unique sequence of Rabbit Globin PolyA.
  • the X axis depicts the basal ganglia and adjacent brain structures whereas the Y axis depicts CN/pg of DNA.
  • rAAV-miR5 there is a dose dependent increase of rAAV-miR5 in the substantia nigra and a relatively low to moderate distribution in adjacent anatomically connected brain regions of Basal Ganglia structures such as the subthalamic nucleus and globus pallidus.
  • Basal Ganglia structures such as the subthalamic nucleus and globus pallidus.
  • the biodistribution analysis of other brain regions of basal ganglia and cortical structures shows that rAAV-miR5 distribution is contained primarily within the target region.
  • FIG. 22D depicts a global SNCA mRNA knockdown profile in critical brain regions by rAAV-miR5.
  • RT-qPCR analysis of SNCA mRNA demonstrated dose-dependent reduction of SNCA mRNA in SN (by >50% at dose 3el0 vg/SN) when compared to the control group (includes diluent treated animals and the misl 1 control group to account for basal variability of endogenous SNCA level) at 2 months post-necropsy.
  • RNA polynucleotide constructs comprising sequences targeting a-synuclein mRNA and encoding nucleic acid.
  • RNA polynucleotide constructs include inhibitory RNA polynucleotides comprising a sequence targeting a-synuclein mRNA, such as pri-miRNA, pre-miRNA, shRNA, miRNA and optionally modified miRNA.
  • the inhibitory RNA polynucleotide is an inhibitory RNA duplex.
  • Reference to an “inhibitory RNA duplex” indicates a double-stranded RNA comprising a guide strand targeting an RNA target region and a passenger strand.
  • the passenger strand is sufficiently complementary to the guide strand to hybridize under physiological conditions.
  • the guide strand in association with an RNA-induced silencing complex (RISC) can inhibit target RNA activity.
  • RISC RNA-induced silencing complex
  • Inhibitory RNA duplexes such as miRNA can be produced, for example, through gene expression producing pri-miRNA, pre-miRNA, or shRNA followed by biogenesis; and by chemical synthesis.
  • Pri-miRNA scaffolds can be embedded with guide and passenger sequences to produce pri-miRNAs.
  • Guide strands can be selected to target different types of RNA sequences or regions such as 5’ UTR, 3’ UTR, or mRNA.
  • Different passenger sequences can be embedded into a pri-miRNA scaffold for a given guide sequence.
  • the passenger strand can be optimized for a particular scaffold and to provide sufficiently complementarity to the guide strand to hybridize under physiological conditions.
  • Constructs comprising a sequence targeting a-synuclein mRNA can be used, for example, in inhibitory RNA or to produce inhibitory RNA, where the inhibitory RNA can inhibit a-synuclein expression and/or treat a synucleinopathy disease or disorder.
  • Inhibiting a- synuclein expression can be carried out, for example, for therapeutic and research purposes. Research purposes include examining the impact of inhibiting a-synuclein expression in an animal model.
  • Reference to “subject” indicates a mammal, such as a human; a non-human primate such as an ape, gibbon, gorilla, chimpanzee, orangutan, or macaques; a domestic animal such as a dog and cat; a farm animal such as poultry, duck, horse, cow, goat, sheep and pig; and an experimental animal such as a mouse, rat, rabbit, or guinea pig.
  • a preferred subject is a human.
  • the inhibitory RNA targeting a-synuclein mRNA are evaluated using transgenic mice expressing human A53T mutant a-synuclein.
  • sequence “identical” or “identity” to a targeting sequence can be calculated by determining the number of identical nucleotides in sequences aligned to provide the maximum identity and dividing by the total number of nucleotides in the targeting sequence and multiplying by 100. Differences between aligned sequences can include one or more deletion, substitution, and/or addition.
  • RNA and the corresponding DNA are considered the same, unless otherwise indicated (e.g., referring to the molecule as DNA or RNA).
  • Corresponding RNA for DNA provides uracil instead of thymine and the ribose backbone instead of the deoxyribose backbone.
  • nucleic acid and “polynucleotide” are used interchangeably herein to refer to different forms of nucleic acid and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) unless otherwise indicated.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Unless indicated otherwise by the context, nucleic acids can be single, double, or triplex, linear or circular, and can be of any length.
  • the polynucleotide is a single-stranded (ssDNA) or a double-stranded DNA (dsDNA) molecule.
  • the dsDNA molecule is a minicircle, a nanoplasmid, open linear duplex DNA or a closed-ended linear duplex DNA (CELiD/ceDNA/doggybone DNA).
  • the ssDNA molecule is a closed circular or an open linear DNA.
  • a “transgene” refers to a nucleic acid that is intended or has been introduced into a cell and operably linked to a promoter. Transgenes include nucleic acid encoding a heterologous polynucleotide sequence and/or a heterologous promoter. [0059] In certain embodiments, encoding polynucleotide constructs are “CpG reduced” or “CpG depleted”. In certain embodiments, regions that are “CpG reduced” or “CpG depleted” are those outside of the pri-miRNA or pre-miRNA sequences.
  • CpG reduced or “CpG depleted” refer to (i) a nucleotide sequence wherein one or more of the CpG dinucleotides (or motifs) are removed from a reference nucleic acid sequence; and/or (ii) the percentage of CpGs in a referred to polynucleotide is 0% to 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%.
  • CpG motifs are reduced in the 5’ and/or 3’ untranslated regions (UTRs), stuffer sequences, promoter, enhancer, polyadenylation signal, 5’ and/or 3’ ITRs, and/or introns.
  • the conjunctive term “and/or” between multiple recited elements encompasses both individual and combined options. For instance, where two elements are conjoined by “and/or”, a first option refers to the applicability of the first option without the second, a second option refers to the applicability of the second option without the first, and a third option refers to the applicability of the first and second options together. Any one of the options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or”. Concurrent applicability of more than one of the options is also understood to fall within the meaning of the term “and/or.”
  • polypeptide can be used interchangeably to refer to an amino acid sequence without regard to function.
  • Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acid acids.
  • the provided amino acids include naturally occurring amino acids and amino acids provided by cellular modification.
  • the term “about” refers to a value within 10% of the underlying parameter (z.e., plus or minus 10%). For example, “about 1 : 10” includes 1.1 : 10.1 or 0.9:9.9, and “about 5 hours” includes 4.5 hours or 5.5 hours. The term “about” at the beginning of a string of values modifies each of the values by 10%. In certain embodiments, the term “about” refers to a value within 10% of the underlying parameter.
  • a dosage of about “0.01 mg/kg to about 10 mg/kg” body weight of a subject includes 0.011 mg/kg, 0.012 mg/kg, 0.013 mg/kg, 0.014 mg/kg, 0.015 mg/kg etc., as well as 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg and so forth.
  • Reference to an integer with more (greater) or less than includes numbers greater or less than the reference number, respectively.
  • reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more; and administration “two or more” times includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times.
  • the instant invention is generally disclosed herein using affirmative language to describe the numerous embodiments of the instant invention.
  • the instant invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.
  • materials and/or method steps are excluded.
  • RNA polynucleotides targeting a particular target region comprise a sequence substantially complementary to the target region.
  • Substantial complementary sequences comprise a region of at least 10 nucleotides that are 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 addition, deletion, and non-complementary bases. Preferably, substantial complementary sequences can hybridize to each other under physiological conditions.
  • substantial complementary sequences comprise a region of 10- 25 nucleotides, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, that when aligned for maximum complementarity are perfectly complementary, at least 70% complementary, at least 80% complementary, or at least 90% complementary.
  • substantial complementary sequences comprise a region of 10- 25 nucleotides, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, that when aligned for maximum complementarity are perfectly complementary except for 1, 2, or 3 nucleotide differences.
  • the construct comprising an a-synuclein mRNA targeting sequence is an inhibitory RNA duplex, pri-miRNA, pre-miRNA, or shRNA.
  • the a-synuclein mRNA targeting sequence present in these constructs provides a guide strand sequence able to hybridize to a-synuclein mRNA.
  • Such constructs also comprise a passenger sequence substantially complementary to the guide sequence. The degree of complementarity of the guide to the passenger sequence can vary, for example, taking into account sufficient complementarity to hybridize and use in a particular scaffold.
  • the guide and passenger stands when aligned for maximum complementarity are perfectly complementary, of have a 1, 2, 3 or 4 nucleotide mismatches from perfect complementarity.
  • Each mismatch can be independently a non-complementary base, addition, and/or deletion.
  • the RNA polynucleotide comprises a targeting sequence that comprises: (1) a region 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 the RNA sequence of any of SEQ ID NOs: 32-62 or 94-100; (2) a sequence differing from any of SEQ ID NOs: 32-62 by 0, 1, 2 or 3 nucleotides; or (4) a sequence differing from any of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 by 0, 1, 2 or 3 nucleotides.
  • the targeting RNA sequence comprises: a) a sequence differing from SEQ ID NO: 34 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 108; b) a sequence differing from SEQ ID NO: 36 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 109; c) a sequence differing from SEQ ID NO: 37 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 110; d) a sequence differing from SEQ ID NO: 38 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 111; e) a sequence differing from SEQ ID NO: 39 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 112; f) a sequence differing from SEQ ID NO: 41 by 0, 1, 2
  • the guide sequence comprises, consists or consist essentially of the targeting sequence and the RNA polynucleotide further comprises a passenger sequence substantially complementary to the guide.
  • the passenger and guide sequences are provided in a combination indicated in Table 2, wherein guide strand comprises, consist essentially of, or consists of the indicated targeting sequences and the passenger sequences comprise, consists essentially of, or consists of the indicated sequences or a sequences differing from the indicated sequence by 1, 2, 3 nucleotide substitutions.
  • the encoding passenger strand and guide strand combination provided in Table 2 is a present in a pri-miRNA, pre-miRNA, shRNA or inhibitory RNA duplex.
  • I A. Inhibitory RNA Duplex
  • RNA-induced silencing complex can inhibit mRNA activity in conjunction with the RNA- induced silencing complex (RISC).
  • RISC RNA- induced silencing complex
  • Reference to RISC includes associated proteins such as the core argonaute (AGO) protein.
  • AGO core argonaute
  • the guide strand is loaded onto an RISC and hybridizes to the target mRNA, wherein hybridization is initiated at the seed region, located at n-2 to approximately n-8 of the guide strand 5’ end.
  • the seed region is perfectly complementary to its mRNA target or different from prefect complementarity by 1 nucleotide.
  • the passenger strand is discarded by the RISC and is degraded.
  • the guide strand ratio to passenger strand ratio is about is about 25: 1, about 15: 1 or about 10: 1.
  • the RISC associated with the guide strand can cause mRNA cleavage or translation repression.
  • Lam et al. Mol. Ther. Nucleic Acids. (2015) 4(9):e252
  • Kobayashi et al. ACS Omega. (2022) 7(2):2398-2410, each of which are hereby incorporated by reference herein in their entirety.
  • the guide strand and the passenger strand are independently 18 to 25 nucleotides in length and each comprises 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 strand 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 are 21, 22, or 23 nucleotides and each overhang is 2 nucleotides.
  • inhibitory RNA duplexes Delivery of inhibitory RNA duplexes to a subject is facilitated using precursor constructs such as pri-miRNA, pre-miRNA and shRNA. These different constructs comprise a stem loop structure, a guide strand sequence and a passenger strand sequence; and make use of intracellular miRNA biogenesis mechanisms to form an inhibitory RNA duplex. Such constructs can be provided, for example, using transgenes encoding for inhibitory RNA.
  • Naturally occurring miRNA production and processing involves pri-miRNA transcription from a miRNA gene and cleavage of 3’ and 5’ regions mediated by drosha- DGCR8, forming pre-miRNA.
  • the resulting pre-miRNA contains a stem with nucleotide overhangs at the 3’ ends and an apical stem loop.
  • Pri-miRNA comprises a 5’ flanking sequence, a guide/passenger sequence, an apical loop, a guide/passenger sequence and 3’ flanking sequence.
  • the guide strand sequence can be either 5’ or 3’ of the loop.
  • the passenger strand sequence is 3’ of the apical loop; and when the guide strand sequence is 3’ of the loop the passenger strand sequence is 5’ of the loop.
  • the 5’ and 3’ flanking regions make up part of a stem region and further comprise single-stranded regions.
  • the length of the flanking regions can independently vary.
  • the 5’ and 3’ pri-miRNA flanking regions are each at least 20 nucleotides, at least 50 nucleotides, or at least 100 nucleotides.
  • a pri-miRNA scaffold refers to pri-miRNA regions excluding guide and passenger sequences.
  • a scaffold can serve as a delivery vehicle for the biogenesis of different inhibitory RNA duplexes, where guide and passenger strand sequences are embedded into the scaffold.
  • Scaffolds able to incorporate guide and passenger strands sequences can be based on naturally occurring pri-miRNA, modification to a naturally occurring miRNA, or artificially designed taking into account pri-miRNA features.
  • pri-miRNA e.g., 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 hereby incorporated by reference herein in their entirety.)
  • Pri-miRNA scaffolds include the stem length, loop size and presence of particular motifs that may enhance biogenesis and/or inhibitory RNA.
  • Pri-miRNA stems may contain different structures such as G-U or U-G wobbles, single base pair mismatches, bulges, and multiple base pair mismatches.
  • the pri-miRNA scaffold comprise in the basal stem a UG motif in the 5’ arm at a position -14 or -13 relative to the drosha 5’ cleavage site; and/or a CNNC in the 3’ arm between positions +14 and +18 relative to the drosha 3’ cleavage site.
  • the pri-miRNA scaffold is a miR-1, miR-26, miR16-l, miR- 30, miR-33, mi-RlOl, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold.
  • eSIBR enhanced miR-155
  • the pri-miRNA comprises a mismatched GHG motif in the 3’ arm of the stem.
  • the pri-miRNA stem length is 33, 34, 35, 36, 37, 38, or 39 nucleotides. [00107] In certain embodiments the pri-miRNA stem length is 34, 35, or 36 nucleotides.
  • the pri-miRNA apical loop is 3 to 23 nucleotides. In a further embodiment the loop is 10 to 23 nucleotides. In certain embodiments the guide and passenger strands do not extend into the loop. In certain embodiments, the guide and/or passenger extend into the loop.
  • the pri-mRNA comprises a 5' 7-methyl guanylate (m7G) cap.
  • the pri-miRNA comprises, consists, or consists essentially of a sequence of any of SEQ ID NOs: 115-121; or a sequences differing from any of SEQ ID NOs: 115-121 by 0, 1, 2, 3, or 4 nucleotides. Nucleotide differences can be introduced in different locations taking into account the guidance provides herein, for example, different pri-miRNA scaffold features, guide complementarity to target, passenger strand complementarity to guide strand and adjustment for particular scaffolds.
  • Pre-miRNA produced by drosha cleavage of pri-miRNA is exported from the nucleus to the cytoplasm, where it is cleaved by dicer to produce an inhibitory RNA duplex.
  • Reference to “pre-miRNA” indicates the RNA polynucleotide, such as those comprising guide and passenger stands described herein, can be cleaved by dicer to produce an inhibitory RNA duplex.
  • the dicer produced inhibitory RNA duplex comprises a guide and passenger strand each about 22 nucleotides in length, wherein each strand contains a nucleotide overhang of 2 bases at the each 3’ end.
  • the pre-miRNA comprises, consists, or consists essentially of a sequence of any of SEQ ID NOs: 122-128; or a sequence differing from any of SEQ ID NOs: 122-128 by 0, 1, 2, 3, or 4 nucleotides. Nucleotide differences can be introduced in different locations such as the guide sequence, loop sequence, and or passenger sequence.
