WO2022104011A1 - Methods for the treatment of neurological disorders - Google Patents

Abstract

The present invention provides methods for treating alpha-synuclein-induced toxicity using phosphatidic acid to phosphoinositide biosynthesis pathway inhibitors, such as a nucleic acid molecule, a small molecule, or a nuclease, among others.

Description

METHODS FOR THE TREATMENT OF NEUROLOGICAL DISORDERS

Sequence Listing

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 12, 2021 is named “51549-021 WO2_Sequence_Listing_1 1_12_21_ST25” and is 314,645 bytes in size.

Field of the Invention

The invention relates to the field of therapeutic treatment of neurological disorders in patients, such as human patients.

Background of the Invention

An incomplete understanding of the molecular perturbations that cause disease, as well as a limited arsenal of robust model systems, has contributed to a failure to generate successful diseasemodifying therapies against common and progressive neurological disorders, such as Parkinson's Disease and Alzheimer's Disease. Progress is being made on many fronts to find agents that can arrest the progress of these disorders. However, the present therapies for most, if not all, of these diseases provide very little relief. Accordingly, a need exists to develop therapies that can alter the course of neurological diseases (e.g., neurodegenerative diseases). More generally, a need exists for better methods and compositions for the treatment of neurological disorders in order to improve the quality of the lives of those afflicted by such diseases.

Summary of the Invention

This disclosure provides methods for modulating the phosphatidic acid (PA) to phosphoinositide (PI) biosynthesis pathway for the treatment of diseases and disorders related to toxicity caused by proteins such as toxicity related to misfolding and/or aggregation of proteins (e.g., alpha-synuclein). In some embodiments, the disease or disorder is a neurological disorder.

In one aspect, the disclosure provides a method of reducing alpha-synuclein-induced toxicity in a subject, the method including administering an effective amount of a PA to PI biosynthesis pathway inhibitor to the subject.

In another aspect, the disclosure provides a method of treating a neurological disorder in a subject, the method including administering an effective amount of a PA to PI biosynthesis pathway inhibitor to the subject.

In another aspect, the disclosure provides a method of suppressing toxicity in a cell related to protein misfolding and/or aggregation in a subject, the method including contacting a cell with a PA to PI biosynthesis pathway inhibitor.

In another aspect, the disclosure provides a PA to PI biosynthesis pathway inhibitor for use in reducing alpha-synuclein-induced toxicity in a subject.

In another aspect, the disclosure provides a PA to PI biosynthesis pathway inhibitor for use in treating a neurological disorder in a subject. In another aspect, the disclosure provides a PA to PI biosynthesis pathway inhibitor for use in suppressing toxicity in a cell related to protein misfolding and/or aggregation in a subject.

In some embodiments of any of the foregoing aspects, the PA to PI biosynthesis pathway inhibitor is a CDP-Diacylglycerol Synthase 1 (CDS1 ) inhibitor, a CDP-DAG inositol 3- phosphatidyltransferase (CDIPT) inhibitor, a diacylglycerol kinase (DGK) activator, a phosphatidate phosphatase lipin (LPIN) inhibitor, or a phospholipase D (PLD) inhibitor.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is a CDS1 inhibitor or a CDIPT inhibitor.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is a CDS1 inhibitor.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is a CDIPT inhibitor.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is a DGK activator.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is a LPIN inhibitor.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is a PLD inhibitor.

In some embodiments of any of the foregoing aspects, administering includes contacting a cell with an effective amount of a PA to PI biosynthesis pathway inhibitor.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is an interfering RNA molecule, such as a short interfering RNA (siRNA), micro RNA (miRNA), or short hairpin RNA (shRNA). The interfering RNA may suppress expression of a mRNA transcript (e.g., CDS1 , CDIPT, LPIN, or a PLD mRNA transcript), for example, by way of (i) annealing to the mRNA or pre-mRNA transcript, thereby forming a nucleic acid duplex; and (ii) promoting nuclease-mediated degradation of the mRNA or pre- mRNA transcript and/or (iii) slowing, inhibiting, or preventing the translation of a mRNA transcript, such as by sterically precluding the formation of a functional ribosome-RNA transcript complex or otherwise attenuating formation of a functional protein product from the target RNA transcript.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is an antisense oligonucleotide.

In some embodiments, the nucleic acid molecule is complementary to a portion of a full-length CDS1 , CDIPT, DGK, LPIN, or PLD nucleic acid.

In some embodiments, the nucleic acid molecule is a CDS1 inhibitor, and the nucleic acid molecule is complementary to a portion of SEQ ID NO: 11 .

In some embodiments, the nucleic acid molecule is a CDIPT inhibitor, and the nucleic acid molecule is complementary to a portion of SEQ ID NO: 13.

In some embodiments, the nucleic acid molecule is a DGK activator, and the nucleic acid molecule is complementary to a portion of any one of SEQ ID NOs: 15, 17, 19, 21 , 23, 25, 27, 29, 31 , or 33.

In some embodiments, the nucleic acid molecule is a LPIN inhibitor, and the nucleic acid molecule is complementary to a portion of any one of SEQ ID NOs: 35, 37, or 39.

In some embodiments, the nucleic acid molecule is a PLD inhibitor, and the nucleic acid molecule is complementary to a portion of any one of SEQ ID NOs: 41 , 43, 45, 47, 49, or 51 .

In some embodiments, the endogenous CDS1 , CDIPT, LPIN or PLD is disrupted by contacting the cells with a nuclease-mediated gene editing system, such as a nuclease that catalyzes cleavage of an endogenous CDS1 , CDIPT, LPIN, or PLD nucleic acid in the cells. In some embodiments, the nuclease is a clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein. In some embodiments, the CRISPR-associated protein is CRISPR-associated protein 9. In some embodiments, the CRISPR-associated protein is CRISPR-associated protein 12a. In some embodiments, the nuclease is a transcription activator-like effector nuclease (TALEN), a meganuclease, or a zinc finger nuclease (ZFN). In some embodiments the PA to PI biosynthesis pathway inhibitor is a guide RNA in the nuclease- mediated gene editing system.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is administered to cells of the subject by transduction with a viral vector selected from the group including an adeno-associated virus (AAV), an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpes virus, a poxvirus, and a Retroviridae family virus.

In some embodiments, the inhibitor is administered systemically to the subject. In some embodiments, the inhibitor is formulated for administration to a subject by way of intravenous injection. In some embodiments, the inhibitor is formulated for administration to the cerebrospinal fluid of the subject. In some embodiments, the inhibitor is formulated for administration to a subject by way of intracerebroventricular injection, intrathecal, stereotactic injection, or a combination thereof. In some embodiments, the inhibitor is formulated for administration by way of intraparenchymal injection. In some embodiments, the composition is formulated for administration to a subject by way of intracerebroventricular injection and intravenous injection.

In some embodiments, the cells are transfected ex vivo to express the PA to PI biosynthesis pathway inhibitor.

In some embodiments, the cells are transfected using an agent selected from the group including a cationic polymer, diethylaminoethyl-dextran, polyethylenimine, a cationic lipid, a liposome, calcium phosphate, an activated dendrimer, and a magnetic bead; or a technique selected from the group including electroporation, NUCLEOFECTION™, squeeze-poration, sonoporation, optical transfection, MAGNETOFECTION™, and impalefection.

In some embodiments of any of the foregoing aspects, toxicity is related to misfolding and/or aggregation of a protein.

In some embodiments of any of the foregoing aspects, toxicity is related to misfolding and/or aggregation of alpha-synuclein.

In some embodiments of any of the foregoing aspects, the cells are neural cells (e.g., neurons or glial cells).

In some embodiments of any of the foregoing aspects, the cells are non-neural cells.

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDS1 RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDS1 RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 1 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 1 ).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 1 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 1 ).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDS1 RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDS1 RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 2 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 2).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 2 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 2).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDS1 RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDS1 RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 3 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 3).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 3 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 3).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDS1 RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDS1 RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 4 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 4).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 4 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 4).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDS1 RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDS1 RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 5 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 5). In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 5 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 5).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDIPT RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDIPT RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 6 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 6).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 6 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 6).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDIPT RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDIPT RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 7 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 7).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 7 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 7).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDIPT RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDIPT RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 8 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9, or 100% identical to the nucleic acid sequence of SEQ ID NO: 8).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 8 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 8).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDIPT RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDIPT RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 9 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 9).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 9 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 9).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains an antisense portion that anneals to a segment of a CDIPT RNA transcript (e.g., mRNA or pre- mRNA transcript), such as a portion that anneals to a segment of a CDIPT RNA transcript having a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 10 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 10).

In some embodiments, the interfering RNA molecule, such as the siRNA, miRNA, or shRNA, contains a sense portion having at least 80% sequence identity to the nucleic acid sequence of a segment of SEQ ID NO: 10 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the nucleic acid sequence of a segment of SEQ ID NO: 10).

In some embodiments of any of the foregoing aspects, neurological disorders include, but are not limited to Alexander disease, Alpers’ disease, Alzheimer’s Disease (AD), amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington disease, Kennedy's disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease, multiple sclerosis, Parkinson's Disease (PD), Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, Ref sum's disease, Sandhoff disease, Schilder's disease, Steele-Richardson-Olszewski disease, tabes dorsalis, frontal temporal dementia, vascular dementia, Down’s syndrome, and Guillain-Barre Syndrome.

In some embodiments of any of the foregoing aspects, the neurological disorder is a proteinopathy (e.g., a synucleinopathy, AD, Alexander disease, ALS, a prion disease (e.g., Creutzfeldt- Jakob disease), Huntington’s disease, Machado-Joseph disease, Pick's disease, or frontotemporal dementia).

In some embodiments of any of the foregoing aspects, the neurological disorder is a synucleinopathy such as PD, dementia with Lewy bodies, pure autonomic failure, multiple system atrophy, incidental Lewy body disease, pantothenate kinase-associated neurodegeneration, Alzheimer's disease, Down's Syndrome, Gaucher disease, or the Parkinsonism-dementia complex of Guam.

In some embodiments of any of the foregoing aspects, the neurological disorder is a neurodegenerative disorder (e.g..Alpers’ disease, ataxia telangectsia, Canavan disease, Cockayne syndrome, corticobasal degeneration, Kennedy’s disease, Krabbe disease, Pelizaeus-Merzbacher disease, primary lateral sclerosis, Refsum’s disease, Sandhoff disease, Schilder's disease, Steele- Richardson-Olszewski disease, tabes dorsalis, vascular dementia, or Guillain-Barre Syndrome).

In some embodiments of any of the foregoing aspects, the neurological disorder is an Apolipoprotein E4 (ApoE)-associated neurodegenerative disorder (AD, vascular cognitive impairment, cerebral amyloid angiopathy, traumatic brain injury, or multiple sclerosis). In some embodiments of any of the foregoing aspects, the subject has an elevated level, or is predicted to have an elevated level of alpha-synuclein, ApoE4, or an undesired form thereof.