  • pri-miRNA is introduced into a cell using a transgene comprising a sequence encoding for the pri-miRNA sequence. Biogenesis of the pri-miRNA leads to the production of an inhibitory RNA duplex. Expression cassettes comprising the transgene can be introduced to a cell using viral or non-viral delivery vehicles.
  • shRNA is introduced into a cell using a transgene comprising a sequence encoding for the shRNA sequence.
  • shRNA is similar in structure and function to pre- miRNA.
  • shRNA comprises an RNA duplex comprising substantially complementary arms and a loop, where the shRNA can be exported from the nucleus to the cytoplasm and be cleaved by dicer to form an inhibitory RNA duplex.
  • the pri-miRNA scaffold is either a S155, S155e, S26, S126 or S33 scaffold.
  • the SI 55 scaffold corresponds to the miR155 scaffold, and was used for miR3, miR5, miR6, miR7, miR8, miRlO, and miRl 1, described in the Examples below.
  • the S 155e scaffold corresponds to an enhanced miRl 55 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 using the miR26 scaffold as a starting point. The different scaffolds can be used to incorporate different guide and passenger strands.
  • the S126 scaffold can be used to incorporate different guide and passenger strands, wherein the scaffold comprises SEQ ID NO: 186 N01N02N03N04N05N06N07N08N09N10N11N12N13N14N15N16N17N18N19N20N21UGUGCAGGUCCCA N22N23N24N25N26N27N28N29N30N31N32N33N34N35N36N37N38N39N40N41N42CG, wherein N01 to N42 are ribonucleotides, N01 is complementary to N42, N02 is not complementary to N41, N03-N10 is complementary to N33-N40, Nn is not complementary to N32, and N12-N21 is complementary to N22-N31.
  • the passenger strand is provided by N01-N21U, where a targeting sequence can be inserted into N01-N21; and the guide strand is provided by N23-N42G.
  • the corresponding DNA can be used to encode the RNA sequence.
  • RNA sequence comprising SEQ ID NO: 186 has the following structure:
  • the RNA sequence comprising SEQ ID NO: 186 can be cut by Dicer; or can be cut by Drosha and Dicer.
  • the RNA sequence further comprises a 5’ flanking region and 3’ flanking region, wherein the polynucleotide comprises the RNA sequence of SEQ ID NO: 187: GUGGCCGN01N02N03N04N05N06N07N08N09N10N11N12N13N14N15N16N17N18N19N20N21UGUGCA GGUCCCAN22N23N24N25N26N27N28N29N30N31N32N33N34N35N36N37N38N39N40N41N42CGGGGA CGC, wherein N01 to N42 are ribonucleotides, N01 is complementary to N42, N02 is not complementary to N41, N03-N10 is complementary to N33-N40, Nn is not complementary to N32, and N12-N21 is complementary to N22-N31.
  • the inhibitory RNA polynucleotide targeting a-synuclein is embedded in a S126 scaffold comprising SEQ ID NOs: 186 or 187, wherein (1) the guide strand comprises the sequence of SEQ ID NO: 95 and the passenger strand comprises a sequence selected from the group consisting of SEQ ID NOs: 189-197; or (2) the guide strand comprises the sequence of SEQ ID NO: 97 and the passenger strand comprises a sequence selected from the group consisting of SEQ ID NOs: 198-206.
  • Tables 3 and 4 illustrate passenger combinations for the guide strands of SEQ ID NO: 95 and SEQ ID NO: 97.
  • the nucleotides shown in bold are located in a bulge created by non-complementary nucleotides.
  • the passenger strand and guide strand combination provided in Tables 3 and 4 are present in a pri-miRNA, pre-miRNA, shRNA or inhibitory RNA duplex.
  • pre-miRNA comprising the sequence combinations of Table 3 in a S126 scaffold
  • examples of pre-miRNA comprising sequence combinations of Table 4 in a S126 scaffold are provided by SEQ ID NOs: 216-224.
  • Longer- length examples of pri-miRNA comprising the sequence combinations of Table 3 in a S126 scaffold are provided by the RNA versions (ribose backbone and U instead T) of SEQ ID NOs: 243-251.
  • Longer-length examples of pri-miRNA comprising the sequence combinations of Table 4 in a S126 scaffold are provided by the RNA versions (ribose backbone and U instead T), of SEQ ID NOs: 252-260.
  • Direct administration can be facilitated using modified RNA.
  • Modified inhibitory RNA duplexes can be produced, for example, by modifying one or more nucleotides of an inhibitory RNA duplex. Modifications can be made to an inhibitory RNA duplex to, for example, improve pharmacokinetics, enhance activity, suppress innate immune activation, improve targeting and reduce off target toxicity.
  • Modifications to inhibitory RNA can be made at different positions such as the 5 ’-end, 3 ’-end, sugar moiety, phosphate group, and nucleobase.
  • the inhibitory RNA comprises one or more modifications selected from: a 5’ phosphate mimic (e.g., 5-(E)-vinylphosphate, 5’-methylene phosphonate, 5’- (R)-m ethyl phosphate, 5’-(S)-methyl phosphate, 5’(R)-MeOCH3 phosphate, 5’-(S)-methyl-F phosphate, 5 deoxy-5’-morpholino-2’O-methyl uridine, and phosphorothioate); internal phosphate modification (e.g., Rp phosphorothioate, Sp phosphorothioate, phosphorodi thioate, methoxyphosphonate, phenylethyl phosphate, 2’-5’ phosphate linkage, and amide linkage); one or more nucleobase modification (e.g., 5’-nitorindole, pseudouridine, 2’-thiouridine, N6’- methyl
  • nucleobase modification
  • the inhibitory RNA duplex is a modified divalent inhibitory RNA duplex.
  • the modified divalent inhibitory RNA duplex is fully chemically stabilized and comprises one or more of 2’-0Me, 2’-F, phosphorothioate, phosphodiester, and 5’vinyl phosphonate. (See, e.g., Alterman et al., Nature Biotechnology (2019) 37:884-894 hereby incorporated by reference herein in its entirety.)
  • Modified RNA can be produced using different techniques such as stepwise synthesis of the RNA and/or modifying a produced RNA sequence.
  • the produced RNA sequence itself can be made by, for example, stepwise synthesis or using encoding nucleic acid.
  • RNA polynucleotides comprising a targeting sequence can be produced from a polynucleotide using a nucleic acid sequence encoding for the RNA polynucleotide.
  • an encoding nucleic acid sequence provides the same sequence as the corresponding the RNA polynucleotide, wherein if the encoding nucleic acid is DNA, the DNA would have the nucleobase thymine instead of uracil, and deoxyribose instead of ribose.
  • the RNA polynucleotide is produced from a template strand, complementary to the encoding strand.
  • the encoding strand can, for example, be provided with the template and/or used to produce the template strand.
  • a polynucleotide comprising a nucleic acid sequence encoding an RNA polynucleotide may comprise additional components such as those facilitating production of the RNA polynucleotide, facilitating delivery of the polynucleotide as a viral vector, and/or providing for additional activity. Additional activity can be provided, for example, by encoding for proteins and/or encoding for sequences providing one or more additional inhibitory RNAs.
  • Sequences providing for additional inhibitory RNAs can be the same or different and can be directed to the same or different target.
  • Examples of different configurations include encoding for two or more pri-miRNA of the same sequence; encoding for two or more pri- mRNA having the same guide strand, but different scaffolds; and/or encoding for two or more two or more pri-miRNA with different guide strands.
  • the different guide strands can be directed to the same target, for example, the same mRNA; or different targets, for example different mRNA.
  • the polynucleotide encodes for 1, 2, 3, 4, or 5 pri-miRNAs where each can be the same or different.
  • the same types of encoding configurations providing pri-mRNA can be used for a polynucleotide encoding for shRNA.
  • the polynucleotide encodes for 1, 2, 3, 4, or 5 shRNA, where each can be the same of different.
  • RNA polynucleotides comprising targeting sequence is facilitated using expression cassettes.
  • the expression cassette comprises a nucleic acid sequence encoding the RNA polynucleotide and one or more expression control elements operably linked to the nucleic acid sequence encoding the RNA polynucleotide.
  • the expression cassette can comprise, for example, nucleic acid sequence encoding for different RNA polynucleotides (e.g., different pri- miRNA or shRNA), which are the same or different, where the different polynucleotides can be operably linked to the same or different expression elements.
  • the same promoter can be linked to a nucleic acid sequence providing for multiple pri-miRNA, or two or more pri- miRNA are operably linked to different promoter.
  • the polynucleotide encoding for an RNA polynucleotide comprises a sequence (1) 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 the sequence of any of SEQ ID NO: 136-142; (2) comprises at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of any of SEQ ID NOs: 136-142; and/or (3) comprises a sequence that differs from SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142
  • the encoding DNA target sequence comprises: a) a sequence differing from SEQ ID NO: 136 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 143; b) a sequence differing from SEQ ID NO: 137 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 144; c) a sequence differing from SEQ ID NO: 138 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 145; d) a sequence differing from SEQ ID NO: 139 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 146; e) a sequence differing from SEQ ID NO: 140 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 147; f) a sequence differing from SEQ ID NO:
  • the targeting sequence is a guide strand sequence and the polynucleotide further comprises a sequence encoding for a passenger sequence substantially complementary to the targeting sequence.
  • the encoding passenger and guide sequences are provided in a combination indicated in Table 6-8, where the encoding guide strand sequence comprises, consist essentially of, or consists of the indicated sequences; and the encoding passenger sequence comprises, consists essentially of, or consists of the indicated sequences or a sequences differing from the indicated sequence by 1, 2, or 3 nucleotides.
  • Table 6 Table 6
  • the encoding passenger strand and guide strand combination are as provided in Tables 6, 7 or 8, and the polynucleotide encodes for a pri-miRNA, pre- miRNA, or shRNA.
  • the polynucleotide encoding for an RNA polynucleotide comprises a sequence (1) 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 the sequence of any of SEQ ID NO: 157-163; (2) comprises at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of any of SEQ ID NOs: 157-163; and/or (3) comprises a sequence that differs from SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, or SEQ ID NO: 163 by 0,
  • the polynucleotide comprises or encodes a sequence (1) 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 the sequence of any of SEQ ID NO: 225-260; (2) at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of any of SEQ ID NO: 225-260; and/or (2) comprises a sequence that differs from any of SEQ ID NO: 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, SEQ ID NO:
  • SEQ ID NO: 239 SEQ ID NO: 240, SEQ ID NO: 241, and SEQ ID NO: 242, SEQ ID NO:
  • SEQ ID NO: 249 SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO:
  • SEQ ID NO: 253 SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO:
  • SEQ ID NO: 259 SEQ ID NO: 260 by 0, 1, 2 or 3 nucleotides.
  • the polynucleotide comprises a sequence encoding for a pre- miRNA or pri-miRNA, where the polynucleotide comprises a guide/passenger combination provided in Table 6 and (1) comprises a sequence 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 the sequence of any of SEQ ID NO: 157-163; and/or (2) comprises a sequence that differs from SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, or SEQ ID NO: 163 by 0, 1, 2 or 3 nucleotides
  • the polynucleotide comprises a sequence encoding for a pre- miRNA or pri-miRNA, where the polynucleotide comprises a guide/passenger combination provided in Table 7 and (1) comprises a sequence 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 the sequence of any of SEQ ID NO: 225-233; and/or (2) comprises a sequence that differs from any of SEQ ID NO: 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
  • the polynucleotide encodes for a pre-miRNA or pri-miRNA polynucleotide, where the polynucleotide comprises a guide/passenger combination provided in Table 8 and (1) comprises a sequence 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 the sequence of any of SEQ ID NO: 234-242; and/or (2) comprises a sequence that differs from any of SEQ ID NO: 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: 24
  • the polynucleotide comprises a sequence encoding for inhibitory RNA targeting a-synuclein mRNA further comprises a nucleic acid sequence encoding a glial cell-derived neurotrophic factor (GDNF).
  • GDNF glial cell-derived neurotrophic factor
  • Reference to GDNF includes full- length or mature and variants thereof.
  • GDNF is a potent neurotropic factor for dopamine neurons.
  • the amino acid sequence for a full-length GDNF is provided by SEQ ID NO: 188, where amino acids 1-19 provide a signal sequence.
  • the GDNF comprises a sequence with a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 188 (full-length), or to amino acids 20-211 of SEQ ID NO: 188 (mature).
  • the mature GDNF further comprises a heterologous signal sequence. Examples of different signal sequences are provided in, for example, International Patent Publications Nos. WO2022155665 and WO2022165027, both of which are incorporated by reference herein in their entirety.
  • An expression cassette comprises a nucleic acid sequence encoding a polynucleotide operably linked to an expression control element.
  • Expression cassettes described herein comprise a nucleotide sequence encoding for an RNA polynucleotide targeting a-synuclein mRNA along with one or more regulatory sequences, and may also comprises additional sequences such as stuffer sequence, and additional encoding sequences such as additional RNA polynucleotide and/or polypeptide sequences.
  • An “expression control element” influences expression of a sequence to which it is operably linked.
  • Protein expression control elements can affect, for example, transcription, translation, splicing, and message stability.
  • Expression control elements impacting production of RNA polynucleotides can affect, for example, the level of transcription.
  • Expression control elements are typically located 5’ (“upstream”) or 3’ (“downstream”) of a transcribed nucleic acid. Expression control elements can also be located within a protein encoding transcript (e.g., in an intron). Expression control elements can be located adjacent to or at a distance away from the transcribed sequence. One or more expression control elements may be present. Examples of expression control elements include a promoter, enhancer, an intron, polyadenylation signal, a Kozak sequence, post-transcriptional regulator elements and a termination sequence.
  • a promoter is a DNA region where transcription is initiated. In general, transcribed nucleic acid are located 3’ of a promoter sequence. In certain embodiments, a promoter sequence is linked to an enhancer. Enhancers are DNA regions that increase promoter transcription. Enhancers can be adjacent to a promoter or can be distal. Typically, enhancers are located upstream of a promoter, but can be located downstream or within a promoter sequence. [00158] Expression control elements such a promoter and an enhancer can be chosen to preferentially drive expression in a particular cell or tissue type. Expression control elements are typically active in particular cells, tissues or organs because they are recognized by transcriptional activator proteins, or other regulators of transcription, providing for activity in a cell, tissue or organ type.
  • tissue specific regulatory elements in expression constructs provide for at least partial tissue tropism for the expression of encoded RNA polynucleotide.
  • Reference to a promoter or enhancer specific for a particular cell type or tissue indicates the promoter or enhancer provides higher levels of expression and/or secretion in the indicated cell or tissue type.
  • CNS specific promoters examples include: neuron specific promoters such as the NSE (neuronal specific enolase), synapsin or NeuN, platelet-derived growth factor (PDGF), platelet-derived growth factor B -chain (PDGF-P), methyl-CpG binding protein 2 (MeCP2), Ca 2 /calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), P-globin minigene nP2, preproenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters; astrocyte specific promoters such as the glial fibrillar acidic protein (GFAP) and EAAT2 promoters; oligodendrocyte specific promoters such as the myelin basic protein (MBP)/myelin-associated glycoprotein and oligodendrocyte
  • Expression control elements also include ubiquitous or promiscuous promotors and promoters/enhancers capable of driving polynucleotide expression in many different cell types.
  • Such elements include the cytomegalovirus (CMV) immediate early promoter/enhancer sequences, the Rous sarcoma virus (RSV) promoter/enhancer sequences, phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer, the chicken beta actin promoter (CBA) and the rabbit beta globin intron) (see, e.g., Boshart et al., (1985) Cell, 41 :521-530), the SV40 promoter, the dihydrofolate reductase promoter, and the cytoplasmic b-actin promoter.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • PKG phosphoglycerate kinase
  • CAG composite of the CMV enhancer,
  • Additional promoters include the superoxide dismutase 1 (SOD1) promoter, U6 promoter, Hl, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, SFFV promoter, rat insulin promoter, TBG promoter, the desmin promoter and similar muscle-specific promoters, synthetic promoters, hybrid promoters, and promoters with multi-tissue specificity.
  • SOD1 superoxide dismutase 1
  • Hl human mammary tumor virus LTR promoter
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • SFFV rat insulin promoter
  • TBG promoter the desmin promoter and similar muscle-specific promoters
  • synthetic promoters synthetic promoters with multi-tissue specificity.
  • the promoter is an EF-la promoter (see, e.g., Wang et al., J. Cell Mol. Med. (2017) 21(11):3044-3054, hereby incorporated by reference herein in its entirety) and/or comprises, consists, or consists essentially of a sequence identity of at least 95%, at least 97%, at least 99% or 100% to SEQ ID NO: 184.
  • Expression control elements also can impact expression in a manner that is regulatable by a signal or stimuli increasing or decreasing expression.
  • a regulatable element increasing expression of transcribed nucleic acid in response to a signal or stimuli is also referred to as an “inducible element” (i.e., is induced by a signal).
  • the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimuli present.
  • Particular examples include zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone- inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (International Patent Publication No.
  • regulatable control elements include those regulated by a specific physiological state such as temperature, acute phase, or development.
  • the expression cassette further comprises one or more introns.
  • introns A variety of different introns can be used. Examples of introns that may be used include the rabbit P-globin intron with splice donor/splice acceptor, SV40 intron with splice donor/splice acceptor, human P-globin introns, intron 2 of the human hemoglobin beta gene, hFIX inti (intron 1 of the human coagulation factor IX gene), CBA-rHHB (synthetic intron derived from the fusion of the intron 1 of the chicken beta actin gene and intron 2 of the rabbit hemoglobin beta), CBA (intron 1 of the chicken beta actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (synthetic intron derived from different portions of the human coagulation factor IX gene and present in the pLIVE vector, Minis Bio, Madison, WI); human hemoglobin subunit beta (HBB2) synthetic intron, and
  • nucleic acid encoding for an RNA polynucleotide comprising a targeting sequence, a pri-miRNA or a shRNA is positioned within an intron.
  • the expression cassette comprises a post-transcriptional regulatory element.
  • Post-translational regulatory elements such as Woodchuck post- transcriptional regulatory element (WPRE) and Hepatitis B regulatory element can increase gene expression. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)
  • Polyadenylation signal sequences provide for the formation of a polyA tail, which facilitates nuclear export, translation and/or mRNA stability, and may also be involved in transcription termination.
  • Examples of polyadenylation signal sequences include SV40 late polyadenylation signal, bovine growth hormone polyA (bGHpA) signal sequence, synthetic polyA, mouse P-globin pA, rabbit P-globin pA (RbG), and H4-based pA, (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197).
  • the expression cassette comprises a Kozak consensus sequence or a variation thereof.
  • Kozak consensus sequences play a role in translation initiation.
  • the Kozak consensus sequence and variations are provided in, for example, McClements et al., (2021) Molecular vision, 27, 233-242.
  • the expression cassette comprises an up-stream promoter and a downstream polyadenylation signal, operably linked to the nucleic acid sequence encoding the RNA polynucleotide.
  • the expression cassette comprises 5’ to 3’, operably linked to the nucleic acid sequence encoding the RNA polynucleotide, a promoter or promoter/enhancer, an intron, the nucleic acid sequence encoding the RNA polynucleotide, and a polyadenylation signal.
  • the expression cassette further comprises a sequence encoding glial cell -derived neurotrophic factor (GDFN).
  • GDFN glial cell -derived neurotrophic factor
  • the GDFN encoding sequence(s) can be operatively linked to the promoter encoding for the RNA polynucleotide comprising an a- synuclein targeting sequence, or to a separate promoter.
  • the expression cassette comprises nucleic acid encoding for GDNF operatively linked to the promoter encoding for the RNA polynucleotide comprising an a-synuclein targeting sequence, where the GDNF encoding sequence is upstream of the RNA polynucleotide encoding sequence.
  • the expression cassettes further comprises a miRNA target sequence, which in further embodiments is incorporated into the 3’ UTR of the expression cassette.
  • a miRNA target sequence is recognized by miRNA present in particular cells or tissues leading to degradation of mRNA transcripts. Based on the presence of certain miRNA in particular cells, incorporating a miRNA target sequence can be used to reduce expression in certain cell or tissue types. Multiple tandem repeats of miRNA target sequences can be used to increase degradation. (Geisle et al., (2016) World Journal of Experimental Medicine 6(2): 37- 54.)
  • the expression cassette not including the sequence encoding for a pri-miRNA or shRNA contains any of 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 contains 0% to 5%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% CpGs.
  • Polynucleotide recombinant viral nucleic acid contain 5’ and/or 3’ viral elements providing for viral packaging and replication, and may provide for additional activities such as promoter activity, genome integration, or episomal concatermerization.
  • the 5’ and 3’ elements are general located at or near the 5’ and 3’ terminal end of the recombinant viral nucleic acid and can be naturally occurring or modified versions of naturally occurring sequences. Examples of 5’ and 3’ elements include adenovirus ITRs, adeno-associated virus ITRs and packaging sequence; and retrovirus 5’ and 3’ long terminal repeats (LTRs) and packaging sequence.
  • recombinant as a modifier of nucleic acid or a vector indicates a combination of elements that does not occur in nature.
  • a recombinant viral vector nucleic acid provides 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.
  • a viral vector such as a rAAV vector may contain a naturally occurring or modified capsid, encapsidating recombinant viral vector nucleic acid.
  • rAAV can be produced comprising ssDNA or dsDNA
  • adenovirus vectors can be produced comprising dsDNA
  • retrovirus vectors can be produced comprising ssRNA.
  • the viral vector nucleic acid outside of the pri-miRNA or shRNA encoding region, contains any of 0-5, 0-10, 0-15, 0-50, 0-100, or 0 to 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 0% to 10%, 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0% CpGs, about 6%, about 7%, about 8%, about 9% or about 10% CpGs.
  • the gene delivery vehicle is a viral vector.
  • Viral vectors comprises a protein capsid encapsidating recombinant viral nucleic acid and can deliver the nucleic acid to cells or tissues.
  • the viral vector may further comprise a viral envelope. Examples of viral vectors that can be used include adenovirus vectors, rAAV, retrovirus vectors and herpes simplex vectors.
  • serotypes exist within different types of viruses.
  • the different serotypes can provide for different activities, such as cell or tissue tropism and likelihood of generating a host immune response.
  • serotype broadly refers to both serologically distinct viruses as well as viruses not serologically distinct that can be within a subgroup or a variant of a given serotype.
  • Serologic distinctiveness can be determined based on the lack of cross-reactivity between antibodies to one capsid as compared to another capsid. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).
  • Adenoviruses are non-enveloped double-stranded DNA viruses.
  • Recombinant adenovirus vectors comprise recombinant adenovirus nucleic acid lacking one or more protein involved in viral replication, and further comprise an adenoviral capsid.
  • Recombinant adenovirus vectors can be produced containing different amounts of adenoviral DNA.
  • the Ad genome is flanked by hairpin-like inverted terminal repeats (ITRs) varying in length from 30- 371 bp at its termini.
  • the ITRs serve as self-priming structures that promote primase- independent DNA replication.
  • the 5’ and 3’ inverted repeats need not be exact inverted repeats.
  • a packaging signal located at the left arm of the genome is required for viral genome packaging.
  • the recombinant adenovirus vector is a third-generation vector, also referred to as “gutless” or “helper-dependent”.
  • Gutless vectors can be produced from recombinant adenoviral nucleic acid where all, or substantially all viral sequences, except for the ITRs and the packaging signal are not present.
  • Gutless adenovirus vectors are high capacity vectors able to accommodate up to about 36 kb of DNA insert.
  • Preferred recombinant adenovirus nucleic acid is about 27 kb to about 37 kb.
  • Stuffer sequences can be added to recombinant adenovirus nucleic acid to increase nucleic acid size and capsid incorporation.
  • Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombination sequences, and immunogenic sequences.
  • recombinant adenovirus vectors can be produced based on rare human serotypes or chimpanzee serotypes.
  • Adenovirus vectors can be produced by supplying viral proteins needed for vector production in trans using for example, appropriate helper viruses or plasmids and cell lines.
  • helper viruses or plasmids and cell lines.
  • a recombinant adeno-associated viral (also referred to herein as “rAAV”) vector is based on the adeno-associated virus.
  • the adeno-associated virus is a single-strand DNA virus containing a 4.7-kb genome flanked by 145-nt ITRs on both ends of the genome. ITR activity is important for self-priming and packaging, and may also provide additional activity such as promoter activity.
  • rAAV 5’ and 3 ITRs can vary in size and the 5’ and 3’ inverted repeats need not be exact inverted repeats.
  • a rAAV vector contains AAV recombinant nucleic acid and a viral capsid.
  • the rAAV recombinant nucleic acid lacks sequence(s) encoding for one or more AAV proteins involved in viral replication.
  • the rAAV vector contains an AAV 5’ and/or 3’ ITR along with a DNA insert.
  • rAAV nucleic acid comprise a 5’ ITR and/or 3’ ITR independently selected from 5’ and 3’ ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh. lO, AAVrh.74 and AAV3B ITRs.
  • 5’ and 3’ ITRs are present, and both ITRs are from the same serotype genome.
  • the 5’ ITR comprises a sequence with a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 182 and the 3’ ITR independently comprises a sequence with a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO: 183.
  • the rAAV nucleic acid is at least about 2.5 kb.
  • stuffer sequence can be used to increase rAAV nucleic acid size and packaging efficiency.
  • the rAAV nucleic acid including 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, recombination sequences, and immunogenic sequences.
  • the rAAV is a self-complementary adeno-associated virus vector (scAAV) or short hairpin adeno-associated virus vector (shAAV).
  • scAAV and shAAV provide for a double-stranded recombinant adeno-associated virus nucleic acid that can be incorporated into AAV capsid.
  • scAAV and shAAV comprise inverted dimeric repeats providing intramolecular double-stranded DNA.
  • scAAV can be produced by mutating an ITR terminal resolution site so that Rep fails to nick the terminal resolution site.
  • shAAV can utilize a short hairpin to produce the dsAAV.
  • scAAV and shAAV being double stranded DNA, provide an advantage in circumventing the DNA synthesis step required for single-stranded rAAV nucleic acid upon entry into a cell.
  • a potential disadvantage of the scAAV and shAAV is the size of DNA inserts that can be incorporated is reduced by about half compared to single-stranded rAAV nucleic acid.
  • Naturally occurring AAV capsids contain viral proteins VP1, VP2 and VP3 in a ratio of about 1 : 1 :10.
  • AAV vectors can be produced where all three viral proteins are based upon a particular serotype or where one, two or all three viral protein are based on different serotypes.
  • Recombinant AAV capsid and nucleic acid can be based on the same serotype (or subgroup or variant), or can be different from each other.
  • a rAAV nucleic acid has the same serotype genome (e.g., ITRs) as the encapsidating capsid protein.
  • the rAAV capsid comprises a protein having a sequence at least 80%, 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%, at least 99.1%, at least 99.2%, at least 99.3%, at least at least 99.4%, at least 99.5%, at least 99.9% or 100% identical to a 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 AAVl/rh.10
  • AAV capsids comprises VP1, VP2 and VP3 each independently having a sequence identity of at least 80%, at least 90%, at least 95% or 100% to a 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, AAVl/rh.10; or VPl of SEQ ID NO: 164 or SEQ ID NO: 167; as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof.
  • variants e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions
  • the capsid comprises VP1 having the sequence of SEQ ID NO: 164; VP2 having the sequence of SEQ ID NO: 165; and VP3 having the sequence of SEQ ID NO: 166.
  • the AAV capsid can cross the blood brain barrier and provide for CNS expression.
  • Examples of such AAV capsids and the design of AAV capsids able to provide for 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 AAVl/rh.10), U.S. Patent No. 9,585,971, and Goertsen etal., (2022) Nat. Neurosci. 25, 106-115 (2022).
  • the AAV genome contains two main genes: rep and cap. Transcription from the rep gene is initiated from two different promoters resulting in the production of nonstructural proteins designated Rep78, Rep68, Rep52, and Rep40.
  • the rep proteins function in genome replication and/or encapsidation.
  • the cap gene encodes for structural proteins making up the capsid (VP1, VP2 and VP3); a non-structural assembly-activating protein (APP), which performs functions related to capsid assembly; and the membrane-associated accessory protein, which may be associated with production phases of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther.
  • AAV requires helper virus functions to complete it replication cycle.
  • Helper virus functions can be supplied by different viruses in permissive cell lines.
  • Permissive cell lines are cell lines able to support viral replication.
  • helper viruses for AAV include adenovirus, HSV-1, HPV-16, and HboVl which can be used in conjunction with, for example, permissive primate cells; and baculovirus which can be used in conjunction with, for example, permissive insect cells such as sf9.
  • helper viruses for AAV include adenovirus, HSV-1, HPV-16, and HboVl which can be used in conjunction with, for example, permissive primate cells; and baculovirus which can be used in conjunction with, for example, permissive insect cells such as sf9.
  • Recombinant AAV can be produced by supplying viral proteins needed for vector production in trans using for example, appropriate helper viruses or plasmids and cell lines.
  • rAAV is produced using a rAAV vector genome plasmid.
  • the plasmid comprises that portion of the rAAV nucleic acid ultimately packaged or encapsidated to form a viral (e.g., rAAV) vector.
  • the “plasmid backbone,” contains elements important for propagation and recombinant virus production. Except for possible 3’ ITR and/or 5’ ITR cloning remnants the plasmid backbone is not itself packaged or encapsidated into virus particles.
  • the vector genome plasmid may contain regions such an origin of replication and a selectable marker. Additional sites that may be present include cloning sites.
  • Recombinant AAV can be produced from different types of cell lines including HeLa, A549, BHK, Vero, and HEK293, or derivatives thereof.
  • HEK293 cells are used (American Type Culture Collection Accession Number ATCC CRL1573).
  • Other host cell lines appropriate for rAAV vector production are described in, for example, Robert et al., (2017) Biotechnol. J. (2017) 12(3), 1600193; and International Application No. PCT/US2017/024951, the disclosures of which are herein incorporated in its entirety.
  • Recombinant AAV can be cultured under a variety of different conditions suitable for providing cell growth and gene expression.
  • References describing rAAV manufacturing 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.; each of which are hereby incorporated by reference herein in their entirety.
  • AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector.
  • a host cell having AAV helper functions can be referred to as a “helper cell” or “packaging helper cell.”
  • AAV helper constructs are sometimes used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions necessary for productive AAV transduction.
  • AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be, for example, in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
  • a number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both rep and cap expression products.
  • a number of other vectors are known which encode rep and/or cap expression products.
  • Recombinant AAV can be produced, for example, as described in US Patent No. 9,408,904; and International Application Nos. PCT/US2017/025396 and PCT/US2016/064414, the disclosures of which are herein incorporated in their entirety.
  • a rAAV vector is produced by a rAAV production cell comprising rAAV helper virus activity.
  • the genome of the rAAV production cell comprises rAAV nucleic acid, the rep gene and the cap gene.
  • a rAAV vector is produced by culturing a rAAV permissive cell comprising an AAV genome plasmid, where the rAAV permissive cell further comprises rep and cap genes provided either as part of the cell genome and/or by one or more separate plasmids; and helper virus activity either as part of the cell genome and/or provided by one or more separate plasmids.