In another aspect, the disclosure provides a method of treating a neurological disorder in a subject, wherein the subject has an elevated level (e.g., the subject has a level about 5%, 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%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more greater as compared to a reference such as the level in a sample from a healthy subject) of a protein or a particular form of a protein (e.g., a misfolded form of a protein) related to a neurological disorder (e.g., alpha- synuclein, ApoE4, or an undesired form thereof), the method including administering an effective amount of a PA to PI biosynthesis pathway inhibitor.

In some embodiments of any of the foregoing aspects, subject is predicted to have an elevated level of alpha-synuclein, ApoE4, and/or an undesired form thereof based on genetic markers.

In some embodiments of any of the foregoing aspects, the method further comprises administering an additional therapeutic agent (small molecule, antibody or fragment thereof, nucleic acid, cognition-enhancing agent, antidepressant agent, anxiolytic agent, antipsychotic agent, sedative, dopamine promoter, an anti-tremor agent) to the subject.

In another aspect, the invention features a kit containing a PA to PI biosynthesis pathway inhibitor. The kit may further contain a package insert, such as one that instructs a user of the kit to perform the method of any of the above aspects or embodiments of the invention. The PA to PI biosynthesis pathway inhibitor in the kit may be a nucleic acid or nuclease described above and herein.

Brief Description of the Drawings

FIGs. 1A and 1B are graphs showing that short interfering RNA (siRNA)-mediated CDP- Diacylglycerol Synthase 1 (CDS1 ) or CDP-DAG inositol 3-phosphatidyltransferase (CDIPT) knockdown, respectively, in Human Bone Osteosarcoma Epithelial Cell (U2OS) cells rescues alpha-synuclein- dependent toxicity, as assessed by decreases in cellular ATP levels. U2OS cells were transfected with 2 pg of aggregation-defective alpha-synuclein (dNAC) as a control or 2 pg wild-type a-Syn. U2OS cells were also co-transfected with 2 pg alpha-synuclein in combination with 10 nM of control scrambled siRNA (SCR); or 2 nM, 10 nM or 50 nM of siRNA against human CDS1 (CDS1 ) or human CDIPT (CDIPT). Bars depict mean values of triplicate determinations; error bars indicate standard deviation. One-way analysis of variance (ANOVA) was utilized to evaluate differences between dNAC alone, scrambled siRNA alone, or alpha-synuclein in combination with siRNA against CDS1 or CDIPT, with Dunnett’s posthoc-test to adjust for multiple comparisons (* p < 0.05, ** p < 0.01 , *** p < 0.001 ). Abbreviations, RLU, relative luminescence units.

FIGs. 2A and 2B are graphs showing that siRNA against CDS1 or CDIPT, respectively, elicited knockdown of CDS1 or CDIPT. Total RNA was extracted from U2OS cells and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify mRNA levels of CDS1 or CDIPT. All samples were normalized to CDS1 or CDIPT level, respectively, in U2OS cells transfected with control scrambled siRNA, which was set to 1 .0. Bars depict mean values of triplicate determinations; error bars indicate standard deviation. Abbreviations: ddCT, the Comparative CT Method. ** p < 0.01 , *** p < 0.001 , **** p < 0.0001. FIGs. 3A, 3B, and 3C are graphs showing that siRNA-mediated CDS1 , CDIPT, or CDP- Diacylglycerol Synthase 2 (CDS2) knockdown, respectively, in BE(2)-M17 (M17) cells rescues alpha- synuclein aggregates found on membranes. In M17 cells, an inducible doxycycline system was used to overexpress reporter alpha-synuclein fused to yellow fluorescent protein (a-Syn-YFP). M17 cells were transfected with 2.5 nM, 10 nM, 25 nM, or 50 nM of siRNA against CDS1 , CDIPT, or CDS2 and the intensity of alpha-synuclein foci were measured. Additionally, cell counts were collected to monitor the potential toxicity of CDS1 , CDIPT, or CDS2 gene knockdown. Knockdown of CDS1 or CDIPT significantly reduced the intensity of a-Syn-YFP inclusions. All samples were normalized to cell count or the relative spot intensity level in M17 cells transfected with control scrambled siRNA, which was set to 100%. Bars depict mean values of triplicate determinations; error bars indicate standard deviation. *“ p < 0.001 , ns = not significant.

FIGs. 4A, 4B, and 4C are graphs showing that siRNA against CDS1 , CDIPT, or CDS2, respectively, elicited knockdown of CDS1 , CDIPT, or CDS2 in M17 cells. Total RNA was extracted from M17 cells and qPCR was performed to quantify mRNA levels of CDS1 , CDIPT, or CDS2. All samples were normalized to respective CDS1 , CDIPT, or CDS2 level in M17 cells transfected with control scrambled siRNA, which was set to 100%.

FIG. 5 is a graph showing that siRNA against CDS1 , CDS2 or CDIPT, respectively, elicited knockdown of CDS1 , CDS2, or CDIPT in in induced pluripotent stem cell (iPSC)-derived human GABAergic cortical neurons (iCELLs). Total RNA was extracted from iCELLss and RT-PCR was performed to quantify mRNA levels of CDS1 , CDS2, or CDIPT. All samples were normalized to CDS1 , CDS2, or CDIPT level, respectively, in iCELLs transfected with control scrambled siRNA, which was set to 1 .0. Bars depict mean values of triplicate determinations; error bars indicate standard deviation.

FIG. 6 is a graph showing that CDS1 or CDIPT knockdown with siRNA, respectively, reduced the cumulative risk of death in iCELLs harboring the alpha-synuclein A53T mutation. iCELLs harboring the alpha-synuclein A53T mutation or an isogenic control line in which the mutation was corected to wild-type were trans-differentiated into neurons. Single cells were evaluted for survival (based on overall morphology) over the course of the 288 hour study. The cumulative risk of neuron death is plotted against time (hrs) for each group as indicated. siRNA against CDS1 (siCDSI ) or CDIPT (siCDIPT), respectively, mediated a significant protection of cell survival in groups treated with alpha-synuclein- A53T, as compared to empty vector scrambled siRNA controls. Cox proportional hazard analysis was used to estimate relative risk of death, or hazard ratio (HR) and the P value was determined by the logrank test. Cl, confidence interval; N, number of neurons. “* p < 0.001 .

FIG. 7 is a table illustrating the consistent results of CDS1 or CDIPT knockdown, respectively, protecting against cumulative risk of death in nine replicate survival studies. Light gray shading illustrates experiments in which siRNA treatment elicited a trend towards the protection of cell survival. Medium gray shading illustrates experiments with siRNA treatment eliciting a significant protection of cell survival, as compared to scrambled siRNA controls. Dark gray shading illustrates experiments in which siRNA treatment did not provide protection of cell survival. In eight of the nine replicate studies, siCDSI significantly enhanced neuronal survival, as compared to scrabled siRNA controls. In five of the nine replicate studies, siCDIPT significantly enhanced neuronal survival, as compared to scrambled siRNA controls. “ p < 0.05.** p < 0.01 , *** p < 0.001 . Definitions

As used herein, “activity” refers to form(s) of a polypeptide which retain a biological activity of the native or naturally-occurring polypeptide, wherein “biological” activity refers to a biological function (e.g., enzymatic function) caused by a native or naturally-occurring polypeptide.

As used herein, the term “activator” refers to substances, such as nucleic acids, nucleases, and small molecules, that enhance the expression, activity, and/or level of a diacylglycerol kinase (DGK) enzyme Activators of this type may, for example, activate enzyme activity by elevating the concentration level and/or stability of DGK mRNA transcripts in vivo, as well as those that enhance the translation of functional DGK enzymes. Examples of activators of this type include nucleic acids encoding DGK and components of nuclease-mediated gene editing systems, such as a nuclease or guide RNA. In addition to encompassing substances that enhance the expression of DGK also encompassed are activators that enhance the activity of the respective enzyme. Activators of this type may, for example, activate enzyme activity by specifically binding the enzyme (e.g., by virtue of the affinity of the activator for the active site). Additional examples of activators that activate the activity of the respective enzyme include substances, such as small molecules, that may bind the enzyme at a site distal from the active site and enhance the binding of endogenous substrates to the enzyme active site by way of a change in the enzyme’s spatial conformation upon binding of the activator. Additional examples of an “activator” are substances, such as small molecules, that enhance the transcription of an endogenous gene encoding DGK.

As used herein, “administration” refers to providing or giving a subject a therapeutic agent (e.g., an inhibitory agent) by any effective route. Exemplary routes of administration are described herein and below (e.g., intracerebroventricular (ICV) injection, intrathecal (IT) injection, intraparenchymal (IP) injection, intravenous (IV) injection, and stereotactic injection). Administration may be systemic or local.

The term “alpha-synuclein” refers to proteins whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring wild-type alpha-synuclein protein as well as proteins whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring mutant alpha-synuclein protein. Alpha-synuclein is also referred to as synuclein alpha (SNCA). Human alpha-synuclein has NCBI Gene ID NO 6622. Alpha-synuclein is considered an intrinsically disordered protein. Naturally occurring mutant alpha-synuclein proteins include A53T, A30P, E46K, H50Q, and G51 D.

As used herein, “alpha-synuclein-induced toxicity” and “alpha-synuclein-mediated toxicity” are used interchangeably to refer to a reduction, impairment, or other abnormality in one or more cellular functions or structures, a reduction in growth or viability, or a combination thereof, occurring as a result of or associated with expression of an alpha-synuclein protein. In the context of a yeast cell, alpha- synuclein-mediated toxicity may be manifested as a reduction in growth or viability, e.g., reduced viability or non-viability, or a reduction, impairment, or other abnormality in one or more cellular functions or structures, e.g., reduction, impairment, or other abnormality in endocytosis or vesicle trafficking. In the context of a neuron or glial cell, e.g., a mammalian neuron or glial cell, alpha-synuclein-mediated toxicity may be manifested as a reduction in growth or viability, e.g., reduced viability or non-viability, or a reduction, impairment, or other abnormality in one or more cellular functions or structures. Cellular functions include any of the biological processes and pathways performed in a cell or by a cell, either itself or together with one or more other cells, in vitro or in vivo (e.g., in the context of a tissue or organ in vivo). In some embodiments, a cellular function is endocytosis, vesicle trafficking, axonal transport, mitochondrial function (e.g., ATP production), neurite outgrowth, neurotransmission, neurogenesis, or maintaining homeostasis. Alpha-synuclein-induced toxicity in vivo may be manifested to a variety of extents and in a variety of ways ranging from cellular dysfunction to death. In some embodiments, alpha- synuclein-mediated toxicity may be evidenced in a subject by development of a synucleinopathy or by an increased propensity to develop a synucleinopathy. In some embodiments, alpha-synuclein-mediated toxicity may be manifested as a decrease or defect in cognition, behavior, or memory, as compared with a normal control. In some embodiments, contacting mammalian cells or treating a mammalian subject with an agent as described herein alleviates one or more manifestations of alpha-synuclein-mediated toxicity.