  • the rAAV permissive cell line is a packaging cell, wherein the genome of the packaging cell comprises the cap gene and the 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.
  • the helper functions are provided by genes encoding for at least UL5, UL8, UL52, and ICP8.
  • the helper functions are provided by genes encoding for at least El A, E1B19K, E1B55K, E2A, E4orf6 and VA RNA.
  • El, E2A and VR RNA functions are provided by a helper plasmid, where additional helper functions are provided by a host strain.
  • rAAV vector is obtained by producing rAAV using methods described herein and purifying the rAAV. Purification of rAAV can be performed using techniques such as gradient-based purification, column-based, and combined methods. (See, e.g., Ayuso et al., Curr Gene Ther. (2010) 10(6):423-36, hereby incorporated by reference herein in its entirety.)
  • Retroviruses are enveloped, single-stranded RNA viruses comprising 5’ and 3’ LTRs, and a signal packaging sequence located just outside of the LTR.
  • Different types of retrovirus vectors can contain different amounts of viral genome.
  • the retrovirus vector is a lentiviral vector based on HIV, retaining all cis-acting sequences needed for viral RNA packaging, reverse transcription and proviral DNA integration, while removing all HIV protein-coding genes.
  • Lentiviral vectors have a packaging capacity of up to about 9 kb. If needed, stuffer sequence can be used to increase rAAV nucleic acid size and packaging efficiency.
  • Lentiviral vectors can be produced by supplying viral proteins needed for vector production in trans using appropriate plasmids and cell lines. (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)
  • the vector is based on a parvovirus virus, other than AAV, such as an Erythroparvovirus, Protoparvovirus, or Tetraparvovirus.
  • a parvovirus virus other than AAV
  • an Erythroparvovirus e.g., human parvovirus Bl 9
  • the Erythroparvovirus typically have a genome size of about 5.6 kb and, therefore, recombinant vectors based on an Erythroparvovirus are expected to accommodate a larger recombinant payload than an AAV vector.
  • Protoparvovirus and Tetraparvovirus have a genome capacity of about 5.3 kb and, therefore, recombinant vectors based on these viruses are also expected to accommodate a larger recombinant payload than an AAV vector.
  • the gene delivery vehicle is a non-viral vector.
  • Preferred non- viral vectors are nanoparticles.
  • a variety of different nanoparticles can be employed including lipid nanoparticles (LNP), polymeric nanoparticles, lipid polymer nanoparticles (LPNP), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes.
  • a nanoparticle can target a cell type using, for example, targeting ligands recognizing a target cell receptor.
  • targeting ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies and protein/peptides (e.g., RGD, epidermal growth factor, and low density lipoprotein) and peptides.
  • carbohydrates e.g., galactose, mannose, glucose, and galactomannan
  • endogenous ligands e.g., folic acid and transferrin
  • antibodies and protein/peptides e.g., RGD, epidermal growth factor, and low density lipoprotein
  • Nanoparticles can be used to deliver inhibitory RNA or encoding polynucleotide constructs to a cell.
  • nanoparticles can deliver additional therapeutic compounds; and one or more additional compounds is provided in different nanoparticles.
  • Reference to compound includes small molecules and large molecules (e.g., therapeutic proteins and antibodies).
  • Factors that may impact small molecule incorporation into a nanoparticle include hydrophobicity and the presence of an ionizable moiety. (See, e.g., Nii and Ishii International Journal of Pharmaceutics (2005) 298, 198; and Chen et al., Journal of Controlled Release (2016) 286, 46.)
  • Lipid-based delivery systems include the use of a lipid as a component. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.
  • a “lipid nanoparticle” or “LNP” refers to a lipid-based vesicle useful for delivery of nucleic acid molecules and having dimensions on the nanoscale. In different embodiments the nanoparticle is from about 10 nm to about 1000 nm, about 50 nm to about 500 nm, or about 50 nm to about 200 nm.
  • DNA is negatively charged.
  • the LNP can be beneficial for the LNP to comprise a cationic lipid such as, for example, an amino lipid.
  • a cationic lipid such as, for example, an amino lipid.
  • Exemplary amino lipids 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 and U.S. Patent Publication Nos.
  • the LNP comprises amino lipids described in U.S. Patent No. 9,512,073, hereby incorporated herein in its entirety.
  • cationic lipid and “amino lipid” are used interchangeably herein to include lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH- titratable amino group (e.g., an alkylamino or dialkylamino group).
  • the cationic lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and is substantially neutral at a pH above the pKa.
  • the cationic lipid can also be titratable cationic lipids.
  • the cationic lipids comprise a protonatable tertiary amine (e.g., pH-titratable) group; Cl 8 alkyl chains, wherein each alkyl chain independently can have one or more double bonds, one or more triple bonds; and ether, ester, or ketal linkages between the head group and alkyl chains.
  • a protonatable tertiary amine e.g., pH-titratable
  • cationic lipids also include 1,2- distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-di oleyloxy -N,N-dimethyl-3- aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[l,3]-dioxolane (Dlin-K- C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[l,3]-dioxolane (Dlin-K-C4-DMA), Dlen- C2K-DMA, y-Dlen-C2K-DMA, and (Dlin-MP-DMA) (also known as 1-B11).
  • DSDMA 1,2- distearyloxy-N,N-dimethyl-3-aminopropane
  • DODMA 1,2-di oleyloxy -N,N
  • Still further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[l,3]-dioxane (Dlin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (Dlin-K-MPZ), 1,2- dilinoleylcarbamoyloxy-3 -dimethylaminopropane (Dlin-C-DAP), 1, 2-dilinoley oxy-3 - (dimethyl amino)acetoxypropane (Dlin-DAC), 1, 2-dilinoley oxy-3 -morpholinopropane (Dlin- MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3- dimethylaminopropane (Dlin-S-DMA), l
  • a number of commercial preparations of cationic lipids can be used, such as, LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECT AMINE® (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN® including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECT AMINE® comprising DOSPA and DOPE, available from GIBCO/BRL
  • Additional ionizable lipids that can be used include Cl 2-200, 306OH0, MC3, cKK-E12, bCKK-E12, Lipid 5, Lipid 9, ATX-002, ATX-003, and Merck-32.
  • cationic lipid can be present in an amount from about 10% by molar ratio of the LNP to about 85% by molar ratio of the LNP, or from about 50% by molar ratio of the LNP to about 75% by molar ratio of the LNP.
  • LNP can comprise a neutral lipid.
  • Neutral lipids can comprise a lipid species existing either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by considerations including particle size and stability.
  • the neutral lipid component can be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacy Iphosphati dy 1 ethanol amine) .
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or can be isolated or synthesized.
  • lipids containing saturated fatty acids with carbon chain lengths in the range of C 14 to C22 can be used.
  • lipids with mono or di -unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 are used.
  • lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • Exemplary neutral lipids include 1,2-dioleoyl-sn- glycero-3 -phosphatidyl -ethanolamine (DOPE), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), or a phosphatidylcholine.
  • DOPE 1,2-dioleoyl-sn- glycero-3 -phosphatidyl -ethanolamine
  • DSPC l,2-distearoyl-sn-glycero-3 -phosphocholine
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine
  • the neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups, such as
  • the neutral lipid can be present in an amount from about 0.1% by weight of the LNP to about 99% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP, e.g., 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%.
  • LNP can contain additional components such as sterols and polyethylene glycol.
  • Sterols can confer fluidity to the LNP.
  • sterol refers to a naturally occurring sterol of plant (phytosterols) or animal (zoosterols) origin as well as non-naturally occurring synthetic sterols, 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.
  • Phytosterols include campesterol, sitosterol, and stigmasterol.
  • Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011/0177156.
  • the sterol is present in an amount from about 1% by weight of the LNP to about 80% by weight of the LNP or from about 10% by weight of the LNP to about 25% by weight of the LNP.
  • Polyethylene glycol is a water-soluble polymer of ethylene PEG repeating units with terminal hydroxyl groups. PEGs are classified by their molecular weights, for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs commercially available from Sigma Chemical Co.
  • MePEG-OH monomethoxypolyethylene glycol
  • MePEG-S monomethoxypolyethylene glycol-succinate
  • MePEG-NHS monomethoxypolyethylene glycol- succinimidyl succinate
  • MePEG-NH2 monomethoxypolyethylene glycol-amine
  • MePEG-TRES monomethoxypolyethylene glycol-tresylate
  • MePEG-IM monomethoxypolyethylene glycol-imidazolyl-carbonyl
  • PEG has an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted by alkyl, alkoxy, acyl or aryl. In further embodiments, the PEG is substituted with methyl at the terminal hydroxyl position. In further embodiments, the PEG has an average molecular weight from about 750 to about 5,000 daltons, or from about 1,000 to about 5,000 daltons, or from about 1,500 to about 3,000 daltons, or from about 2,000 daltons, or from about 750 daltons.
  • PEG-modified lipids include the PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397.
  • PEG-modified lipids can have a variety of “anchoring” lipid portions to secure the PEG portion to the surface of the lipid vesicle.
  • suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which are described in U.S. Patent No.
  • the PEG-modified lipid can be PEG-modified diacylglycerols and dialkylglycerols.
  • the PEG can be in an amount from about 0.1% by weight of the LNP to about 50% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.
  • LNPs prior to encapsulating nucleic acid, have a size range from about 10 nm to 500 nm, or from about 50 nm to about 200 nm, or from 75 nm to about 125 nm.
  • the LNP is described by Billingsley et al., Nano Lett. 2020, 20, 1578; Billingsley et al., International Patent Publication No. WO 2021/077066; or U.S. Patent Application No. 2022/396556 (each of which are hereby incorporated by reference herein in their entirety). These publications describe LNPs containing lipid-anchored PEG, cholesterol, phospholipid and ionizable lipids.
  • the LNP contains a C14-4 polyamine core and/or has a particle size of about 70 nm.
  • C14-4 has the following structure.
  • the LNP is made up of a cationic lipid or lipopeptide described by U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374 or WO2021/077066 (each of which is hereby incorporated by reference herein in its entirety).
  • the LNP contains a cationic lipid, a cholesterol-based lipid, and/or one or more PEG-modified lipids.
  • the LNP contains cKK-E12 (Dong et al., PNAS (2014) 111(11), 3955):
  • the LNP comprises a modified form of cKK-E12 referred to herein as “bCKK-E12,” having the following structure:
  • the LNP comprises Lipid 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described by Sabnis et al., Molecular Therapy 2018, 26:6, 1509-1519 (hereby incorporated by reference herein in its entirety). In certain embodiments the LNP comprises Lipid 5, 8, 9, 10, or 11 described in Sabnis et al.
  • Lipid 5 of Sabnis et al. has the structure:
  • Lipid 9 of Sabnis et al. has the structure:
  • Additional lipids that may be utilized include those described by Roces et al., Pharmaceutics, 2020, 12,1095; Jayaraman etal, 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.
  • the nanoparticle is an LNP.
  • the LPN in mol% comprises, consists essentially, or consists, of the following components: (1) one or more cationic lipids from about 20% to about 65%, one or more phospholipids from about 1% to about 50%, one or more PEG-conjugated lipids from about 0.1 % to about 10%, and cholesterol from 0% to about 70%; and (2) one or more cationic lipids from about 20% to about 50%, one or more phospholipids from about 5% to about 20%, one or more PEG-conjugated lipids from about 0.1 % to about 5%, and cholesterol from about 20% to about 60%.
  • the phospholipid lipid is a neutral lipid; and the phospholipid lipid is DOPE or DSPC.
  • the LNP in mole %, comprises, consists essentially, or consists of the following components: (1) cKK-E12 about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (2) bCKK-E12 about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (3) Lipid 9 about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC about 10%; (4) Lipid 5 about 50%; C14-PEG2000 about 1.5%; cholesterol about 38.5%; and DSPC about 10%; (5) ionizable lipid, about 50%; DSPC, about 10%; cholesterol, about 37.5%; and stabilizer (PEG-Lipid), about 2.5%; or (6) is GenVoy-ILMTM LNP (Precision NanoSystems).
  • Polymer-based delivery systems can be made from a variety of different natural and synthetic materials. DNA and other compounds can be entrapped into the polymeric matrix of polymeric nanoparticles or can be adsorbed or conjugated on the surface of the nanoparticles.
  • Examples of commonly used polymers for nucleic acid delivery include poly(lactic-co-glycolic acid) (PLGA), poly lactic acid (PLA), poly(ethylene imine) (PEI) and PEI derivatives, chitosan, dendrimers, poly anhydride, polycaprolactone, polymethacrylates, poly-L-lysine, pullulan, dextran, hyaluronic acid, and poly-P-aminoesters.
  • Polymeric-based nanoparticles can have different sizes, ranging from about 1 nm to about 1000 nm, from about 10 nm to about 500 nm, from about 50 nm to about 200 nm, from about 100 nm to about 150 nm, and from about 150 nm or less.
  • Lipid polymer nanoparticles are hybrid nanoparticles providing both a lipid component and a polymer component, and as such can be considered to be an LNP or LPNP.
  • the LPNP configuration can provide an outer polymer and inner lipid or an outer lipid and inner polymer.
  • the presence of two different types of material facilitates designing nanoparticles to provide for delayed release of a component. Different lipid and polymer components can be selected taking into account the material to be delivered.
  • Protein and peptide-based systems can employ a variety of different proteins and peptides. Examples of proteins that can be employed include gelatin and elastin. Peptide-based systems can employ, for example, CPPs.
  • CPPs are short peptides (6-30 amino acid residues) potentially capable of intracellular penetration to deliver therapeutic molecules.
  • the majority of CPPs consists mainly of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphiphilic, anionic, or hydrophobic.
  • CPPs can 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).
  • CPPs examples include cationic CPPs (highly positively charged) such as the Tat peptide, penetratin, protamine, poly-L-lysine, and polyarginine; amphipathic CPPs (chimeric or fused peptides, constructed from different sources, containing both positively and negatively charged amino acid sequences), such as transportan, VT5, bactenecin-7 (Bac7), proline-rich peptide (PPR), SAP (VRLPPPjs, TP 10, pep-1, and MPG); membranotropic CPPs (exhibits both hydrophobic and amphipathic nature simultaneously, and comprise both large aromatic residues and small residues) such as H625, SPIONs-PEG-CPP and NPs; and hydrophobic CPPs (contain only non-polar motifs or residues) such as SG3, PFVYLI, pep-7, and fibroblast growth factors.
  • cationic CPPs highly positively charged
  • Protein and peptide nanoparticles can be provided in different sizes, for example, ranging from about 1 nm to about 1000 nm, from about 10 nm to about 500 nm, from about 50 nm to about 200 nm, from about 100 nm to about 150 nm, or from about 150 nm or less.
  • IV.E Peptide Cage Nanoparticles
  • Peptide cage-based delivery systems can be produced from proteinaceous material able to assemble into a cage-like structure forming a constrained internal environment.
  • Peptide cages can comprise a proteinaceous shell that self-assembles to form a protein cage (e.g., a structure with an interior cavity that is either naturally accessible to the solvent or can be made so by altering solvent concentration, pH, or equilibria ratios).
  • the monomers of the protein cages can be naturally occurring or variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments).
  • Protein cages can be produced using viral coat protein(s) (e.g., from the Cowpea Chlorotic Mottle Virus protein coat), as well non-viral proteins (e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Publication No. 20040028694, and U.S. Patent Publication No. 20090035389, each of which is incorporated by reference herein in their identity).
  • viral coat protein(s) e.g., from the Cowpea Chlorotic Mottle Virus protein coat
  • non-viral proteins e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Publication No. 20040028694, and U.S. Patent Publication No. 20090035389, each of which is incorporated by reference herein in their identity).