As used herein, “Alzheimer’s disease” and “AD” refer to a late-onset neurodegenerative disorder presenting as cognitive decline, insidious loss of short- and long-term memory, attention deficits, language-specific problems, disorientation, impulse control, social withdrawal, anhedonia, and other symptoms. Brain tissue of AD patients exhibits neuropathological features such as extracellular aggregates of amyloid-p protein and neurofibrillary tangles of hyperphosphorylated microtubule- associated tau proteins. Accumulation of these aggregates is associated with neuronal loss and atrophy in a number of brain regions including the frontal, temporal, and parietal lobes of the cerebral cortex as well as subcortical structures like the basal forebrain cholinergic system and the locus coeruleus within the brainstem. AD is also associated with increased neuroinflammation characterized by reactive gliosis and elevated levels of pro-inflammatory cytokines.

As used herein, “apolipoprotein E” and “ApoE” refer to proteins whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring wild-type ApoE protein as well as proteins whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring allelic variant ApoE protein. Human APOE has NCBI Gene ID NO 348. APOE has three common alleles in humans: APOE £2 (frequency -8%), APOE £3 (frequency -80%), and APOE £4 (frequency -14%). The proteins encoded by the three common APOE alleles differ at two amino acids, located at positions 112 and 158 in the mature protein ApoE2 has cysteine at residues 112 and 158; ApoE3 has cysteine at residue 112 and arginine at residue 158; and ApoE4 has arginine at residues 112 and 158. Human ApoE protein is naturally synthesized as a precursor polypeptide of 317 amino acids, including an 18 amino acid signal sequence, which is cleaved to produce the mature 299 amino acid polypeptide. The sequence of human ApoE3 precursor polypeptide is found under NCBI RefSeq Acc. No. NP_000032.1 . Naturally occurring ApoE mutations include ApoE4(L28P), which confers on carriers an increased risk for late-onset AD that remains significant even after adjusting for the effect of ApoE4 itself (Kamboh et al. Neurosci Lett. 263(2-3):129-32, 1999). Other variants include E13K, R136C, G196S, Q248E, R251 G, and G278W (Tindale et al., Neurobiology of Aging 35, 727e1 - 727e3, 2014).

An “ApoE-associated neurodegenerative disorder” refers to a neurodegenerative disorder that is associated with and/or mediated at least in part by an ApoE protein (e.g., ApoE4). Exemplary ApoE- associated neurodegenerative disorders include, e.g., AD, dementia with Lewy bodies (DLB; also referred to as “Lewy body dementia”), mild cognitive impairment (MCI), frontotemporal dementia (FTD), cerebral amyloid angiopathy (CAA), CAA-associated intracerebral hemorrhage, vascular cognitive impairment, PD, multiple sclerosis (MS), traumatic brain injury (TBI), or Fragile X-associated tremor/ataxia syndrome.

The term “CDS1 ” refers to CDP-Diacylglycerol Synthase 1 . As used herein, the term CDS1 encompasses full-length, unprocessed CDS1 , as well as any form of CDS1 resulting from processing in the cell, as well as any naturally occurring variants of CDS1 (e.g., splice variants or allelic variants). Human CDS1 has NCBI Gene ID NO 1040. An exemplary wild-type human CDS1 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001263.4 (SEQ ID NO: 11 ), and an exemplary wild-type CDS1 amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001254.2 (SEQ ID NO: 12).

The term “CDIPT” refers to CDP-DAG inositol 3-phosphatidyltransferase. As used herein, the term CDIPT encompasses full-length, unprocessed CDIPT, as well as any form of CDIPT resulting from processing in the cell, as well as any naturally occurring variants of CDIPT (e.g., splice variants or allelic variants). Human CDIPT has NCBI Gene ID NO 10423. An exemplary wild-type human CDIPT nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001286585.2 (SEQ ID NO: 13), and an exemplary wild-type CDIPT amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001273514.1 (SEQ ID NO: 14).

As used herein, a “combination therapy” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection while a second therapeutic agent of the combination may be administered orally.

By “determining the level of a nucleic acid” is meant the detection of a nucleic acid (e.g., mRNA) by methods known in the art either directly or indirectly. Methods to measure mRNA level generally include, but are not limited to, northern blotting, nuclease protection assays (NPA), in situ hybridization (ISH), RT-PCR, and RNA sequencing (RNA-Seq).

By “determining the level of a protein” is meant the detection of a protein by methods known in the art either directly or indirectly. Methods to measure protein level generally include, but are not limited to, western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of a protein including, but not limited to, enzymatic activity or interaction with other protein partners.

The term “DGK” refers to diacylglycerol kinase. As used herein, the term DGK encompasses full- length DGK (e.g., DGK alpha, DGK beta, DGK delta, DGK epsilon, DGK eta, DGK gamma, DGK iota, DGK kappa, DGK theta, and DGK zeta), unprocessed DGK (e.g., DGK alpha, DGK beta, DGK delta, DGK epsilon, DGK eta, DGK gamma, DGK iota, DGK kappa, DGK theta, and DGK zeta), as well as any form of DGK resulting from processing in the cell, as well as any naturally occurring variants of DGK (e.g., splice variants or allelic variants of DGK alpha, DGK beta, DGK delta, DGK epsilon, DGK eta, DGK gamma, DGK iota, DGK kappa, DGK theta, and DGK zeta). Human DGK alpha has NCBI Gene ID No. 1060. Human DGK beta has NCBI Gene ID No. 1607. Human DGK delta has NCBI Gene ID No. 8527. Human DGK epsilon has NCBI Gene ID No. 8526. Human DGK eta has NCBI Gene ID No. 160851 . Human DGK gamma has NCBI Gene ID No. 1608. Human DGK iota has NCBI Gene ID No. 9162. DGK kappa has NCBI Gene ID No. 139189. Human DGK theta has NCBI Gene ID No. 1609. Human DGK zeta has NCBI Gene ID No. 8525. An exemplary wild-type human DGK alpha nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001345.5 (SEQ ID NO: 15), and an exemplary wild-type DGK alpha amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001336.2 (SEQ ID NO: 16). An exemplary wild-type human DGK beta nucleic acid sequence is provided in NCBI RefSeq Acc. No.

NM 001350705.1 (SEQ ID NO: 17), and an exemplary wild-type DGK beta amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001337634.1 (SEQ ID NO: 18). An exemplary wild-type human DGK delta nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_003648.3 (SEQ ID NO: 19), and an exemplary wild-type DGK delta amino acid sequence is provided in NCBI RefSeq Acc. No. NP_003639.2 (SEQ ID NO: 20). An exemplary wild-type human DGK epsilon nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_003647.3 (SEQ ID NO: 21 ), and an exemplary wild-type DGK epsilon amino acid sequence is provided in NCBI RefSeq Acc. No. NP_003638.1 (SEQ ID NO: 22). An exemplary wild-type human DGK eta nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001204504.3 (SEQ ID NO: 23), and an exemplary wild-type DGK eta amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001191433.1 (SEQ ID NO: 24). An exemplary wild-type human DGK gamma nucleic acid sequence is provided in NCBI RefSeq Acc. No NM 001080744.2 (SEQ ID NO: 25), and an exemplary wild-type DGK gamma amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001074213.1 (SEQ ID NO: 26). An exemplary wild-type human DGK iota nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001321708.2 (SEQ ID NO: 27), and an exemplary wild-type DGK iota amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001308637.1 (SEQ ID NO: 28). An exemplary wild-type human DGK kappa nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_001013742.4 (SEQ ID NO: 29), and an exemplary wild-type DGK kappa amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001013764.1 (SEQ ID NO: 30). An exemplary wild-type human DGK theta nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_001347.4 (SEQ ID NO: 31 ), and an exemplary wild-type DGK theta amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001338.2 (SEQ ID NO: 32). An exemplary wild-type human DGK zeta nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_001105540.2 (SEQ ID NO: 33), and an exemplary wild-type DGK zeta amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001099010.1 (SEQ ID NO: 34).

As used herein, the term “disrupt,” with respect to a gene, refers to preventing the formation of a functional gene product. A gene product is functional if it fulfills its normal (wild-type) functions. Disruption of the gene prevents expression of a functional factor encoded by the gene and may contain an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and/or a promoter and/or an operator that is necessary for expression of the gene in the animal. The disrupted gene may be disrupted by, e.g., removal of at least a portion of the gene from a genome of the animal, alteration of the gene to prevent expression of a functional factor encoded by the gene, an interfering RNA, or expression of a dominant negative factor by an exogenous gene. The disruption of endogenous CDS1 , CDIPT, LPIN, or PLD can be accomplished e.g., by using nucleic acid molecules, siRNA, shRNA, miRNA, antisense oligonucleotide, and gRNA, nucleases, meganuclease, a transcription activator-like effector nuclease, a zinc-finger nuclease, a CRISPR associated protein 9, and a CRISPR-associated protein 12a. Exemplary materials and methods for genetically modifying cells so as to disrupt the expression of one or more genes are detailed in US 8,518,701 ; US 9,499,808; and US 2012/0222143, the disclosures of each of which are incorporated herein by reference in their entirety (in case of conflict, the instant specification is controlling).

In the practice of the methods of the present invention, an “effective amount” of any one of the compounds or a combination of any of the compounds or a pharmaceutically acceptable salt thereof, is administered via any of the usual and acceptable methods known in the art, either singly or in combination.

As used herein, the term “endogenous” describes a molecule (e.g., a metabolite, polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).

As used herein, the terms “induced pluripotent stem cell,” “I PS cell,” and “iPSC” refer to a pluripotent stem cell that can be derived directly from a differentiated somatic cell. Human iPS cells can be generated by introducing specific sets of reprogramming factors into a non- pluripotent cell that can include, for example, Oct3/4, Sox family transcription factors (e.g., Sox1 , Sox2, Sox3, Soxl5), Myc family transcription factors (e.g., c-Myc, 1 -Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1 , KLF2, KLF4, KLF5), and/or related transcription factors, such as NANOG, LIN28, and/or Glisl . Human iPS cells can also be generated, for example, by the use of miRNAs, small molecules that mimic the actions of transcription factors, or lineage specifiers. Human iPS cells are characterized by their ability to differentiate into any cell of the three vertebrate germ layers, e.g., the endoderm, the ectoderm, or the mesoderm. Human iPS cells are also characterized by their ability propagate indefinitely under suitable in vitro culture conditions. See, for example, Takahashi and Yamanaka, Cell 126:663 (2006).