  • Examples of protein cages derived from non-viral proteins include: eukaryotic or prokaryotic derived ferritins and apoferritins such as 12 and 24 subunit ferritins; and heat shock proteins (HSPs), such as the class of 24 subunit heat shock proteins that form an internal core space, the small HSP of Methanococcus jannaschii. the dodecameric Dsp HSP of E. colv. and the MrgA protein.
  • HSPs heat shock proteins
  • Protein cages can have different core sizes, such as ranging from about 1 nm to about 1000 nm, from about 10 nm to about 500 nm, from about 50 nm to about 200 nm, from about 100 nm to about 150 nm, or from about 150 nm or less.
  • Exosomes are small biological membrane vesicles. Exosome have been utilized to deliver various cargoes including small molecules, peptides, proteins and nucleic acids. Exosomes generally range in size from about 30 nm to 100 nm and can be taken up by a cell and deliver its cargo (e.g., expression cassette comprising nucleic acid encoding for RNA polynucleotide comprises an a-synuclein targeting sequence). Cargoes can be associated with exosome surface structure or may be encapsulated within the exosome bilayer.
  • exosomes facilitating cargo delivery and cell targeting include structures for associating with cargoes such as protein scaffolds and polymers.
  • Modifications for cell targeting include targeting ligands and modifying surface charge.
  • Publications describing production, modification, and use of exosomes for delivery of different 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 which is hereby incorporated by reference herein in their entirety).
  • compositions can be used to facilitate storage and/or delivery of an agent being administered to a subject.
  • the pharmaceutical composition comprises: (a) an RNA polynucleotide comprising a sequence targeting a-synuclein mRNA, (b) an optionally modified inhibitory RNA duplex targeting a-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding an a-synuclein mRNA targeting sequence, or (d) a delivery vehicle comprising (a), (b) or (c); and a pharmaceutically acceptable carrier.
  • compositions do not cause substantial undesirable biological effects at the amount utilized.
  • Pharmaceutically acceptable carriers can contain different components such as one or more pharmaceutically acceptable excipients such a salt, sugar, buffer, solvent, preservative, protein and surfactant. A particular excipient can have more than one function. Examples of pharmaceutically acceptable excipients and carriers that can be used for viral vectors are provided in, for example, International Patent Publication Nos. WO2021/071835 and WO2024/138129 (hereby incorporated by reference herein in their entireties).
  • compositions can be formulated to be compatible with a particular route of administration or delivery.
  • Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • Illustrative examples include water, buffered saline, Hanks’ solution, Ringer’s solution, dextrose, fructose, ethanol, animal vegetable and synthetic oils.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the pharmaceutical composition contains a formulation capable of injection into a subject.
  • injectable formulation components include isotonic, sterile, saline solutions, salts (e.g., monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and mixtures of such salts), buffered saline, sugars e.g., dextrose), and water for injection.
  • Pharmaceutical compositions include dry, for example, freeze-dried compositions which upon addition of sterilized water or physiological saline, permit the constitution of solutions suitable for administration.
  • suspensions can be prepared as appropriate oil injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension can also contain suitable stabilizers or agents which increase compound solubility facilitating the preparation of concentrated solutions.
  • an “effective amount” or “sufficient amount” refers to an amount providing an indicated or desired effect.
  • the effective amount can be administered, in single or multiple doses, alone or in combination, with one or more other compositions (e.g., additional therapeutic or immunosuppressive agents), treatments, protocols, or therapeutic regimens; and provide for a long or short term response.
  • compositions comprising transgenes encoding an RNA polynucleotide targeting a-synuclein mRNA can be delivered to a subject, so as to allow production of the encoded polynucleotide. Delivery can be in vivo or ex vivo. In certain embodiments, pharmaceutical compositions comprise sufficient genetic material to enable a recipient to produce a therapeutically effective amount in the subject.
  • a “therapeutically effective amount” refers to an amount that elicits the desired or indicated biological or medicinal response in a subject.
  • a therapeutically effective amount can be determined based on observed symptoms and/or through the use of biomarkers associated with a particular disease or disorder. Selection of a particular effective dose can be optimized taking into account different factors, including the disease to be treated or prevented, the symptoms involved, the targeted disease or disorder, safety and effectiveness in animal models, the patient’s body mass, and the patient’s immune status.
  • the optimal dose to be employed in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and can be evaluated depending upon patient’s circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a pharmaceutical composition comprising a rAAV vector comprises empty AAV capsids.
  • the ratio of the empty AAV capsids to the rAAV vector is within or between about 100: 1-50: 1, from about 50: 1-25: 1, from about 25: 1- 10: 1, from about 10: 1-1 :1, from about 1 :1-1 : 10, from about 1 : 10-1 :25, from about 1 :25-1 :50, or from about 1 :50-1 :100.
  • the ratio of the empty AAV capsids to the rAAV vector is about 2:1, 3: 1, 4:1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • compositions and delivery systems are provided in, for example, Remington: The Science and Practice of Pharmacy (2020) 23th ed., University of the Science in Philadelphia, published by Elsevier; The Merck Index (2013) 15 th ed., Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; and Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD.
  • RNA polynucleotides comprising an inhibitory sequence targeting a-synuclein can be used in methods for reducing a-synuclein expression and/or treating a synucleinopathy disease or disorder in a subject.
  • Such methods can, for example, comprise administration of (a) an RNA polynucleotide comprising a sequence targeting a-synuclein mRNA, (b) an optionally modified inhibitory RNA duplex targeting a-synuclein mRNA, (c) a polynucleotide, expression cassette, or recombinant viral nucleic acid comprising a sequence encoding for an a-synuclein mRNA targeting sequence or (d) a delivery vehicle comprising (a), (b) or (c) and a pharmaceutically acceptable carrier.
  • Treatment can be administered to a subject, preferably a human subject, to provide for prophylactic treatment reducing the likelihood or severity of a disease or order and/or treating a diagnosed disease or disorder. Treating a diagnosed disease or disorder includes improvement of one or more symptoms and/or delaying the onset of one or more symptoms.
  • the particular therapeutic agent, route of administration, and/or pharmaceutical composition is selected taking into account the particular disease or disorder being treated.
  • Subjects having a particular disease or disorder, or at an increased risk of a particular disease or disorder can be identified, for example, based on symptoms, a-synuclein, biomarkers and/or genetic markers. Treatment can be carried, for example, on subjects at increased risk for developing a synucleinopathy disease or disorder in a subject.
  • markers include SNCA A53T, and SNCA A30P substitutions.
  • diagnose involves an a-synuclein seed amplification assay.
  • a-synuclein seed amplification assay (Siderowf et al., Lancet Neurol. 2023 May;22(5):407-417, hereby incorporated by reference herein in its entirety.)
  • treatment is for a Lewy body disease or disorder.
  • the Lewy body disease or disorder is either Parkinson’s disease, Parkinson’s disease dementia, dementia with Lewy bodies, infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, adult-onset dystonia-parkinsonism, autosomal recessive early-onset parkinsonism, POLG-associated neurodegeneration, Niemann-Pick type Cl, or Krabbe disease.
  • Certain embodiments are directed to treating Parkinson’s disease, Parkinson’s disease dementia, or dementia with Lewy bodies.
  • Parkinson’s disease symptoms include resting tremor, bradykinesia, gait, speech difficulties, hypophonia, muscle dystrophy, instability and postural deformities. Parkinson’s disease may also be accompanied by non-motor symptoms, such as rapid eye movement, sleep behavior disorder (RBD), hyposmia, depression, and autonomic failure. Early-stage symptoms include pain, stiffness or numbness in limbs, bradykinesia, tremors, and a decline in facial expression. Late-stage symptoms include motor fluctuations, dyskinesia, gait freezing, and falling. Initial diagnosis can be based on evaluation of clinical features of patient history and examination. Responsiveness to dopamine agents may also be used in the diagnosis of Parkinson’s disease over time.
  • Dementia with Lewy bodies symptoms include dementia, fluctuating cognition, visual hallucinations, rapid eye movement, sleep behavior disorder, and parkinsonism. (Koga et al. Molecular Neurodegeneration (2021) 16:83.)
  • diagnosis, patient monitoring, and/or clinical assessments involves the use of a questionnaire, scale, and/or assessment tool used by a clinician in the treatment and clinical outcome assessment of movement disorders such as Parkinson’s Disease.
  • Such clinical outcome assessments can be found, for example, at the International Parkinson and Movement Disorder website which is incorporated by reference (hypertext transfer protocol://www. movementdisorders. org/MDS/MDS-Clinical-Outcome- Assessment.htm).
  • Parkinson’s disease progression is evaluated 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 hereby incorporated by reference herein in their entirety.)
  • MDS-UPDRS Unified Parkinson's Disease Rating Scale
  • Parkinson’s disease progression can be evaluated using imaging biomarkers.
  • imaging biomarkers and techniques to monitor Parkinson’s disease pathology and progression include: SV2A and VMAT2 PET to investigate changes in dopamine terminal integrity in the caudate and putamen; structural MRI to investigate volumetric loss in the substantia nigra, as well as quantitative susceptibility mapping to evaluate structural change based on iron accumulation in the substantia nigra; biofluid biomarkers for tracking disease progression such as CSF levels of total a-Syn, total and phosphorylated tau, dopa decarboxylase, and 3,4 dihydroxyphenylacetic acid; and a -Syn protein knockdown can be measured in neuronal-derived exosomes isolated from plasma.
  • a Roche Parkinson’s Disease Mobile Application DHT can be used which includes a suite of performance outcome (“PerfO”) assessments designed to measure the progression of the major motor features in people with early-stage Parkinson’s disease.
  • PerfO performance outcome
  • the DHT provides a measurement of impairments underlying the core motor symptoms of Parkinson’s Disease including upper and lower body bradykinesia, gait dysfunction, postural instability, tremor, and speech and voice difficulties. These can be derived from 7 motor DHT PerfO tests: Hand Turning, Dexterity, U-turn, Rest Tremor, Postural Tremor, Speech, and Voice.
  • MSA is characterized by a variable combination of autonomic failure, levodopa (L-DOPA)-unresponsive parkinsonism, cerebellar ataxia, pyramidal signs, and nonmotor symptoms.
  • Non-motor symptoms may include RBD, sleep-disordered breathing, dysphagia, severe dysphonia and dysarthria. (Koga et al. Molecular Neurodegeneration (2021) 16:83.)
  • Administration can be by different routes, such as, subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intranasally, intraperitoneally, intravenously, intra-pleurally, intraarterially, intracavitary, orally, via the portal vein, intramuscularly, intraparenchymal, intracistemal or intraventricular administration.
  • administration to a patient is via infusion in a pharmaceutical carrier.
  • administration is direct to the CNS, for example, intraparenchymal, intracistemal or intraventricular administration.
  • the administration route and/or vector targets the caudate- putamen and/or the substantia nigra.
  • the caudate-putamen + substantia nigra is targeted for treating MSA; or the substantia nigra is targeted for treating Parkinson’s disease.
  • administration is direct to the CNS, for example, by intraparenchymal, intracistemal or intraventricular administration.
  • CNS administration is carried out by direct administration to the brain using a needle or catheter.
  • CNS administration is performed by convection enhanced delivery.
  • Convection enhanced delivery comprises surgical exposure of the brain, placement of a catheter directly into the target area, followed by infusion of a therapeutic agent.
  • intraparenchymal administration is performed via IP infusions to the caudate and putamen, or substantia nigra, by bilateral infusion using convection-enhanced delivery.
  • administration employes bilateral stereotactic infusions.
  • administration is carried under MRI-guidance, wherein the rAAV is provided in a formulation comprising an imaging agent.
  • Different devices are available for infusion including, for example, the ClearPoint® SmartFlow cannula via convection-enhanced delivery (CED) under MRI-guidance and ClearPoint® Neuro Navigation System.
  • the presence of inhibitory nucleic acid encoded by a transgene is measured in cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the presence of inhibitory nucleic acid in the CSF can be monitored at different time points, for example at baseline, and months 3, 6, 12, 18, and 24.
  • a vector encoding miR5 is administered and presence of miR5 is measured in cerebrospinal fluid (CSF). Measuring inhibitory nucleic acid in CSF can be used, for example, as a durability marker.
  • CNS delivery devices, systems and techniques also include those described in, for example, U.S. Patent No. 8128600, U.S. Patent Publication No. 2020/0324089, U.S. Patent No. 11129643, U.S. Patent No. 11154377, U.S. Patent Publication No. 2021/0343397, U.S. Patent Publication No. 2021/0282866, U.S. Patent No. 9572928, U.S. Patent No. 8337458, U.S. Patent No. 10722265, and US Patent publication No. 2021/214749, each of which are incorporated by reference herein in their entirety.
  • CNS administration can also be carried out using techniques facilitating transport across the blood brain barrier including disruption of the blood brain barrier and making use of blood brain barrier carriers.
  • Techniques facilitating crossing the blood brain barrier can be utilized on viral and non-viral delivery vehicles.
  • CNS delivery is achieved using techniques and/or agents that facilitate the crossing of the blood brain barrier.
  • CNS delivery is achieved by use of techniques to bypass the blood brain barrier.
  • CNS delivery is achieved by direct administration to the brain.
  • Techniques facilitating transport across the blood brain barrier include disruption of the blood brain barrier, use of blood brain barrier carriers and the use of vectors able to cross the blood brain barrier.
  • General techniques facilitating crossing the blood brain barrier can be utilized on viral and non-viral delivery vehicles.
  • particular delivery vehicles such as certain AAV serotypes can facilitate crossing the blood brain barrier.
  • an AAV capsid facilitating CNS entry is used.
  • 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 AAVl/rh.10), U.S. Patent No. 9,585,971, U.S. Patent Publication Nos. US2021/214749 and US2021/107947, U.S. Patent No. 11,518,787, and International Patent Publication No. WO2019/158619.
  • the AAV capsid comprises an insert targeting CNS cells such as those present in the caudate and/or putamen and/or the substantia nigra and/or the ventral tegmental area.
  • CNS cells such as those present in the caudate and/or putamen and/or the substantia nigra and/or the ventral tegmental area.
  • Examples of targeting sequences are provided in U.S. Patent Publication No US2021/214749 and International Patent Publication No. WO2019/158619, both of which are hereby incorporated by reference herein in their entirety.
  • Targeting peptides can be inserted into different AAV capsid locations.
  • AAV capsid insertion sites are outside the lipase domain of VP1 and outside the assembly-activating protein (AAP).
  • the AAV capsid comprises a targeting peptide insert comprising an amino acid sequence described in U.S. Patent Publication No. US2021/214749 or International Patent Publication No. WO2019/158619.
  • the capsid is an AAV2 capsid comprising a peptide insert comprising SEQ ID NO: 279, 280, 281, or 282; and the insert is centered around amino acid residue 587 of AAV2 VP1 or the insert is between N587 and R588 of the AAV2 VP1 capsid protein.
  • CNS delivery involves the use of focused ultrasound and microbubbles.
  • Focused ultrasound combined with microbubbles can temporarily disrupt the blood brain barrier facilitating entry of therapeutic agents, including transgene delivery vehicles.
  • Focused ultrasound can deposit energy to a selected area of the human anatomy, and its use can be facilitated by magnetic resonance imaging (MRI) guidance.
  • MRI magnetic resonance imaging
  • Different types of microbubbles can be used with focused ultrasound to temporarily disrupt the blood brain barrier.
  • Microbubbles are typically injected intravenously.