As used herein, the term “interfering RNA” refers to a RNA, such as a siRNA, miRNA, or shRNA that suppresses the expression of a target RNA transcript, for example, by way of (i) annealing to the target RNA transcript, thereby forming a nucleic acid duplex; and (ii) promoting the nuclease-mediated degradation of the RNA transcript and/or (iii) slowing, inhibiting, or preventing the translation of the RNA transcript, such as by sterically precluding the formation of a functional ribosome-RNA transcript complex or otherwise attenuating formation of a functional protein product from the target RNA transcript. Interfering RNAs as described herein may be provided to a patient, such as a human patient having a neurological disorder described herein, in the form of, for example, a single- or double-stranded oligonucleotide, or in the form of a vector (e.g., a viral vector) containing a transgene encoding the interfering RNA. Exemplary interfering RNA platforms are described, for example, in Lam et al., Molecular Therapy - Nucleic Acids 4:e252 (2015); Rao et al., Advanced Drug Delivery Reviews 61 :746- 769 (2009); and Borel et al., Molecular Therapy 22:692-701 (2014), the disclosures of each of which are incorporated herein by reference in their entirety.

As used herein, the term “IRES” refers to an internal ribosome entry site. In general, an IRES sequence is a feature that allows eukaryotic ribosomes to bind an mRNA transcript and begin translation without binding to a 5' capped end. An mRNA containing an IRES sequence produces two translation products, one initiating form the 5' end of the mRNA and the other from an internal translation mechanism mediated by the IRES.

By “level” is meant a level of a protein or nucleic acid (e.g., mRNA), as compared to a reference. The reference can be any useful reference, as defined herein. By a “decreased level” or an “increased level” of a protein or nucleic acid (e.g., mRNA) is meant a decrease or increase in protein or nucleic acid (e.g., mRNA) level, as compared to a reference (e.g., a decrease or an increase by about 5%, 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%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or an increase of more than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%, as compared to a reference; a decrease or an increase by less than about 0.01 - fold, about 0.02-fold, about 0.1 -fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase by more than about 1 .2-fold, about 1 .4-fold, about 1 .5-fold, about 1 .8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). A level of a protein may be expressed in mass/vol (e.g., g/dL, mg/mL, pg/mL, ng/mL) or percentage relative to total protein or nucleic acid (e.g., mRNA) in a sample.

The term “LPIN” refers to phosphatidate phosphatase lipin. As used herein, the term LPIN encompasses full-length LPIN (e.g., LPIN1 , LPIN2, or LPIN3), unprocessed LPIN (e.g., LPIN1 , LPIN2, or LPIN3), as well as any form of LPIN resulting from processing in the cell, as well as any naturally occurring variants of LPIN1 , LPIN2, or LPIN3 (e.g., splice variants or allelic variants of LPIN1 , LPIN2, or LPIN3). Human LPIN1 has NCBI Gene ID NO 643418. Human LPIN2 has NCBI Gene ID NO 9663. Human LPIN3 has NCBI Gene ID NO 64900. An exemplary wild-type human LPIN1 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001261427.3 (SEQ ID NO: 35), and an exemplary wild-type lipin-1 amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001248356.1 (SEQ ID NO: 36). An exemplary wild-type human LPIN2 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_014646.2 (SEQ ID NO: 37), and an exemplary wild-type lipin-2 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_055461.1 (SEQ ID NO: 38). An exemplary wild-type human LPIN3 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001301860.2 (SEQ ID NO: 39), and an exemplary wild-type lipin-3 amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001288789.1 (SEQ ID NO: 40).

A “neurodegenerative disorder” refers to a disorder characterized by progressive loss of the number (e.g., by cell death), structure, and/or function of neurons. In some instances, a neurodegenerative disease may be associated with protein misfolding, defects in protein degradation, genetic defects, programmed cell death, membrane damage, or other processes. Exemplary, nonlimiting neurodegenerative disorders include AD, PD, ApoE-associated neurodegenerative disorders, Alpers’ disease, ataxia telangectsia, Canavan disease, Cockayne syndrome, corticobasal degeneration, Kennedy’s disease, Krabbe disease, Pelizaeus-Merzbacher disease, primary lateral sclerosis, Refsum’s disease, Sandhoff disease, Schilder's disease, Steele-Richardson-Olszewski disease, tabes dorsalis, vascular dementia, and Guillain-Barre Syndrome. A “neurological disorder,” as used herein, refers to a disorder of the nervous system, for example, the central nervous system (CNS). Examples of neurological disorders include, without limitation, proteinopathies (e.g., synucleinopathies, tauopathies, prion diseases, and amyloidosis (e.g., Ap- amyloidosis) and/or neurodegenerative disorders (e.g., ApoE-associated neurodegenerative disorders).

“Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

The term “pharmaceutical composition,” as used herein, represents a composition containing a nucleic acid or nuclease described herein, formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a subject.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

As used herein, the term “phosphatidic acid (PA) to phosphoinositide (PI) biosynthesis pathway inhibitor” refers to a molecule that directly inhibits the expression, activity, and/or level of CDS1 , CDIPT, LPIN, or PLD; or that indirectly inhibits the expression, activity, and/or level of CDS1 , CDIPT, LPIN, or PLD through the modulation of a molecule (e.g., an upstream or downstream molecule) that modulates CDS1 , CDIPT, LPIN, or PLD. As used herein, the term “inhibitor” refers to substances, such as nucleic acids, nucleases, and small molecules, that suppress the expression, activity, and/or level of an CDS1 , CDIPT, LPIN, or PLD enzyme Inhibitors of this type may, for example, inhibit enzyme activity by reducing the concentration level and/or stability of CDS1 , CDIPT, LPIN, or PLD mRNA transcripts in vivo, as well as those that suppress the translation of functional CDS1 , CDIPT, LPIN, or PLD enzymes. Examples of inhibitors of this type are interfering RNA molecules, such as siRNA, miRNA, and shRNA, and components of nuclease-mediated gene editing systems, such as a nuclease or guide RNA (gRNA). In addition to encompassing substances that inhibit the expression of CDS1 , CDIPT, LPIN, or PLD, also encompassed are inhibitors that suppress the activity of the respective enzyme. Inhibitors of this type may, for example, competitively inhibit enzyme activity by specifically binding the enzyme (e.g., by virtue of the affinity of the inhibitor for the active site), thereby precluding, hindering, or halting the entry of one or more endogenous substrates into the enzyme’s active site. Additional examples of PA to PI biosynthesis pathway inhibitors that suppress the activity of the respective enzyme include substances, such as small molecules, that may bind the enzyme at a site distal from the active site and attenuate the binding of endogenous substrates to the enzyme active site by way of a change in the enzyme’s spatial conformation upon binding of the inhibitor (e.g., VU0155069, FIPI, Halopemide, ML-298, VU0364739, CAY10594, ML-200, and VU0359595). Additional examples of an “inhibitor” are substances, such as small molecules, that attenuate the transcription of an endogenous gene encoding CDS1 , CDIPT, LPIN, or PLD.

As used herein, the term “plasmid” refers to a to an extrachromosomal circular double stranded DNA molecule into which additional DNA segments may be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked.

The term “PLD” refers to phospholipase D. As used herein, the term PLD encompasses full- length (e.g., PLD1 , PLD2, PLD3, PLD4, PLD5, or PLD6), unprocessed PLD (e.g., PLD1 , PLD2, PLD3, PLD4, PLD5, or PLD6), as well as any form of PLD (e.g., PLD1 , PLD2, PLD3, PLD4, PLD5, or PLD6) resulting from processing in the cell, as well as any naturally occurring variants of PLD (e.g., splice variants or allelic variants of PLD1 , PLD2, PLD3, PLD4, PLD5, or PLD6). Human PLD1 has NCBI Gene ID NO 5337. Human PLD2 has NCBI Gene ID NO 53378. Human PLD3 has NCBI Gene ID NO 23646. Human PLD4 has NCBI Gene ID NO 122618. Human PLD5 has NCBI Gene ID NO 200150. Human PLD6 has NCBI Gene ID NO 201164. An exemplary wild-type human PLD1 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_002662.5 (SEQ ID NO: 41 ), and an exemplary wild-type PLD1 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_002653.1 (SEQ ID NO: 42). An exemplary wild-type human PLD2 nucleic acid sequence is provided in NCBI RefSeq Acc. No.

NM 001243108.2 (SEQ ID NO: 43), and an exemplary wild-type PLD2 amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001230037.1 (SEQ ID NO: 44). An exemplary wild-type human PLD3 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001031696.4 (SEQ ID NO: 45), and an exemplary wild-type PLD3 amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001026866.1 (SEQ ID NO: 46). An exemplary wild-type human PLD4 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM 001308174.2 (SEQ ID NO: 47), and an exemplary wild-type PLD4 amino acid sequence is provided in NCBI RefSeq Acc. No. NP 001295103.1 (SEQ ID NO: 48). An exemplary wildtype human PLD5 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_001195811 .2 (SEQ ID NO: 49), and an exemplary wild-type PLD5 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001182740.1 (SEQ ID NO: 50). An exemplary wild-type human PLD6 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_178836.4 (SEQ ID NO: 51 ), and an exemplary wild-type PLD6 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_849158.2 (SEQ ID NO: 52).

A “proteinopathy” is a disorder that is characterized by structural abnormalities of proteins (e.g., protein misfolding and/or protein aggregation) that disrupt the function of cells, tissues, and/or organs of a subject. In some cases, misfolding can lead to loss of a protein’s usual function. In other cases, a misfolded protein can gain toxic functions. In some cases, proteins can be induced to have structural abnormalities by exposure to the same (or a similar) protein that has folded into a disease-causing conformation (e.g., amyloid beta, tau, alpha-synuclein, superoxide dismutase-1 , polyglutamine, prion, and TAR DNA-binding protein-43). Exemplary, non-limiting proteinopathies include AD, Parkinson’s disease, Alexander disease, ALS, a prion disease (e.g., Creutzfeldt-Jakob disease), Huntington’s disease, Machado-Joseph disease, Pick's disease, or frontotemporal dementia.

By a “reference” is meant any useful reference used to compare protein or nucleic acid (e.g., mRNA) levels related to neurological disorders. The reference can be any sample, standard, standard curve, or level that is used for comparison purposes. The reference can be a normal reference sample or a reference standard or level. A “reference sample” can be, for example, a control, e.g., a predetermined negative control value such as a “normal control” or a prior sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject not having neurological disorders; a sample from a subject that is diagnosed with a neurological disorder; a sample from a subject that has been treated for neurological disorders; or a sample of a purified protein (e.g., any described herein) at a known normal concentration. By “reference standard or level” is meant a value or number derived from a reference sample. A “normal control value” is a pre-determined value indicative of non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range (“between X and Y”), a high threshold (“no higher than X”), or a low threshold (“no lower than X”). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as “within normal limits” for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject not having a neurological disorder. In preferred embodiments, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of levels of a purified protein, e.g., any described herein, within the normal reference range can also be used as a reference.

As used herein, the term “subject” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition. In preferred embodiments, the subject is a human.