  • microbubbles are either DefinityTM (Lantheus Medical Imaging, North Billerica, MA, USA) containing perflutren lipid microspheres (mean diameter of 1.1-3.3 pm with a maximum diameter of 20 pm); SonoVueTM (Bracco Imaging, Milan, Italy) a suspension of phospholipid microspheres containing sulfur hexafluoride gas (mean diameter of ⁇ 2.5 pm; more than 90% of the bubbles are smaller than 8 pm); and OptisonTM (GE Healthcare, Princeton, NJ, USA) made up of a sterile non-pyrogenic suspension of microspheres of human serum albumin with perflutren (mean diameter of 3.0-4.5 pm with a maximum diameter of 32 pm).
  • DefinityTM Lantheus Medical Imaging, North Billerica, MA, USA
  • SonoVueTM Billracco Imaging, Milan, Italy
  • OptisonTM GE Healthcare, Princeton, NJ, USA
  • microbubbles are produced and administrated as described in U.S. Patent No. 10,322,178, hereby incorporated by reference herein in its entirety.
  • rAAV vectors are selected, or engineered, to facilitate use with focused ultrasound delivery.
  • rAAV vectors comprise an acoustic targeting peptide described in U.S. Patent Publication No. 2023/0047753 and/or International Patent Publication No. W02023/004416.
  • intranasal administration is used to achieve CNS vector delivery.
  • Intranasal delivery bypasses the blood brain barrier and reduces systemic exposure.
  • intranasal delivery comprises the use of focused ultrasound and microbubbles. The use of focused ultrasound and microbubbles in combination with intranasal delivery has been indicated to enhance penetration of therapeutic agents already at the perivascular space beyond the blood brain barrier. (Ye et al., The Lancet (2022) Oct: Vol 84: 1- 14, hereby incorporated by reference in its entirety.)
  • the polynucleotide expressing an RNA polynucleotide comprises the PGK promoter, CBh promoter, and/or EFl -a promoter.
  • Optimal doses can vary depending upon different factors such as the particular therapeutic, and desired endpoint. The dose amount, number, frequency or duration can be proportionally increased or reduced, taking into account adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject.
  • a “unit dosage form” refers to a physically discrete unit containing a predetermined effective amount of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Unit dosage forms can be provided within, for example, ampules and vials, which can include a pharmaceutically acceptable carrier, or a composition in a freeze-dried or lyophilized state. In the case of a freeze-dried or lyophilized state, a sterile liquid carrier can be added prior to administration. Individual unit dosage forms can be included in multi-dose kits or containers.
  • An “effective amount” achieves the desired or indicated effect. For example, an effective amount for treatment decreases one or more adverse symptoms, reduces the likelihood of one or more symptoms associated with a disease or disorder, or reduces disease or disorder progression. Preferred effective amounts for treatment are effective to decrease multiple or all adverse symptoms.
  • a-Syn is a naturally occurring protein, some amount of the protein may be important or helpful for certain functions. While the biological functions of a-Syn are not well characterized, a-Syn-null animals in which the SNCA gene has been experimentally deleted develop normally and remain healthy throughout the lifespan (Chandra et al., Proc Natl Acad Sci USA 2004; 101(41): 14966-71) although subtle learning deficits have been reported in 1 strain of a-Syn-deficient mice (Kokhan et al., Behav Brain Res 2012;231(l):226-30.).
  • a pharmaceutical composition comprising a viral or non-viral vector is administered to a subject at a dose suitable to decrease a-synuclein or a-synuclein expression.
  • a-synuclein is decreased by at least 15%, at least 20% least 50%, at least 60%, or at least 70%; and/or a-synuclein or a-synuclein expression is decreased 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%; relative to baseline levels in the transduced brain region (e.g., substantia nigra).
  • a suitable dosage is from about 0.01 mg/kg to about 10 mg/kg of vector per kg body weight of a subject, about 0.01 mg/kg to about 0.1 mg/kg of vector per kg body weight of a subject, about 0.1 mg/kg to about 1.0 mg/kg of vector per kg body weight of a subject, or about 1.0 mg/kg to about 10 mg/kg of vector per body weight of a subject.
  • rAAV vector doses range from at least IxlO 8 vector genomes per kilogram (vg/kg) of the weight of the subject, or more, for example, IxlO 9 , IxlO 10 , IxlO 11 , IxlO 12 , IxlO 13 or IxlO 14 , or more, vector genomes per kilogram (vg/kg) of the weight of the subject, to achieve a therapeutic effect.
  • the rAAV dose is about 5xl0 n rAAV vg/kg or greater than about 5xl0 n rAAV vg/kg; about IxlO 12 rAAV vg/kg or greater than about IxlO 12 rAAV vg/kg; about 2xl0 12 rAAV vg/kg or greater than about 2xl0 12 rAAV vg/kg; about 3xl0 12 rAAV vg/kg or greater than about 3xl0 12 rAAV vg/kg; about 4xl0 12 rAAV vg/kg or greater than about 4xl0 12 rAAV vg/kg; about 5xl0 12 rAAV vg/kg or greater than about 5xl0 12 rAAV vg/kg; about IxlO 13 rAAV vg/kg or greater than about IxlO 13 rAAV vg/kg; about 2xl
  • Examples of dose ranges of rAAV vg/kg include a dose range from about 5xl0 n to about 6xl0 13 rAAV vg/kg; a dose range from about 5xl0 n to about 5.5xl0 n rAAV vg/kg; a dose range from about 5.5xl0 n to about 6xlO n rAAV vg/kg; a dose range from about 6xlO n to about 6.5xlO n rAAV vg/kg; a dose range from about 6.5xlO n to about 7xlO n rAAV vg/kg; a dose range from about 7xlO n to about 7.5xlO n rAAV vg/kg; a dose range from about 7.5xlO n to about 8xl0 n rAAV vg/kg; a dose range from about 8xl0 n to about 8.5xl0
  • rAAV vg/kg are administered at a dose of about 5x10 11 vg/kg, about 6xlO n vg/kg, about 7xlO n vg/kg, about 8xl0 n vg/kg, about 9xlO n vg/kg, about IxlO 12 vg/kg, about 2x10 12 vg/kg, about 3x10 12 vg/kg, about 4x10 12 vg/kg, about 5x10 12 vg/kg, about 6xl0 12 vg/kg, about 7xl0 12 vg/kg, about 8xl0 12 vg/kg, about 9xl0 12 vg/kg, about IxlO 13 vg/kg, about 2xl0 13 vg/kg, about 3xl0 13 vg/kg, about 4xl0 13 vg/kg, about 5xl0 13 vg/kg, or about
  • dose and dose ranges for other viral vectors is as provided herein with respect to rAAV.
  • the dose and dose range for recombinant adenovirus vectors, recombinant retrovirus vectors (e.g., lentivirus), and recombinant herpes simplex virus vectors is the same as illustrated above with respect to rAAV.
  • the provided vector genome dose is based on the target region, such as the caudate, putamen and/or substantia nigra (SN).
  • the provided dose is 3e8 vg/targeted brain region (TBR) to 3el 1 vg/TBR, about 3e8 vg/TBR, about 3e9 vg/TBR, about 3el0 vg/TBR, or about 3el 1 vg/TBR; or 3e8 vg/SN to 3el 1 vg/SN, about 3e8 vg/SN, about 3e9 vg/SN, about 3el0 vg/SN, or about 3el 1 vg/SN.
  • TBR vg/targeted brain region
  • the polynucleotide constructs, viral vectors and non-viral vectors described herein are administered in combination with additional compounds or treatments for a particular disease of disorder; and/or in combination with a compound decreasing an immune response generated against the polynucleotide, delivery vehicle and/or produced protein.
  • Additional compounds or treatments can be provided in different modalities such as administered separately; and administered or performed prior to, substantially contemporaneously with or following administration of the polynucleotide constructs, viral vectors and non-viral vectors described herein.
  • administration of polynucleotide constructs, viral vectors and non-viral vectors described herein is in combination with one or more additional treatments for a synucleinopathy disease or disorder.
  • Treatments for Parkinson’s Disease include, for example, dopaminergic medications (e.g., levodopa); monoamine oxidase inhibitors and antagonists; and glial cell line derived neurotrophic factor (GDNF).
  • dopaminergic medications e.g., levodopa
  • monoamine oxidase inhibitors and antagonists e.g., monoamine oxidase inhibitors and antagonists
  • GDNF glial cell line derived neurotrophic factor
  • treatment is carried out in combination with (a) one or more additional compounds used for treating Parkinson’s Disease selected from a dopamine decarboxylase inhibitor/dopamine precursor (e.g., carbidopa-levodopa); a cathechol-o- methyltransferase inhibitor, inhibits breakdown of levodopa (e.g., entacapone, tolcapone, or opicapone); a dopamine agonist (e.g., pramipexole, ropinirole, apomorphine, or rotigotine); a monoamino oxidase-B inhibitor, inhibits breakdown of dopamine (e.g., selegiline, rasagiline, or safinamide); a mixed mechanism inhibitor, including N-methyl-D-aspartate antag
  • a dopamine decarboxylase inhibitor/dopamine precursor e.g., carbidopa-levodopa
  • treatment is carried out in combination with (a) one or more additional compounds used for treating Lewy body dementia selected from a cholinesterase inhibitor (e.g. rivastigmine, donepezil, and galantamine), N-methyl-D-aspartate antagonists (e.g. memantine and amantadine), dopamine decarboxylase inhibitor/dopamine precursor (e.g., carbidopa-levodopa), and antipsychotics (e.g. clozapine and quetiapine), and/or (b) the patient has been or is undergoing treatment with (a).
  • a cholinesterase inhibitor e.g. rivastigmine, donepezil, and galantamine
  • N-methyl-D-aspartate antagonists e.g. memantine and amantadine
  • dopamine decarboxylase inhibitor/dopamine precursor e.g., carbidopa-levodopa
  • the patient has been or is undergoing treatment with (a) and has a decreased responsiveness to treatment involving (a).
  • multiple system atrophy is treatment is carried in combination with one or more additional compounds or other treatments directed at particular symptoms.
  • additional compounds and treatment include blood pressure raising medicine (e.g., fludrocortisone, midodrine, pyridostigmine, and droxidopa); medicines reducing Parkinson’s disease like symptoms (e.g., levodopa and carbidopa); and medicines treating erectile dysfunction (e.g., sildenafil).
  • an RNA polynucleotide comprising an inhibitory sequence targeting a-synuclein is used in combination with GDNF.
  • GDNF or nucleic acid encoding for GDNF is administered. Nucleic acid encoding GDNF can be provided on the same or a different polynucleotide, as the polynucleotide encoding an RNA inhibitory sequence targeting a-synuclein.
  • administration of polynucleotide constructs, viral vectors and non-viral vectors described herein is in combination with an immunosuppressive agent or regimen.
  • agents and regimens can be utilized, as needed, to achieve immune tolerance or mitigate the immune response to the provided polynucleotides or delivery vehicles.
  • immunosuppressive agents and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particle (SVP)-rapamycin (rapamycin encapsulated in a biodegradable nanoparticle)), ImmTOR-ILTM (ImmTOR with Treg- selective IL-2 agonist), B-cell depletion, immunoadsorption, and plasmapheresis.
  • ImmTOR® synthetic vaccine particle (SVP)-rapamycin (rapamycin encapsulated in a biodegradable nanoparticle)
  • ImmTOR-ILTM ImmTOR with Treg- selective IL-2 agonist
  • B-cell depletion immunoadsorption
  • plasmapheresis examples include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particle (
  • the polynucleotide construct, viral vector or non-viral vector is administered in conjunction with one or more immunosuppressive agents, where one or more immunosuppressive agent is administered prior to, substantially at the same time as, or after, administering the polynucleotide construct, viral vector or non-viral vector.
  • the one or more immunosuppressive agent is administered concomitantly with the polynucleotide construct, viral vector or non-viral vector.
  • the one or more immunosuppressive agent is 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 prior to polynucleotide construct, viral vector or non-viral vector administration.
  • the one or more immunosuppressive agent is 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; following polynucleotide construct, viral vector or non-viral vector administration.
  • the immunosuppressive agent is an anti-inflammatory agent.
  • the immunosuppressive agent is a steroid, e.g., a corticosteroid.
  • the immunosuppressive agent is prednisone, prednisolone, a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, e.g., CellCept®, Myfortic®), a CD52 inhibitor (e.g., alemtuzumab), a CTLA4-Ig (e.g., abatacept, belatacept), an anti-CD3 mAb, an anti-LFA-1 mAb (e.g., efalizumab), an anti-CD40 mAb (e.g., ASKP1240), an anti-CD22 mAb (e.g., epratuzumab), an anti-CD20 m
  • a calcineurin inhibitor e.g
  • Empty capsids used as decoy probes can be provided in different ratios to viral vectors.
  • the decoy probes is provided contemporaneously with the rAAV. Amounts of empty capsids administered can be calibrated based upon the amount (titer) of antibodies produced in a particular subject.
  • the ratio of the empty AAV capsids to the rAAV vector is within or between about 100: 1-50: 1, from about 50: 1 to 25 to 1, from about 25: 1 to 10: 1, from about 10: 1 to 1 : 1, from about 1 : 1 to 1 : 10, from about 1 : 10 to 1 :25, from about 1 :25 to 1 :50, or from about 1 : 50 to 1 : 100.
  • the ratio of the administered empty AAV capsids to rAAV vector is about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • the serotype of the empty capsids is the same as the rAAV serotype.
  • viral vehicles are delivered using methods bypassing the bloodstream and viral antibodies. Examples of such techniques include deliver to the liver via the hepatic artery; and endoscopic retrograde cholangiopancreatography (ERCP) deliver to the liver.
  • ERCP endoscopic retrograde cholangiopancreatography
  • Other ductal systems such as the ducts of the submandibular gland, can also be used as portals for delivering viral vectors into a subject that develops or has preexisting anti -antibodies to the viral vector.
  • Additional strategies to reduce humoral immunity to rAAV include methods to remove, deplete, capture, and/or inactivate AAV antibodies, commonly referred to as apheresis and more particularly, plasmapheresis where blood products are involved.
  • Apheresis or plasmapheresis is a process in which a human subject’s plasma is circulated ex vivo (extracorporal) through a device that modifies the plasma through addition, removal and/or replacement of components before its return to the patient.
  • Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from a blood product (e.g., plasma).
  • This procedure can be employed to deplete, capture, inactivate, reduce or remove immunoglobulins (antibodies) that bind AAV thereby reducing the titer of AAV antibodies in the treated subject that can contribute to rAAV neutralization.
  • An example is using a device composed of an AAV capsid affinity matrix column, and passing blood product (e.g., plasma) through an AAV capsid affinity matrix resulting in binding of AAV antibodies of different isotypes. (See, e.g., Bertin et al., 2020, Sci. Rep. 10, 864, hereby incorporated by reference herein in its entirety.)
  • the polynucleotide constructs, viral vectors and non-viral vectors can be used in combination with an agent that blocks, inhibits, or reduces the interaction of IgG with the neonatal Fc receptor (FcRn), such as an anti-FcRn antibody, to reduce IgG recycling and enhance IgG clearance in vivo-, and/or an agent that decreases circulating antibodies that binds to a recombinant viral vector, or that binds to a nucleic acid or a polypeptide, protein or peptide encoded by a polynucleotide encapsidated by a recombinant viral vector, or that binds to the polynucleotide.
  • antibody binding to a viral vector is reduced or inhibited by an agent that reduces interaction of IgG with FcRn, a protease or a glycosidase.
  • the polynucleotide constructs, viral vectors and non-viral vectors described herein can be used in combination with an endopeptidase (e.g., IdeS from Streptococcus pyogenes) or a modified variant thereof, or an endoglycosidase (e.g., S. pyogenes EndoS) or a modified variant thereof.