A “synucleinopathy” is a disorder characterized by misfolding and/or abnormal accumulation of aggregates of alpha-synuclein in the central nervous system (e.g., in neurons or glial cells). Exemplary, non-limiting synucleinopathies include PD, dementia with Lewy bodies, pure autonomic failure, multiple system atrophy, incidental Lewy body disease, pantothenate kinase-associated neurodegeneration, Alzheimer's disease, Down's Syndrome, Gaucher disease, or the Parkinsonism-dementia complex of Guam.

It is to be understood that the above lists are not all-inclusive, and that a disorder or disease may fall within various categories. For example, Alzheimer’s disease can be considered a neurodegenerative disease, a proteinopathy, and, in some instances, may also be considered a synucleinopathy. Likewise, Parkinson’s disease can be considered a neurodegenerative disease and a proteinopathy.

As used herein, the terms “transduction” and “transduce” refer to a method of introducing a viral vector construct or a part thereof into a cell and subsequent expression of a transgene encoded by the vector construct or part thereof in the cell.

As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, diethylaminoethyl (DEAE)-dextran transfection, NUCLEOFECTION™, squeeze-poration, sonoporation, optical transfection, MAGNETOFECTION™, impalefection, and the like.

As used herein, the terms “treat,” “treated,” or “treating” mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e. , not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus, or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/011026; incorporated herein by reference as it pertains to vectors suitable for the expression of a gene of interest. Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of PA to PI biosynthesis pathway inhibitors as described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of PA to PI biosynthesis pathway inhibitors contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an IRES, and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

Detailed Description

The present disclosure provides methods for the treatment of neurological disorders, e.g., by suppressing toxicity in cells related to protein misfolding and/or aggregation. Membranes are structurally diverse assemblies containing thousands of different lipids. Cytidine diphosphate diacylglycerol (CDP- DAG) is involved is involved in two major lipid synthetic pathways. The CDP-DAG synthases (CDS) catalyze the conversion of phosphatidic acid (PA) to cytidine diphosphate diacylglycerol (CDP-DAG). The two human CDS genes encode for two enzymes, CDP-Diacylglycerol Synthase 1 (CDS1 ) and CDP- Diacylglycerol Synthase 2, which differ in their expression patterns throughout the body. The cytidine diphosphate diacylglycerol inositol 3-phosphatidyltransferase (CDIPT) enzyme catalyzes the conversion of cytidine diphosphate diacylglycerol (CDP-DAG) to phosphoinositide (PI). The diacylglycerol kinase (DGK) is a family of enzymes that catalyzes the conversion of diacylglycerol (DAG) to PA. In some embodiments, the DGK is DGK beta, gamma, kappa, or theta. In some embodiments, the DGK is DGK beta. In some embodiments, the DGK is DGK gamma. In some embodiments, the DGK is DGK kappa. In some embodiments, the DGK is DGK gamma. Phosphatidate phosphatase lipin (LPIN) acts as an enzyme which catalyzes the conversion of PA to DAG. Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine, yielding PA and choline. In some embodiments, the PLD is PLD1 , PLD2, or PLD6. In some embodiments, the PLD is PLD1 . In some embodiments, the PLD is PLD2. In some embodiments, the PLD is PLD6. The present inventors have discovered that inhibition of CDS1 and CDIPT is capable of suppressing toxicity in cells related to protein misfolding and/or aggregation. Accordingly, inhibition of the PA to PI biosynthesis pathway may provide new methods for the treatment of diseases and disorders related to toxicity caused by protein misfolding and/or aggregation.

PA to PI Biosynthesis Pathway Inhibitors

The present disclosure provides PA to PI biosynthesis pathway inhibitors for use in the methods described herein. Any suitable PA to PI biosynthesis pathway inhibitor described herein or known in the art may be used.

A number of approaches are known in the art for determining whether a compound modulates expression or activity of CDS1 , CDIPT, DGK, LPIN, or PLD, for example, to determine whether a compound is a PA to PI biosynthesis pathway inhibitor. The PA to PI biosynthesis pathway activity assay may be cell-based, cell-extract-based (e.g., a microsomal assay), a cell-free assay (e.g., a transcriptional assay), or make use of substantially purified proteins. For example, identification of compounds as PA to PI biosynthesis pathway inhibitors can be performed using a PA to PI biosynthesis pathway liver microsomal assay, for example, as described by Shanklin et al. Proc. Natl. Acad. Sci. USA 88:2510-2514, 1991 or Miyazaki et al. J. Biol. Chem. 275:30132-30138, 2000. In some instances, liquid- chromatography/mass spectrometry (LC/MS)-based approaches can be used to measure CDS1 , CDIPT, DGK, LPIN, or PLD activity, for example, as described by Dillon et al. Anal. Chim. Acta. 627(1 ):99-104, 2008. A high-throughput assay can be used, for example, as described by Soulard et al. Anal. Chim. Acta. 627(1 ):105-111 , 2008. Still further approaches to measure CDS1 , CDIPT, DGK, LPIN, or PLD activity are described in U.S. Patent No. 7,790,408.

Any suitable method can be used to determine whether a compound binds to CDS1 , CDIPT, LPIN, DGK, or PLD, for instance, mass spectrometry, surface plasmon resonance, or immunoassays (e.g., immunoprecipitation or enzyme-linked immunosorbent assay).

Any suitable method can be used to determine whether a compound modulates expression of CDS1 , CDIPT, DGK, LPIN, or PLD, for instance, Northern blotting, Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), mass spectrometry, or RNA sequencing.

Agent modalities

A PA to PI biosynthesis pathway inhibitor can be selected from a number of different modalities. A PA to PI biosynthesis pathway inhibitor can be a nucleic acid molecule (e.g., DNA molecule or RNA molecule, e.g., mRNA or inhibitory RNA molecule (e.g., short interfering RNA (siRNA), micro RNA (miRNA), or short hairpin RNA (shRNA)), or a hybrid DNA-RNA molecule), a small molecule (e.g., a CDS1 , CDIPT, LPIN or PLD small molecule inhibitor; or a DGK small molecule activator), an inhibitor of a signaling cascade, an activator of a signaling cascade, or an epigenetic modifier), or a nuclease (e.g., clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9), CRISPR-associated protein 12 (Cas12a), meganuclease, transcription activator-like effector nuclease (TALEN), or zinc finger nuclease (ZFN)). Any of these modalities can be a PA to PI biosynthesis pathway inhibitor directed to target (e.g., to inhibit or activate) CDS1 , CDIPT, DGK, LPIN, or PLD function; CDS1 , CDIPT, DGK, LPIN, or PLD expression; CDS1 , CDIPT, DGK, LPIN, or PLD binding; or CDS1 , CDIPT, DGK, LPIN, or PLD signaling. The nucleic acid molecule, small molecule, or nuclease can be modified. For example, the modification can be a chemical modification, e.g., conjugation to a marker, e.g., fluorescent marker or a radioactive marker. The modification can also include conjugation to an antibody to target the agent to a particular cell or tissue. Additionally, the modification can be a chemical modification, packaging modification (e.g., packaging within a nanoparticle or microparticle), or targeting modification to enable the agent to cross the blood brain barrier.

I. Nucleic acids

IA. Inhibitory RNA

In some embodiments, the PA to PI biosynthesis pathway inhibitor is an inhibitory RNA molecule, e.g., that acts by way of the RNA interference (RNAi) pathway. An inhibitory RNA molecule can decrease the expression level (e.g., protein level or mRNA level) of CDS1 , CDIPT, LPIN, or PLD. For example, an inhibitory RNA molecule includes a siRNA, shRNA, and/or a miRNA that targets full-length CDS1 , CDIPT, LPIN, or PLD. An siRNA is a double-stranded RNA molecule that typically has a length of about 19-25 base pairs. An shRNA is an RNA molecule containing a hairpin turn that decreases expression of target genes via RNAi. shRNAs can be delivered to cells in the form of plasmids (e.g., viral or bacterial vectors), by transfection, electroporation, or transduction. A microRNA is a non-coding RNA molecule that typically has a length of about 22 nucleotides. miRNAs bind to target sites on mRNA molecules and silence the mRNA, e.g., by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA. In some embodiments, the inhibitory RNA molecule decreases the level and/or activity of CDS1 , CDIPT, LPIN, or PLD function. In other embodiments, the inhibitory RNA molecule decreases the level and/or activity of an inhibitor of a positive regulator of function.

An inhibitory RNA molecule can be modified, e.g., to contain modified nucleotides, e.g., 2’-fluoro, 2’-o-methyl, 2’-deoxy, unlocked nucleic acid, 2’-hydroxy, phosphorothioate, 2’-thiouridine, 4’-thiouridine, or 2’-deoxyuridine. Without being bound by theory, it is believed that certain modification can increase nuclease resistance and/or serum stability or decrease immunogenicity.

In some embodiments, the inhibitory RNA molecule decreases the level and/or activity or function of CDS1 , CDIPT, LPIN, or PLD. In some embodiments, the inhibitory RNA molecule inhibits expression of CDS1 , CDIPT, LPIN, or PLD. In other embodiments, the inhibitory RNA molecule increases degradation of CDS1 , CDIPT, LPIN, or PLD. The inhibitory RNA molecule can be chemically synthesized or transcribed in vitro. The making and use of inhibitory therapeutic agents based on non-coding RNA such as ribozymes, RNAase P, siRNAs, and miRNAs are also known in the art, for example, as described in Sioud, RNA Therapeutics: Function, Design, and Delivery (Methods in Molecular Biology). Humana Press 2010.

IB. Antisense

In one approach, the invention provides a single-stranded oligonucleotide having a nucleobase sequence with at least 6 contiguous nucleobases complementary to an equal-length portion within a CDS1 , CDIPT, LPIN, or PLD target nucleic acid. This approach is typically referred to as an antisense approach. Without wishing to be bound by theory, this approach involves hybridization of an oligonucleotide to a target nucleic acid (e.g., CDS1 , CDIPT, LPIN, or PLD pre-mRNA, transcript 1 , or transcript 2), followed by ribonuclease H (RNase H) mediated cleavage of the target nucleic acid. Alternatively and without wishing to be bound by theory, this approach involves hybridization of an oligonucleotide to a target nucleic acid (e.g., CDS1 , CDIPT, LPIN, or PLD pre-mRNA, transcript 1 , or transcript 2), thereby sterically blocking the target nucleic acid from binding cellular post-transcription modification or translation machinery and thus preventing the translation of the target nucleic acid. In some embodiments, the single-stranded oligonucleotide may be delivered to a patient as a double stranded oligonucleotide, where the oligonucleotide is hybridized to another.

IC. Exemplary Inhibitory Nucleic Acids

In some embodiments, the nucleic acid is a CDS1 inhibitor, such as an siRNA, miRNA, or shRNA, comprising a total of 3 to 200 interlinked nucleotides and having a nucleobase sequence comprising at least 3 contiguous nucleobases complementary to an equal-length portion of a CDS1 target nucleic acid (e.g., SEQ ID NO: 11 ). For example, the CDS inhibitor may include the nucleic acid sequence of any one of SEQ ID NOs: 1 , 2, 3, 4, or 5, or a nucleic acid sequence that includes a sequence that is at least 80% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 2, 3, 4, or 5 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1 , 2, 3, 4, or 5).