  • an endopeptidase e.g., IdeS from Streptococcus pyogenes
  • an endoglycosidase e.g., S. pyogenes EndoS
  • Such treatment can, for example, be carried out to reduce or clear neutralizing antibodies against the gene delivery vehicle (e.g., viral vector capsid) and enable treatment of patients previously viewed as not eligible for gene therapy or that develop antibodies resulting from gene therapy.
  • Such strategies are described in, for example, Leborgne et a!., (2020) Nat. Med.,
  • kits with packaging material and one or more components therein typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
  • a kit can contain a collection of such components, e.g., polynucleotide construct, a viral or a non-viral vector, and optionally a second active, such as another compound, agent, drug or composition.
  • a kit refers to a physical structure housing one or more components.
  • Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes such as paper, corrugated fiber, glass, plastic, foil, ampules, vials, and tubes.
  • Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacture location and date, and expiration dates. Labels or inserts can include information on a disease for which a kit component can be used.
  • Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.
  • Labels or inserts can include information on one or more benefits a component can provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that can be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.
  • Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component.
  • Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD- ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
  • RNA polynucleotide comprising a targeting RNA sequence having a sequence identity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the sequence of any of SEQ ID NOs: 36, 38, 32-35, 37, or 39-62; and/or differs from any of SEQ ID NOs: 36, 38, 32-35, 37, or 39-62 by 1 or 2 nucleotides.
  • RNA polynucleotide of 1, wherein the targeting RNA sequence comprises: a sequence selected from the group consisting of: a) a sequence differing from SEQ ID NO: 34 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 108; b) a sequence differing from SEQ ID NO: 36 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 109; c) a sequence differing from SEQ ID NO: 37 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 110; d) a sequence differing from SEQ ID NO: 38 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 111; e) a sequence differing from SEQ ID NO: 39 by 0, 1, 2 or 3 nucleotides, wherein the targeting sequence comprises the sequence of SEQ ID NO: 112; f)
  • RNA polynucleotide of 1, wherein said targeting RNA sequence comprises the sequence of SEQ ID NO: 36 or SEQ ID NO: 38.
  • RNA polynucleotide of 3, wherein said targeting RNA sequence comprises the sequence of SEQ ID NO: 95 or SEQ ID NO: 97.
  • RNA polynucleotide of any of 1-4 wherein said RNA polynucleotide further comprises a second RNA sequence, wherein the second RNA sequence is substantially complementary to the targeting RNA sequence.
  • RNA polynucleotide of 5 wherein said RNA polynucleotide is a pre-miRNA, wherein said pre-miRNA comprises a pre-miRNA scaffold, an embedded guide sequence and an embedded passenger sequence, wherein said guide sequence comprises said targeting sequence and said passenger sequence comprises said second sequence.
  • RNA polynucleotide of 6 wherein the RNA polynucleotide comprises a sequence having a sequence identity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to any of SEQ ID NOs: 122-128 and 207-224.
  • RNA polynucleotide of 5 wherein said RNA polynucleotide is a shRNA, wherein said shRNA comprise a shRNA scaffold, an embedded guide sequence and an embedded passenger sequence, wherein said guide sequence comprises said targeting sequence and said passenger sequence comprises said second sequence.
  • RNA polynucleotide of 5 wherein said RNA polynucleotide is a pri-miRNA, wherein said pri-miRNA comprise a pri-miRNA scaffold, an embedded guide sequence and an embedded passenger sequence, wherein said guide sequence comprises said targeting sequence and said passenger sequence comprises said second sequence.
  • RNA polynucleotide of 10 wherein the scaffold is a miR-1, miR-26, miR16-l, miR- 30, miR-33mi-R101, miR-64, miR-122, miR-125, miR-135, miR-155, enhanced miR-155 (eSIBR), or miR-451 scaffold.
  • RNA polynucleotide of 10 wherein the scaffold is an S155e scaffold, S26, S126 scaffold, S33 scaffold or SI 55 scaffold.
  • the scaffold comprises SEQ ID NOs: 186 or 187.
  • RNA polynucleotide of 10 wherein said RNA polynucleotide comprises a sequence having a sequence identity of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to any of SEQ ID NOs: 115-121, 207-224 or the RNA version (ribose backbone and U instead of T) of 243-260.
  • the RNA polynucleotide comprises a passenger/guide combination as provided in Tables 2, 3, or 4.
  • RNA polynucleotide of 11 wherein the RNA polynucleotide comprises the sequence of SEQ ID NO: 116 or SEQ ID NO: 118.
  • An optionally modified inhibitory RNA comprising (a) a guide strand able to hybridize to the target sequence of any of SEQ ID NOs: 129-135; 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 the RNA polynucleotide of any of 1-14.
  • An expression cassette comprising a nucleic acid sequence encoding the RNA polynucleotide of any one of 1-14, operably linked to one or more expression control elements.
  • the expression cassete of any one of 17-24 further comprises a nucleic acid sequence encoding a glial cell-derived neurotrophic factor (GDFN) that is either operatively linked to said promoter or is operatively linked to a second promoter.
  • GDFN glial cell-derived neurotrophic factor
  • a recombinant viral vector nucleic acid comprising the expression cassette of any one of 17-25, and 5’ and 3’ viral elements providing for viral packaging and/or replication.
  • the recombinant viral vector nucleic acid of 26 wherein the recombinant viral vector nucleic acid is a recombinant DNA and comprises an adeno-associated virus (AAV) inverted repeat (ITR) flanking the 5’ terminus of the recombinant viral vector nucleic acid and an AAV ITR flanking the 3’ terminus of the recombinant viral vector nucleic acid.
  • AAV adeno-associated virus
  • ITR inverted repeat
  • the recombinant viral vector nucleic acid of 27 or 30, comprising a sequence having a sequence identity of at least 90%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the sequence of any of SEQ ID NOs: 175-181.
  • the recombinant viral vector nucleic acid of 27, comprising a sequence having a sequence identity of at least 95% at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NOs: 176 or 178.
  • a delivery vehicle comprising a viral or a non-viral vector and the RNA polynucleotide of any one of 1-14, the inhibitory RNA of 15, the polynucleotide of 16, the expression cassette of any one of 17-25 or the recombinant viral vector nucleic acid of any one of 26-32.
  • the viral vector is a recombinant AAV, a recombinant lentivirus vector, or a recombinant adenovirus vector.
  • the viral vector is a recombinant AAV comprising the recombinant viral vector nucleic acid of any one of 26-32 and a capsid.
  • the recombinant AAV vector comprises a capsid comprising a VP1, VP2 or VP3 having a sequence identity of at least 90% to a 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 AAVl/rh.10; or VP1 of SEQ ID NO: 164 or SEQ ID NO: 167.
  • capsid is an 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 AAVl/rh.10 capsid; or the capsid comprises VP1 of SEQ ID NO: 164 or SEQ ID NO: 167.
  • the delivery vehicle of 36, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166.
  • capsid is an AAV2 capsid comprising the targeting sequence of SEQ ID NOs: 279, 280, 281, or 282 inserted between a location corresponding to N587 and R588 of VP1.
  • the delivery vehicle of 39 or 40, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequences of SEQ ID NOs: 137 and 151.
  • the delivery vehicle of 41, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 158.
  • the delivery vehicle of 41, wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 169.
  • the delivery vehicle of 41 wherein the recombinant viral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 176.
  • 45. The delivery vehicle of any one of 41-44, wherein the capsid comprises VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166.
  • capsid is an AAV2 capsid comprising the targeting sequence of SEQ ID NOs: 279 or 282 inserted between a location corresponding to N587 and R588 of VPl .
  • the delivery vehicle of 33 wherein the delivery vehicle is a nanoparticle selected from the group consisting of a lipid nanoparticle (LNP), a polymeric nanoparticle, a lipid polymer nanoparticle (LPNP), a protein or peptide-based nanoparticle, a DNA dendrimer or DNA-based nanocarrier, a carbon nanotube, a microparticle, a microcapsule, an inorganic nanoparticle, a peptide cage nanoparticle, and an exosome.
  • LNP lipid nanoparticle
  • LPNP lipid polymer nanoparticle
  • a recombinant adeno associated vector comprising rAAV nucleic acid encapsidated in a capsid, wherein said rAAV nucleic acid comprises (a) a 5’ ITR of SEQ ID NO: 182; (b) an EFla promoter of SEQ ID NO: 184; (c) a pri-miRNA encoding sequence comprising the sequence of SEQ ID NO: 158, (d) a poly adenylation signal comprising the sequence of SEQ ID NO: 185, and (e) a 3’ ITR of SEQ ID NO: 183, wherein (c) is operably linked to (b) and (d).
  • the recombinant AAV vector of 49 comprising a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or comprising, the sequence of SEQ ID NO: 169
  • a pharmaceutical composition comprising the RNA polynucleotide of any one of 1-14, the inhibitory RNA of 15, the polynucleotide of 16, the expression cassette of any one of 17-25, the recombinant viral vector nucleic acid of any one of 26-32, the delivery vehicle of any one of 33-48, or the rAAV vector of any one of 49-51 and a pharmaceutically acceptable carrier.
  • a method of reducing or inhibiting a-synuclein aggregation in a cell or subject comprising administering to the cell or subject the RNA polynucleotide of any one of 1-14, the inhibitory RNA of 15, the polynucleotide of 16, the expression cassette of any one of 17-25, the recombinant viral vector nucleic acid of any one of 26-32, the delivery vehicle of any one of 33- 48, the rAAV vector of any one of 49-51, or the pharmaceutical composition of 52.
  • a method of treating a subject for a synucleinopathy disease or disorder comprising administering to the subject the RNA polynucleotide of any one of 1-14, the inhibitory RNA of 15, the polynucleotide of 16, the expression cassette of any one of 17-25, the recombinant viral vector nucleic acid of any one of 26-32, the delivery vehicle of any one of 33-48, the rAAV vector of any one of 49-51, or the pharmaceutical composition of 52.
  • synucleinopathy disease or disorder is either a Lewy body disease or multisystem atrophy.
  • synucleinopathy disease or disorder is either infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, adult-onset dystonia-parkinsonism, autosomal recessive early- onset parkinsonism, POLG-associated neurodegeneration, Niemann- Pick type Cl, or Krabbe disease.
  • a method of treating a disease or disorder associated with low dopamine comprising administering to the subject the RNA polynucleotide of any one of 1-14, the inhibitory RNA of 15, the polynucleotide of 16, the expression cassette of any one of 17-25, the recombinant viral vector nucleic acid of any one of 26-32, the delivery vehicle of any one of 33-48, the rAAV vector of any one of 49-51, or the pharmaceutical composition of 52.
  • administering comprises direct intraparenchymal, intracistemal or intraventricular administration.
  • An AAV vector genome plasmid comprising the recombinant viral nucleic acid of any one of 26-32.
  • a method of producing a rAAV vector comprising the step of culturing a rAAV packaging cell line comprising a rAAV helper virus activity, wherein the genome of said production cell comprises the nucleic acid of any of 26-32, a rep gene and a cap gene, wherein said rAAV vector is produced.
  • a method of producing rAAV vector comprising the step of culturing a rAAV permissive cell comprising the rAAV genome plasmid of 64, wherein the rAAV permissive cell further comprises (a) rep and cap genes provided either 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 provided by one or more separate plasmids.
  • rAAV permissive cell is a packaging cell, wherein the genome of said packaging cell comprises a cap gene and a rep gene.
  • a method of obtaining a rAAV vector comprising the steps of (a) producing the rAAV vector using the method of any one of 66-69 and (b) purifying the rAAV vector.
  • Table 9 provides different nucleic acid and amino acid sequences.
  • a polynucleotide comprises a nucleic acid sequence having a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to any of the nucleic acid sequences provided in Table 9; and a polypeptide comprises an amino acid sequence having a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to any of the amino acid sequences provided in Table 9.
  • Table 10 references different miR constructs and provides the SEQ ID NOs: for the guide RNA, the passenger, the seed region, pri-miRNA, and pre-miRNA; and provides the encoding/ corresponding DNA (except for the pre-miRNA).
  • a polynucleotide comprises a nucleic acid sequence having a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to any of the sequences provided in Table 10.
  • Example 1 siRNA Reduction of SNCA Expression in HEK293 Cells
  • RNAiMAX 0.3 pL containing 1 nM, 10 nM, or 100 nM siRNA were transfected into cells having a cell density of about 1.5 x 10 4 .
  • SNCA expression was determined by measuring a-synuclein mRNA using PCR, 72 hours after transfection. Cell survival was measured using Presto blue staining.
  • the siRNA sequences contained two 5’ deoxythymidines.
  • FIG. 1 illustrates the ability of different constructs to reduce SNCA expression, expressed as RQ.
  • the center concentration is 10 nM
  • the concentration to the left is 1 nM
  • the concentration to the right is 100 nM.
  • Table 12 indicates an siRNA designation and provides inhibition data for some of the constructs at 100 mM.
  • FIGs. 2A, 2B and 2C illustrate percent survival at different siRNA concentrations.
  • FIG. 2 A illustrates survival at 1 nM siRNA
  • FIG. 2B illustrates survival at 10 nM siRNA
  • FIG. 2C illustrates percent survival at different siRNA concentrations.
  • 2C illustrates survival at 100 nM siRNA.
  • Example 2 siRNA Reduction of SNCA Expression in HEK293 Cells
  • Example 3 siRNA Inhibition of SNCB and SNCG Expression
  • SNCB encodes for P-synuclein
  • SNCG encodes for y-synuclein.
  • P-synuclein and y-synuclein have a similar structure to a-synuclein.
  • FIG. 4A and FIG. 4B illustrate the impact of siRNAs on off-target genes SNCB and SNCG at 72 hours post-transfection using 1 nM (left value), 10 nM (center value), and 100 nM (right value) of siRNA.
  • the siRNA constructs are summarized in Table 11.
  • FIG. 4A illustrates expression of SNCB at 1 nM, 10 nM, or 100 nM siRNA.
  • FIG. 4B illustrates expression of SNCG at 1 nM, 10 nM, or 100 nM siRNA.
  • Example 4 siRNA Inhibition of SNCA Expression in SH-SY5Y
  • siRNA constructs are summarized in Table 11. Cells were transduced with 10 nM or 100 nM of siRNA, as described in Example 1 and expression was evaluated by measuring the SNCA mRNA transcripts using qPCR. Expression was measured 72 hours after transduction.
  • siRNA treated cells were compared to the siGLO control.
  • the data confirms the ability of the tested siRNAs to inhibit SNCA expression in SH-SY5Y cells.
  • Example 6 Pri-miRNA Inhibition of SNCA
  • Pri-miRNA sequences were designed by modifying selected siRNA sequences, based on inhibition activity, impact on cell survival, and off-target affect.
  • the siRNA guide strand was modified by replacing the 3’ terminal dTs with nucleotides complementary to the target, and embedded into a S155 scaffold.
  • the siRNA passenger strand was modified by replacing the 3’ terminal dTs with nucleotides complementary to the guide strand, and removing two internal nucleotides for embedding in the SI 55 scaffold.
  • Nucleic acid encoding for different pri-miRNA were inserted to a plasmid comprising a reporter gene (mCherry driven by CMV promoter), and placing the pri-miRNA encoding sequence under control of a CBH promote.
  • the resulting plasmid comprised CBH-miRNA- CMV-mCherry.
  • Plasmids encoding the human a-synuclein-GFP fusion protein were inserted into HEK293 cells.
  • GFP signal provided a measure of SCNA expression
  • mCherry (Red) positive cells indicated cells transfected with nucleic acid expressing pri-miRNA.
  • a flow cytometry assay (CytoFLEX) was performed. The provided controls were naive (no transfection), and mock (transfected with only reagent, no plasmid).