In some embodiments, the nucleic acid is a CDIPT inhibitor, such as an siRNA, miRNA, or shRNA, comprising a total of 3 to 200 interlinked nucleotides and having a nucleobase sequence comprising at least 3 contiguous nucleobases complementary to an equal-length portion of a CDIPT target nucleic acid (e.g., SEQ ID NO: 13). For example, the CDS inhibitor may include the nucleic acid sequence of any one of SEQ ID NOs: 6, 7, 8, 9, or 10, or a nucleic acid sequence that includes a sequence that is at least 80% identical to the nucleic acid sequence of any one of SEQ ID NOs: 6, 7, 8, 9, or 10 (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the nucleic acid sequence of any one of SEQ ID NOs: 6, 7, 8, 9, or 10).

In some embodiments, the nucleic acid is a LPIN inhibitor, such as an siRNA, miRNA, or shRNA, comprising a total of 3 to 200 interlinked nucleotides and having a nucleobase sequence comprising at least 3 contiguous nucleobases complementary to an equal-length portion of a LPIN target nucleic acid (e.g., SEQ ID NOs: SEQ ID NOs: 33, 35, or 37).

In some embodiments, the nucleic acid is a PLD inhibitor, such as an siRNA, miRNA, or shRNA, comprising a total of 3 to 200 interlinked nucleotides and having a nucleobase sequence comprising at least 3 contiguous nucleobases complementary to an equal-length portion of a PLD target nucleic acid (e.g., SEQ ID NOs: SEQ ID NOs: 39, 41 , 43, 45, 47, or 49).

In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 6 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 10 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 12 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 14 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 16 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 18 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 20 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 22 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 24 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 26 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 28 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 30 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 40 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 50 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 60 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 70 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 80 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 90 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 100 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 110 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 120 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 130 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 140 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 150 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 160 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 170 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 180 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising at least 190 contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the nucleic acid comprises a nucleobase sequence comprising 200 of fewer contiguous nucleobases complementary to a region comprising a sequence selected from the group consisting of a CDS1 , CDIPT, LPIN, or a PLD target nucleic acid.

IIA. Nucleic acids encoding DGK

In some examples, the DGK activator is a nucleic acid encoding DGK (e.g., DGK beta, gamma, kappa, or theta), Nucleic acids encoding DGK may be overexpressed in a cell using any suitable approach. For example, in some embodiments, overexpressing a nucleic acid encoding DGK in a mammalian cell (e.g., neurons, glial cells, or non-neural cells, such as colon and kidney cells) comprises expressing a protein encoded by the gene in the cell or organism, wherein the protein is encoded by a nucleic acid (e.g., an expression construct) that has been introduced into the cell. The nucleic acid may be operably linked to a promoter and/or an enhancer, and any other suitable control elements. The nucleic acid may be introduced into a cell using any suitable approach, including any approach described herein.

II. Nuclease-mediated gene regulation

The PA to PI biosynthesis pathway inhibitor may be a nuclease or gRNA. Any suitable nuclease may be used. In some embodiments, the PA to PI biosynthesis pathway inhibitor is a component of a nuclease-mediated gene editing system. For example, the PA to PI biosynthesis pathway inhibitor introduces an alteration (e.g., insertion, deletion (e.g., knockout), translocation, inversion, single point mutation, or other mutation) in CDS1 , CDIPT, DGK, LPIN, or PLD. Exemplary gene editing systems include the CRISPR system, meganucleases, the ZFNs, and TALENs. CRISPR-based methods, ZFNs, and TALENs are described, e.g., in Gaj et al. Trends Biotechnol.31 .7(2013):397-405.

For example, a useful tool for the disruption and/or integration of target genes into the genome of a cell is the CRISPR/Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and a Cas protein (e.g., Cas9 or Cas12a). This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a gRNA, which can subsequently anneal to a target sequence and localize the Cas nuclease to this site. In this manner, highly site-specific Cas- mediated DNA cleavage can be caused in a foreign polynucleotide because the interaction that brings Cas within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can design a CRISPR/Cas system to cleave a target DNA molecule of interest (e.g., endogenous CDS1 , CDIPT, DGK, LPIN, or PLD). This technique has been exploited in order to edit eukaryotic genomes (Hwang et al. Nature Biotechnology 31 :227 (2013), the disclosure of which is incorporated herein by reference) and can be used as an efficient means of site-specif ically editing cell genomes in order to cleave DNA prior to the incorporation of a gene encoding a target gene. The use of CRISPR/Cas to modulate gene expression has been described in, e.g., US 8,697,359, the disclosure of which is incorporated herein by reference.

The CRISPR system has been modified for use in gene editing (e.g., changing, silencing, and/or enhancing certain genes) in eukaryotes. See, e.g., Wiedenheft et al., Nature 482: 331 , 2012. For example, such modification of the system includes introducing into a eukaryotic cell a plasmid containing a specifically designed CRISPR and one or more appropriate Cas proteins. The CRISPR locus is transcribed into RNA and processed by Cas proteins (e.g., Cas9) into small RNAs that contain a repeat sequence flanked by a spacer. The RNAs serve as guides to direct Cas proteins to silence specific DNA/RNA sequences, depending on the spacer sequence. See, e.g., Horvath et al., Science 327: 167, 2010; Makarova et al., Biology Direct 1 :7, 2006; Pennisi, Science 341 : 833, 2013. In some examples, the CRISPR system includes the Cas9 protein, a nuclease that cuts on both strands of the DNA.

In some embodiments, in a CRISPR system for use described herein, e.g., in accordance with one or more methods described herein, the spacers of the CRISPR are derived from a target gene sequence, e.g., from a CDS1 , CDIPT, DGK, LPIN, or PLD sequence.

In some embodiments, the PA to PI biosynthesis pathway inhibitor includes a gRNA for use in a CRISPR system for gene editing. In embodiments, the PA to PI biosynthesis pathway inhibitor contains a meganuclease, or an mRNA encoding a meganuclease, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) of CDS1 , CDIPT, DGK, LPIN, or PLD. In embodiments, the PA to PI biosynthesis pathway inhibitor contains a ZFN, or an mRNA encoding a ZFN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) of CDS1 , CDIPT, DGK, LPIN, or PLD. In some embodiments, the PA to PI biosynthesis pathway inhibitor contains a TALEN, or an mRNA encoding a TALEN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) of CDS1 , CDIPT, DGK, LPIN, or PLD. In some embodiments, the PA to PI biosynthesis pathway inhibitor contains a Cas (e.g., Cas9), or an mRNA encoding a Cas (e.g., Cas9), that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) of CDS1 , CDIPT, DGK, LPIN, or PLD.

In certain embodiments, the CRISPR system is used to edit (e.g., to add or delete a base pair) a target gene, e.g., CDS1 , CDIPT, DGK, LPIN, or PLD. In other embodiments, the CRISPR system is used to introduce a premature stop codon, e.g., thereby decreasing the expression of a target gene. In yet other embodiments, the CRISPR system is used to turn off a target gene in a reversible manner, e.g., similarly to RNA interference. In some embodiments, the CRISPR system is used to direct Cas (e.g., Cas9) to a promoter of a target gene, e.g., CDS1 , CDIPT, DGK, LPIN, or PLD, thereby blocking an RNA polymerase sterically.

In some embodiments, a CRISPR system can be generated to edit CDS1 , CDIPT, DGK, LPIN, or PLD using technology described in, e.g., U.S. Publication No. 20140068797; Cong, Science 339: 819, 2013; Tsai, Nature Biotechnol., 32:569, 2014; and U.S. Patent Nos.: 8,871 ,445; 8,865,406; 8,795,965; 8,771 ,945; and 8,697,359.

In some embodiments, the CRISPR interference (CRISPRi) technique can be used for transcriptional repression of specific genes, e.g., the gene encoding CDS1 , CDIPT, LPIN or PLD. In CRISPRi, an engineered Cas9 protein (e.g., nuclease-null dCas9, or dCas9 fusion protein, e.g., dCas9- KRAB or dCas9-SID4X fusion) can pair with a sequence specific guide RNA (sgRNA). The Cas9-5 gRNA complex can block RNA polymerase, thereby interfering with transcription elongation. The complex can also block transcription initiation by interfering with transcription factor binding. The CRISPRi method is specific with minimal off-target effects and is multiplexable, e.g., can simultaneously repress more than one gene (e.g., using multiple gRNAs). Also, the CRISPRi method permits reversible gene repression.

In some embodiments, CRISPR-mediated gene activation (CRISPRa) can be used for transcriptional activation, e.g., of one or more genes described herein, e.g., a gene that inhibits CDS1 , CDIPT, LPIN or PLD, or that activates DGK. In the CRISPRa technique, dCas9 fusion proteins recruit transcriptional activators. For example, dCas9 can be used to recruit polypeptides (e.g., activation domains) such as VP64 or the p65 activation domain (p65D) and used with sgRNA (e.g., a single sgRNA or multiple sgRNAs), to activate a gene or genes, e.g., endogenous gene(s). Multiple activators can be recruited by using multiple sgRNAs, which can increase activation efficiency. A variety of activation domains and single or multiple activation domains can be used. In addition to engineering dCas9 to recruit activators, sgRNAs can also be engineered to recruit activators. For example, RNA aptamers can be incorporated into a sgRNA to recruit proteins (e.g., activation domains) such as VP64. In some examples, the synergistic activation mediator (SAM) system can be used for transcriptional activation. In SAM, MS2 aptamers are added to the sgRNA. MS2 recruits the MS2 coat protein (MCP) fused to p65AD and heat shock factor 1 (HSF1 ). The CRISPRi and CRISPRa techniques are described in greater detail, e.g., in Dominguez et al., Nat. Rev. Mol. Cell Biol. 17:5, 2016, incorporated herein by reference.

In some embodiments, the gRNA or Cas (e.g., Cas9) can be used in a CRISPR system to engineer an alteration in a gene (e.g., CDS1 , CDIPT, DGK, LPIN, or PLD). In other examples, the meganuclease, ZFN, and/or TALEN can be used to engineer an alteration in a gene (e.g., CDS1 , CDIPT, DGK, LPIN, or PLD). Exemplary alterations include insertions, deletions (e.g., knockouts), translocations, inversions, single point mutations, or other mutations. The alteration can be introduced in the gene in a cell, e.g., in vitro, ex vivo, or in vivo. In some embodiments, the alteration decreases the level and/or activity of (e.g., knocks down or knocks out) CDS1 , CDIPT, LPIN, or PLD, e.g., the alteration is a negative regulator of function. In some embodiments, the alteration increases the level and/or activity of (e.g., overexpression) DGK, e.g., the alteration is a positive regulator of function. In yet another example, the alteration corrects a defect (e.g., a mutation causing a defect), in CDS1 , CDIPT, DGK, LPIN, or PLD.

Alternative methods for disruption of a target DNA by site-specifical ly cleaving genomic DNA prior to the incorporation of a gene of interest in a cell include the use of meganucleases, ZFNs, and TALENs. Unlike the CRISPR/Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. The use of meganucleases, ZFNs, and TALENs in genome editing applications is described, e.g., in Urnov et al. Nature Reviews Genetics 11 :636 (2010); and in Joung et al. Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosure of both of which are incorporated herein by reference. In some embodiments, the endogenous CDS1 , CDIPT, LPIN, or PLD may be disrupted in the cells containing the CDS1 , CDIPT, LPIN, or PLD transgene using these gene editing techniques described herein.

In some embodiments, the PA to PI biosynthesis pathway inhibitor is a component of a nuclease- mediated gene regulation system, such as a nuclease or gRNA.

In some embodiments, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 3 contiguous nucleotides complementary to an equal-length portion of a CDS1 target nucleic acid (e.g., SEQ ID NO: 11 ).

In some embodiments, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 3 contiguous nucleotides complementary to an equal-length portion of a CDIPT target nucleic acid (e.g., SEQ ID NO: 13).

In some embodiments, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 3 contiguous nucleotides complementary to an equal-length portion of a DGK target nucleic acid (e.g., SEQ ID NOs: 15, 17, 19, 21 , 23, 25, 27, 29, or 31 ).

In some embodiments, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 3 contiguous nucleotides complementary to an equal-length portion of a LPIN target nucleic acid (e.g., SEQ ID NOs: SEQ ID NOs: 33, 35, or 37).

In some embodiments, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 3 contiguous nucleotides complementary to an equal-length portion of a PLD target nucleic acid (e.g., SEQ ID NOs: SEQ ID NOs: 39, 41 , 43, 45, 47, or 49). In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 6 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 8 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 10 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 12 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 14 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 16 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 18 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 20 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 22 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 24 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 26 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 28 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 30 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 40 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 50 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 60 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 70 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 80 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 90 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 100 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 110 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 120 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 130 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 140 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 150 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 160 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 170 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 180 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 190 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. In some embodiments of any of the foregoing aspects, the DNA-binding domain of the nuclease or the gRNA binds to a nucleotide sequence comprising at least 200 contiguous nucleotides complementary to an equal-length portion of a sequence selected from the group consisting of a CDS1 , CDIPT, DGK, LPIN, or a PLD target nucleic acid. Delivery

I. Viral vectors for expression of therapeutic PA to PI biosynthesis pathway inhibitors

Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell (e.g., neurons, glial cells, or non-neural cells, such as colon and kidney cells). Viral genomes are particularly useful vectors for gene delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors are a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses are: avian leukosis-sarcoma, avian C-type viruses, mammalian C- type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996)). Other examples are murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (US 5,801 ,030), the teachings of which are incorporated herein by reference.

IA. Retroviral vectors

The delivery vector used in the methods and compositions described herein may be a retroviral vector. One type of retroviral vector that may be used in the methods and compositions described herein is a lentiviral vector. Lentiviral vectors (LVs), a subset of retroviruses, transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the transgene. An overview of optimization strategies for packaging and transducing LVs is provided in Delenda, The Journal of Gene Medicine 6: S125 (2004), the disclosure of which is incorporated herein by reference.

The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the transgene of interest is accommodated. In particular, the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1 ) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of an heterologous nature; and (3) the transfer vector, consisting in the viral complimentary DNA (cDNA) deprived of all open reading frames, but maintaining the sequences required for replication, encapsidation, and expression, in which the sequences to be expressed are inserted. A LV used in the methods and compositions described herein may include one or more of a 5'- Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 -alpha promoter and 3'-self inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in US 6,136,597, the disclosure of which is incorporated herein by reference as it pertains to WPRE. The lentiviral vector may further include a pHR' backbone, which may include for example as provided below.

The Lentigen LV described in Lu et al., Journal of Gene Medicine 6:963 (2004) may be used to express the DNA molecules and/or transduce cells. A LV used in the methods and compositions described herein may a 5'-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 - alpha promoter and 3'-self inactivating L TR (SIN-LTR). It will be readily apparent to one skilled in the art that optionally one or more of these regions is substituted with another region performing a similar function.

Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency. The LV used in the methods and compositions described herein may include a nef sequence. The LV used in the methods and compositions described herein may include a cPPT sequence which enhances vector integration. The cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome. The introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells. The LV used in the methods and compositions described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to LV results in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein may include both a cPPT sequence and WPRE sequence. The vector may also include an IRES sequence that permits the expression of multiple polypeptides from a single promoter.

In addition to IRES sequences, other elements which permit expression of multiple polypeptides are useful. The vector used in the methods and compositions described herein may include multiple promoters that permit expression more than one polypeptide. The vector used in the methods and compositions described herein may include a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow expression of more than one polypeptide are described in Klump et al., Gene Ther. 8:811 (2001 ), Osborn et al., Molecular Therapy 12:569 (2005), Szymczak and Vignali, Expert Opin Biol Ther. 5:627 (2005), and Szymczak et al., Nat Biotechnol. 22:589 (2004), the disclosures of which are incorporated herein by reference as they pertain to protein cleavage sites that allow expression of more than one polypeptide. It will be readily apparent to one skilled in the art that other elements that permit expression of multiple polypeptides identified in the future are useful and may be utilized in the vectors suitable for use with the compositions and methods described herein. The vector used in the methods and compositions described herein may be a clinical grade vector.

IB. Adeno-associated viral vectors

Nucleic acids of the compositions and methods described herein may be incorporated into recombinant adeno-associated virus (rAAV) vectors and/or virions in order to facilitate their introduction into a cell (e.g., a neuron, glial cell, or non-neural cell, such as colon and kidney cells). Adeno-associated virus (AAV) vectors can be used in the central nervous system, and appropriate promoters and serotypes are discussed in Pignataro et al., J Neural Transm., 125: 575 (2018), the disclosure of which is incorporated herein by reference as it pertains to promoters and AAV serotypes useful in CNS gene therapy. rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs (e.g., nucleic acids capable of expression in neurons, glial cells, or non-neural cells, such as colon and kidney cells) that include (1 ) a heterologous sequence to be expressed and (2) viral sequences that facilitate integration and expression of the heterologous genes. The viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional inverted terminal repeat sequences (ITR)) of the DNA into a virion. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

The nucleic acids and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1 , VP2, and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for example, in US 5,173,414; US 5,139,941 ; US 5,863,541 ; US 5,869,305; US 6,057,152; and US 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery. rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 and rh74. For targeting cells located in or delivered to the central nervous system, AAV2, AAV9, and AAV10 may be particularly useful. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001 ); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001 ), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10, among others). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662 (2001 ); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001 ).

AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001 ).

II. Methods for the delivery of exogenous nucleic acids to target cells

Techniques that can be used to introduce a polynucleotide, such as DNA or RNA (e.g., mRNA, transfer RNA, siRNA, miRNA, shRNA, chemically modified RNA), including codon-optimized DNA or RNA, into a mammalian cell (e.g., neurons, glial cells, or non-neural cells, such as colon and kidney cells) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids (e.g., nucleic acids capable of expression in e.g., neurons, glial cells, or non-neural cells, such as colon and kidney cells). Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, NUCLEOFECTION™, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. NUCLEOFECTION™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.

An additional technique useful for the transfection of target cells is the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., Journal of Visualized Experiments 81 :e50980 (2013), the disclosure of which is incorporated herein by reference.

Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in US 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids are contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and DEAE-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1 :9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference.

Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.

Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107:25 1870 (2010), the disclosure of which is incorporated herein by reference.

MAGNETOFECTION™ can also be used to deliver nucleic acids to target cells. The principle of MAGNETOFECTION™ is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications. Their association with the gene vectors (DNA, siRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in US2010/0227406, the disclosure of which is incorporated herein by reference.

Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein. For example, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the sitespecific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

Pharmaceutical Compositions

The PA to PI biosynthesis pathway inhibitors (e.g., small molecules, and nucleic acid molecules or nucleases) described herein can be formulated into pharmaceutical compositions for administration to a patient, such as a human patient exhibiting or at risk of developing alpha-synuclein aggregation, in a biologically compatible form suitable for administration in vivo. A pharmaceutical composition containing, for example, a PA to PI biosynthesis pathway inhibitor described herein, such as an interfering RNA molecule, typically includes a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and a nucleic acid. The sterile saline is typically a pharmaceutical grade saline. A pharmaceutical composition may include (e.g., consist of), e.g., sterile water and a nucleic acid. The sterile water is typically a pharmaceutical grade water. A pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and a nucleic acid. The sterile PBS is typically a pharmaceutical grade PBS.

In certain embodiments, pharmaceutical compositions include one or more PA to PI biosynthesis pathway inhibitors and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, PA to PI biosynthesis pathway inhibitors may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions including a PA to PI biosynthesis pathway inhibitor encompass any pharmaceutically acceptable salts of the inhibitor, esters of the inhibitor, or salts of such esters. In certain embodiments, pharmaceutical compositions including a PA to PI biosynthesis pathway inhibitor, upon administration to a subject (e.g., a human), are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of inhibitors, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, prodrugs include one or more conjugate group attached to a PA to PI biosynthesis pathway inhibitor, wherein the conjugate group is cleaved by endogenous nucleases within the body.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue. In certain embodiments, pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, pharmaceutical compositions include a co-solvent system. Certain of such co-solvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intraocular (e.g., intravitreal), intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular, etc.). In certain of such embodiments, a pharmaceutical composition includes a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

Kits

The compositions described herein can be provided in a kit for use in treating a neurological disorder. The kit may include one or more PA to PI biosynthesis pathway inhibitors as described herein. The kit can include a package insert that instructs a user of the kit, such as a physician, to perform any one of the methods described herein. The kit may optionally include a syringe or other device for administering the composition. In some embodiments, the kit may include one or more additional therapeutic agents. Combination therapies

A PA to PI biosynthesis pathway inhibitor described herein can be administered in combination with a one or more additional therapeutic agents for treatment of neurological disorders. The one or more additional therapeutic agents may include a cognition-enhancing agent (e.g., donepezil, rivastigmine tartrate, galantamine HBr, memantine, and modafinil), an antidepressant agent (e.g., sertraline, fluoxetine, citalopram, escitalopram, paroxetine, and fluvoxamine), an anxiolytic agent (e.g., alprazolam, chlordiazepoxide, clobazepam, clonazepam, clorazepate, diazepam, estazolam, and flurazepam), an antipsychotic agent (e.g., aripiprazole, asenapine, cariprazine, clozapine, lurasidone, olanzapine, quetiapine, and risperidone), a sedative (e.g., alprazolam, chloral hydrate, chlordiazepoxide, clorazepate, clonazepam, diazepam, and estazolam), a dopamine promoter (e.g., selegiline, pramipexole and levodpa), or an anti-tremor agent (e.g., propranolol, primidone, gabapentin, and topiramate), or a combination thereof.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Example 1. CDP-Diacylglycerol Synthase 1 and CDP-DAG Inositol 3-Phosphatidyltransferase Inhibition Rescues Alpha-Synuclein-lnduced Cell Toxicity in Heterologous Cell Disease Models