  • Table 13 provides pri-miRNA designations and summarizes the encoding guide DNA sequence, encoding passenger DNA sequence, encoding pri-miRNA DNA, and provides reference to parent siRNA.
  • Parent siRNA indicates the siRNA whose guide and passenger sequences were modified and embedded into the SI 55 scaffold.
  • Example 7 In vitro Inhibition of q-Synuclein Aggregation and Expression
  • the miRNAs are summarized in Table 14.
  • Recombinant AAV vectors were produced by incorporating rAAV nucleic acid into capsids comprising VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 or SEQ ID NO: 166. Production of rAAV was carried out using triple transfection.
  • AAVl-CMV-A53Ta-Syn-WPRE (comprises a recombinant viral vector encoding a-synuclein with an A53T substitution) at a multiplicity of infection (MOI) of 10,000 and rAAV encoding miR at a MOI of 10,000 and 100,000;
  • MOI multiplicity of infection
  • PFF human a- synuclein preformed fibrils
  • test constructs are summarized in Table 14.
  • the pri-miRNA was operatively linked to an EFl promoter (SEQ ID NO: 184).
  • Control constructs were rAAV-CAG-EGFP containing the CAG promoter linked to EGFP; rAAV-EFl-misl 1 containing a missense miRNA sequence; and rAAVl/2-3XmiR-GFP (AAVl/2-CAG-Human-SNCA-3xmiR/GFP-WPRE- BGH-polyA) encoding 3 copies of a miRNA targeting a-synuclein encoding mRNA.
  • AAV1/2- CAG-Human-SNCA-3xmiR/GFP-WPRE-BGH-polyA was obtained from Charles River).
  • FIG. 8A illustrates the percentage of a-synuclein intensity normalized to the a- synuclein control. The different miRs significantly inhibited a-synuclein expression.
  • FIG. 8B illustrates the percentage of pS129 intensity, as a measure of aggregation.
  • Aggregate formation was determined by measuring fluorescent intensity of psi 29 staining and taking images using Opera Phenix 20X air objective. The different miRs significantly inhibited a-synuclein aggregation.
  • Example 8 In Vivo Inhibition in Co-Transduction Model
  • IPa refers to intraparenchymal administration into either both hemispheres (two values) or one hemisphere.
  • FIG. 9A illustrates the fold change of a-synuclein relative to a rAAVl/2-A53T aSyn + rAAV-misl 1 (misl 1) control.
  • Recombinant AAV encoding miR5 and miR7 significantly reduced SCNA expression at doses of 5e9 and lelO vg/hemisphere at 7 weeks post-injection in mouse striatum.
  • AAVl/2-3XmiRNA was included as positive control of knock-down.
  • FIG. 9D The effect of the viral vectors on endogenous murine SNCA (yaSCNA) is show in FIG. 9D.
  • miR7 has a one based mismatch (non-complementary at position 6) to the murine a- synuclein mRNA, while miR5 has a two-base mismatch (non-complementary at positions 7 and 10) to the murine a-synuclein mRNA.
  • miR7 reduced a-synuclein in the striatum, while miR5 did not have a significant effect.
  • Example 9 Inhibition of endogenous Expression in Substantia Nigra
  • FIG. 10A illustrates biodistribution in substantia nigra (SN) and striatum (Str). Recombinant AAV-miR5 and rAAV-miR7 vector injected into substantia nigra were detected in the striatum, at a lower VCGN, suggesting antegrade movement of viral vector.
  • FIG. 10B illustrates m5GV4 expression in the substantia nigra (SN) and striatum (Str).
  • Mouse endogenous a-synuclein mRNA level in striatum was significantly higher compared to substantia nigra.
  • FIG. 10C illustrates mSCNA expression in the substantia nigra (SN).
  • Mouse endogenous a-synuclein mRNA in substantia nigra was significantly reduced by rAAV-miR7 (1 mismatch), but not by rAAV-miR5 (two mismatches).
  • FIG. 10D illustrates m5 V4 expression in the striatum (Str).
  • Mouse endogenous a- synuclein mRNA in striatum was significantly reduced by both rAAV-miR7 (1 mismatch) and rAAV-miR5 (two mismatches).
  • Example 11 miRNA Processing
  • FIG. 11 illustrates guide and passenger processing for miR5 and miR7.
  • NGS Next generation sequencing
  • IP intraparenchymal
  • C57BL/6 mice were co-injected with rAAVl/2-A53T aSyn (expressing human SNCA with the A53T mutation) plus rAAV-misl 1 (encodes missense miRNA), or rAAV-miR5 or rAAV-miR7 at two doses into mouse brain striatum. Seven weeks later, tissue was collected and processed for NGS sequencing of small RNAs.
  • the amounts of guide RNA and passenger RNA generated from two different doses of two different rAAV -miRNA vectors are indicative of a favorable guide to passenger ratio (where the guide is the predominant species) driving specific degradation of the target mRNA (FIG. 11).
  • Example 12 Ten Week rAAV-miR5 Studies
  • rAAV vectors comprising viral nucleic acids encoding miR5 bilaterally delivered into the substantia nigra of C57BL6 mice were evaluated ten weeks after administration. Different parameters were measured including body weight, biodistribution, a- synuclein knockdown, dopamine levels, and dopamine turnover. In addition, miRNA processing and the impact on endogenous miRNA machinery was evaluated.
  • Recombinant AAV vectors were produced by incorporating rAAV nucleic acid into capsids comprising VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166. Production of rAAV was carried out using triple transfection.
  • mice were administered a fixed dose of human aSyn (AAVl/2-A53TaSyn) to induce loss of dopamine, as seen in synucleinopathies including Parkinson’s disease, and treatment animals received 5e7, 5e8, or 5e9 vector genomes (vg) doses of rAAV-miR5. Controls were administered diluent, or misl 1 (missense construct) at 5e9 vg + aSyn.
  • FIG. 12 illustrates the statistical significance of the end-point body weight.
  • the endpoint body weight analysis indicated a significant loss of body weight in the human aSyn (AAVl/2-A53TaSyn) and control miRNA (rAAV-CAG-misl 1) treated group compared to diluent treated animals.
  • rAAV-miR5 at 5e7 vg showed a loss of body weight.
  • FIGs. 13A and 13B Vector presence was assessed using specific primers designed based on the BgH and RbG polyA sequences.
  • the AAVl/2-A53TaSyn vector has a BgH polyA; and the rAAV-miR5 and rAAV- CAG-misl 1 have a RbG PolyA.
  • FIG. 13 A illustrates measurement of BgH polyA.
  • FIG. 13B illustrates measurement of RbG polyA.
  • FIG. 14 The ability of rAAV-miR5 to knockdown a-Syn, as measured by qRT-PCR is illustrated in FIG. 14.
  • Substantia nigra brain tissue was analyzed by qRT-PCR using a-Syn primers. No signal was observed in the diluent injected group, illustrating the specificity for human a-Syn.
  • a statistically significant knockdown of human a-Syn was observed in rAAV- miR5 treated groups at doses of 5e9 (highest dose) and 5e8 (mid dose) vg, but not with 5e7 (lowest dose) when compared to the rAAV-CAG-misl 1 (control miRNA).
  • Statistics were done by Ordinary 1-way Anova with Tukey’s multiple comparison test.
  • FIG. 15 The ability of rAAV-miR5 to knockdown aSyn in the cortex, as measured by JESS assay is illustrated in FIG. 15.
  • the first diluents lacks human a-Syn while the second diluent contains a-Syn.
  • a statistically significant knockdown of human a-Syn was observed in rAAV- miR5 treated groups at all doses 5e9 vg (highest dose), 5e8 vg (mid dose) and 5e7 vg (lowest dose), when compared to the rAAV-CAG-misl 1.
  • Parkinson’s disease the dopaminergic neurons of substantia nigra project into the striatum, where they release the neurotransmitter dopamine. Dopamine level and turnover were measured to evaluate whether rAAV-miR5 treatment inhibited dopamine loss.
  • FIG. 16 The ability of rAAV-miR5 to inhibit dopamine loss caused by A53T a-Syn is illustrated in FIG. 16. A significantly greater dopamine loss was observed in the striatum upon administration of rAAV encoding human A53TaSyn (AAVl/2-A53TaSyn) and negative control miRNA (rAAV-CAG-misl 1), compared to the diluent group.
  • Impaired dopamine turnover was observed upon treatment with AAV1/2- A53TaSyn and negative control miRNA (rAAV-CAG-misl 1). Protection of impaired dopamine turnover in rAAV-miR5 treated groups at mid and low dose, but not at the higher dose was observed. This is indicative of an impaired dopamine machinery because of total viral load at the highest dose, despite good a-Syn knockdown and protection of dopamine loss.
  • FIG. 18 illustrates an evaluation of microRNA processing by small RNA sequencing.
  • Small RNA sequencing after rAAV-miR5 indicates that the miRNA guide was favorably expressed over the passenger strand.
  • FIG. 19 summarizes results of small RNA-sequencing. Endogenous miRNA machinery was not perturbed by overexpression of the rAAV-miR5. Even at the highest dose, 5e9 vg, the expressed exogenous matured miRNA was 1% of the total endogenous miRNA pool. The mid and lowest dose 5e8 and 5e7 vg, accounted for ⁇ 0.1% of the total endogenous miRNA pool. Diluent treated group is the negative control group, which had no miRNA.
  • SNCA-OVX mice were dosed with rAAV-miR5 at 5e8 vg/SN and 5e9vg/SN.
  • SNCA- OVX mice are genetically altered where both copies of the murine SNCA gene have been genetically deleted; and containing a BAC construct providing one copy of the human SNCA gene under control of its endogenous promoter providing expression of approximately 1.6-1.9x normal levels of a-Syn. (Janezic et al., Proc Natl Acad Sci USA 2013; 110(42):E4016-25). Human SNCA mRNA levels were measured by RT-qPCR.
  • Recombinant AAV vectors were produced by incorporating rAAV nucleic acid into capsids comprising VP1 of SEQ ID NO: 164, VP2 of SEQ ID NO: 165, and VP3 of SEQ ID NO: 166. Production of rAAV was carried out using triple transfection. Classical hematoxylin and eosin (H&E) staining was performed to assess potential histopathology.
  • H&E Classical hematoxylin and eosin
  • the primary objective of the study was to evaluate brain biodistribution of a gene therapy AAV vector, following delivery by MRI-guided, convection-enhanced, bilateral intraparenchymal (IP) infusion to the substantia nigra pars compacta of male Chlorocebus sabaeus, followed by an 8-week observation period.
  • IP intraparenchymal
  • rAAV-miR5 Dose-dependent distribution and expression of rAAV-miR5 was observed, which corresponded to a reduction in SNCA mRNA for 2 months (longest timepoint tested) after bilateral infusion of rAAV-miR5 into the substantia nigra of NHPs (FIGs 21A, 21B, and 21C).
  • Global SNCA mRNA data also indicated that rAAV-miR5-mediated reduction in SNCA mRNA was observed primarily in the substantia nigra (>40%).
  • Quantification of immunohistochemical intensity staining of cytoplasmic a-syn protein in TH positive DA neurons of the SN indicates a dose-dependent rAAV-miR5-mediated reduction of cytoplasmic a-Syn protein (FIG. 21D).
  • Distribution of rAAV-miR5, administered at doses of 3el0 vg/SN (FIG. 22A), 3e9 vg/SN (FIG. 22B), and 3e8 vg/SN (FIG. 22C) was primarily contained in the substantia nigra with limited spread to connected basal ganglia structures of the subthalamic nucleus and globus pallidus.
  • SNCA mRNA levels primarily showed a dose-dependent reduction in the SN and in some adjoining basal ganglia regions (Fig. 22D).
  • RT-qPCR analysis of SNCA mRNA was conducted using a validated primer-probe combination, and demonstrated dosedependent reduction of SNCA mRNA in the substantia nigra (by >50% at dose 3el0 vg/SN) when compared to the control group (includes diluent treated animals and the misl 1 control group to account for basal variability of endogenous SNCA level) at 2 months post-necropsy. Very minimal spread to spinal cord regions was observed and no spread of the vectors to any peripheral tissues outside the CNS was observed. Expression of transgene product miR5 followed the distribution pattern of rAAV-miR5, showing significant presence only in the substantia nigra, with limited expression in adjacent basal ganglia structures only.
  • NHP study demonstrated that rAAV-miR5 induced a stable and dose-dependent reduction of both SNCA mRNA and a-Syn protein when administered via neurosurgical delivery to NHP brain.
  • a 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.
  • Doses are planned to be administered once via direct convection-enhanced IP infusion to the substantia nigra, using a ClearPoint OrchestraTM neurosurgical frame paired with a ClearPoint SmartFlow Neuro Ventricular cannula and targeting conducted using ClearPoint Neuro navigation software.
  • An MRI (3T Siemens Skyra) is used for imaging.
  • Various parameters and endpoints such as one or more, or all of the following can be examined: observations, body weight, physical examinations, neurological FOB, ophthalmic examinations, blood pressure, electrocardiography, clinical pathology evaluations, organ weights, and macroscopic and microscopic examination; tissue samples (multiple brain regions, DRGs [cervical, thoracic, and lumbar], adrenal gland, liver, lung, heart, kidney, spleen, testis, and ovary) collected at necropsy, as well as blood and CSF at selected timepoints, for biodistribution and/or biomarker analysis.
  • tissue samples multiple brain regions, DRGs [cervical, thoracic, and lumbar], adrenal gland, liver, lung, heart, kidney, spleen, testis, and ovary
  • Tissues for microscopic examination can include brain regions, spinal cord including nerve roots (cervical, thoracic and lumbar), dorsal root ganglion (“DRG”) (cervical, thoracic, and lumbar), sciatic nerve, bone, eye, liver, spleen, heart, lung, kidney, skeletal muscle, stomach, testis, epididymis, ovary, uterus, and lymph node (cervical and submandibular).
  • nerve roots cervical, thoracic and lumbar
  • DRG dorsal root ganglion
  • sciatic nerve bone, eye, liver, spleen, heart, lung, kidney, skeletal muscle, stomach, testis, epididymis, ovary, uterus, and lymph node (cervical and submandibular).
  • rAAV-miR5 genomic DNA and miRNA transgene and cynomolgus SNCA mRNA levels can be examined in the CNS to determine the extent of biodistribution and magnitude of SNCA mRNA reduction (pharmacodynamics) at each dose level.
  • Levels of a-Syn protein can be measured using multiple methods, including enhanced chemiluminescence and immunohistochemistry detection methods.
  • Dosing data from non-human primates can be used as a starting point for extrapolating to human brain targets, for example, doses targeting the substantia nigra to achieve a 25% to 75% reduction in a-syn.
  • NHP doses can be scaled to humans, for example, based on volumetric differences between the adult cynomolgus macaque substantia nigra and substantia nigra volumes determined from Parkinson’s disease patients at the same stage of disease as in target patient population (modified Hoehn & Yahr Stage 1-2.5).
  • Substantia nigra MRI volumetric data is available through the Parkinson’s Progression Markers Initiative database and associated published reports (Langley et al, medRxiv [Preprint], 2023.08.19.23294281).

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Abstract

La présente invention concerne des constructions polynucléotidiques comprenant des séquences ciblant l'ARNm de l'α-synucléine. Des constructions comprenant des séquences ciblant l'ARNm de l'α-synucléine et/ou des séquences codantes peuvent être utilisées, par exemple, pour inhiber l'expression de l'ARNm de l'α-synucléine et/ou traiter une maladie ou un trouble de type synucléinopathie.
PCT/US2024/051907 2023-10-20 2024-10-18 Acide nucléique inhibiteur ciblant l'expression de l'alpha synucléine Pending WO2025085713A1 (fr)

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