Introduction

To assess the relevance of targets in ameliorating alpha-synuclein-induced toxicity, various disease-relevant cellular events were modeled in two heterologous cell models. Based upon the fact that the multiplication of genes that encodes alpha-synuclein causes familial Parkinson’s Disease (see, e.g., Singleton et al., Science, 302: 841 , 2003), we used overexpression of alpha-synuclein (a-Syn) in Human Bone Osteosarcoma Epithelial Cell (U2OS) cells to model alpha-synuclein-induced toxicity. In a second heterologous cell model, we took advantage of recent findings that mutations that shift alpha-synuclein equilibrium from proposed physiological tetramers to monomers initiate pathological processes. See, e.g., Dettmer et al., Nature Communications, 6: 1 , (2015). One of these mutations, the E46K familial SNCA mutation, has been amplified to include additional lysine (K) residues in two adjacent KTKEGV motifs (final construct designated as 3K) to further exacerbate the tetramer to monomer shift. This alpha- synulcein-3K variant drives the formation of inclusions composed of aggregated alpha-synuclein-bound to endo-lysosomal membrane and vesicle structures as observed by electron microscopy and are suggestive of an early Lewy-like pathology. See, e.g., Dettmer et al., Nature communications, 6: 1 , (2015); Dettmer et al., Human molecular genetics, 26: 3466, (2017). Furthermore, it has been shown that the inclusions formed by 3K overexpression can be inhibited or reversed by known modulators of alpha- synuclein toxicity, as well as inhibitors of stearoyl-CoA desaturase. See, e.g., Imberdis et al., Proceedings of the National Academy of Sciences, 116: 20760, (2019). Therefore, we used this assay in BE(2)-M17 (M17) cells, as a secondary heterologous system to allow the interrogation of whether a target of interest is affecting alpha-synuclein toxicity-related interaction with membranes.

Materials and Methods

Molecular Biology and Compound Sources

For survival assay, expression constructs for alpha-synuclein A53T and empty vector control were obtained from the Whitehead Institute (Massachusetts Institute of Technology, Cambridge, MA). For 3K aSyn assay in M17 cells, the synthesis of the vector encoding the human a-synuclein-3K-YFP was outsourced to GenScript USA Inc. (New Jersey, USA). The a-synuclein-3K-YFP sequence, provided and designed by Yumanity, was synthesized and inserted into the pLIX-402 backbone that had previously been provided to GenScript by Yumanity. “SMARTpool” short interfering RNAs (siRNAs) for CDP- Diacylglycerol Synthase 1 (CDS1 ), CDP-Diacylglycerol Synthase 2 (CDS2), and CDP-DAG inositol 3- phosphatidyltransferase (CDIPT) were purchased from GE Dharmacon.

Cell Culture

U2OS cells (Sigma-Aldrich) between passages 12 to 22 were cultured in McCoy’s 5A medium (ATCC) supplemented with 10% heat inactivated fetal bovine serum (Thermo Fisher).

The a-synuclein-3K M17D cell line Clone #2 was generated by transduction of naive M17D cells with lentiviral particles generated from the lentiviral vector pLIX402 with an insertion of the a-synuclein- 3K-yellow fluorescent protein (YFP) sequence. Limiting dilution cloning was performed to isolate the highest a-synuclein-3K-YFP expressing cells. Clone #2 was among the clones chosen for high target protein expression and was selected for development of the a-synuclein assays.

The naive M17D cell line was maintained in 1 :1 F-12:EMEM supplemented with 10% tetracycline- tested fetal bovine serum. The transformed M17D cell line was maintained in the same media as naive cells with the addition of puromycin selection. Cells were passaged weekly in tissue culture treated T75 or T150 flasks. Splitting cells for either assay purposes or propagation was achieved by dissociating cells with TrypLE and plating them in either normal maintenance media for assays (onto PDL-coated plates), or normal maintenance media with puromycin for culture maintenance (in tissue culture treated culture flasks). Cells were maintained in a 37° incubator with 5% CO2.

U2OS Cell Transfection

U2OS cell transfection was performed essentially as performed in PCT Publication No. WO 2018/129403A1 . Briefly, U2OS cells were trypsinized using 0.25% trypsin-EDTA (Thermo Fisher) for 5 min at 37°C followed by centrifugation at 800 rpm for 5 min at room temperature. Cell pellets were resuspended in SE solution (Lonza Biologies, Inc.) at a density of 1 x10⁴ cells/pL. Alpha-synuclein wild-type or aggregation-defective alpha synuclein (dNAC) plasmids were transfected at a ratio of 10 mg per 1 ,000,000 cells. siRNA control scrambled (SCR) siRNA, siRNA against CDS1 , CDS2, or siRNA against CDIPT were transfected at a ratio of 10 mg per 1 ,000,000 cells (SMARTpool siRNAs from Dharmacon, Lafayette, CO). NUCLEOFECTION™ was performed using 4D-NUCLEOFECTOR™ System (Lonza Biosciences, Inc.) under program code CM130 in either 20 pL Nucleocuvette™ strips or 100 pl single Nucleocuvettes™. Cells recovered at room temperature for 10-15 minutes after NUCLEOFECTION™ before further handling. Pre-warmed medium was added, and cells were thoroughly but gently mixed to a homogenous suspension before plating. Cells were seeded at 2x10⁴ cells/100 pl/well into 96 well PLD- coated white plates (Corning, Inc.) using a customized semi-automated pipetting program (VIAFLO 384/96, Integra Biosciences).

U2OS ATP Assay

The U2OS ATP assay was performed essentially as performed in PCT Publication No. WO 2018/129403A1 . Briefly, plates were gently rocked, and plates were incubated for 72 h after transfection. Plates were sealed with MicroClime® lids (Labcyte Inc.) to reduce evaporation and variability. ATP content was then measured using the CellTiter-Glo® kit (Promega) with luminescence signals measured on an EnVision multimode plate reader (Perkin Elmer).

RNA Purification and Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) RT-PCR was performed essentially as performed in PCT Publication No. WO 2018/129403A1 . Briefly, U2OS and M17 cells were rinsed with ice-cold PBS (pH 7.4). Total RNA was purified using an RNEasy® Mini Kit following the manufacturer’s instructions (Qiagen). Reverse transcription was performed with 150 ng RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher) in a MASTERCYCLER® Pro thermal cycler (Eppendorf). Real-time PCR analyses of 2 pL cDNA products in a total reaction volume of 20 pL were carried out in duplicates using TaqMan® Fast Advanced Master Mix in a StepOnePlus™ Real-Time PCR System (Thermo Fisher). The primer pairs and probes for real-time amplification of CDS1 , CDIPT, and CDS2 were predesigned TaqMan® gene expression assays (Applied Biosystems Hs00181633_m1 , Hs00197004_m1 , and Hs00197004_m1 , respectively). Human beta-actin was used as an endogenous housekeeping control (Applied Biosystems #4310881 E). The relative quantity of gene transcript abundance was calculated using the AACt method.

3K alpha-synuclein-YFP Inclusion Assay

24 hours after alpha-synuclein-3K-YFP expression induction, cells were stained with nuclear and plasma membrane markers followed by fluorescence imaging of the alpha-synuclein-3K inclusions. Images were acquired on the Operetta CLS high-content analysis system (Perkin Elmer, Waltham, MA). The “Experiment Protocol” designed for image acquisition was “BC_BioCoat PDL 20Xair BF 405 488 647 M17D 96w”. Images were acquired in brightfield, Hoechst 33342, AlexaFluor 488 and AlexaFluor 647 channels. Acquired images were analyzed with the Harmony software using Image Analysis protocol “BC 3K analysis_M17D 20Xair”. Briefly, the image analysis protocol detected and measured the number of nuclei as well as their intensity and morphological properties using the 405 nm channel, performed cell segmentation using the 647 nm channel, and detected and measured the number of spots as well as their intensity and morphological properties using the 488 nm channel.

Results

To investigate the cellular events related to alpha-synuclein-induced toxicity, an assay was developed to measure the effects of alpha-synuclein expression on cellular ATP content in transfected U2OS cells, which is a general proxy for cell health and viability. U2OS cells transfected with alpha- synuclein exhibited a significant reduction in cellular ATP levels relative to cells transfected with the empty vector control. To assess the relevance of targets in ameliorating alpha-synuclein-induced toxicity, U2OS were co-transfected with control scrambled siRNA or 2 nM, 10 nM or 50 nM of siRNA against CDS1 (siCDSI ) or CDIPT (siCDIPT), which are the enzymes that, respectively, regulate the amount of PI available for signaling by catalyzing the conversion of PA to CDP-DAG and catalyze the conversion of CDP-DAG to PI. Co-transfection with siCDSI and alpha-synuclein in U2OS cells revealed that cellular ATP levels were significantly higher in the 2 nM and 10 nM siCDSI groups, as compared to the control scrambled siRNA group (FIG. 1 A). Co-transfection with siCDIPT and alpha-synuclein in U2OS cells revealed that cellular ATP levels were significantly higher in the 10 nM and 50 nM siCDIPT groups, as compared to the control scrambled siRNA group (FIG. 1 B). siRNA mediated CDS1 or CDIPT transcript knockdown were confirmed by significantly reduced mRNA levels of CDS1 or CDIPT, respectively in all siCDSI and siCDIPT dosage groups, as compared to the control scrambled siRNA group (FIGs. 2A and 2B). This rescue of ATP levels demonstrates that knocking down CDS1 or CDIPT ameliorated alpha- synuclein-induced toxicity in the heterologous U2OS cell model.

To investigate the ability of CDS1 , CDIPT, and CDS2 knockdown to reduce cellular events related to alpha-synuclein-induced toxicity in a second heterologous cell model, we developed a stably transformed alpha-synuclein-reporter cell-based model in M17 cells. To test whether perturbing the transcript level of CDS1 , CDIPT, or CDS2 resulted in the decrease of alpha-synuclein inclusions, M17 cells were transfected with control scrambled siRNA or 2.5 nM, 10 nM, 25 nM or 50 nM of siCDSI siCDIPT, or siCDS2, respectively. CDS1 and CDIPT knockdown significantly reduced alpha-synuclein inclusions, as measured by relative spot intensity of the alpha-synuclein reporter (FIGs. 3A and 3B). siRNA mediated CDS1 and CDIPT transcript knockdown, respectively, was confirmed by significantly reduced mRNA levels of CDS1 or CDIPT in all siCDSI and siCDIPT dosage groups, as compared to the control scrambled siRNA group (FIGs. 4A and 4B). Taken together, these results demonstrate that CDS1 and CDIPT knockdown ameliorated aberrant interactions of alpha-synuclein with the membrane in the heterologous M17 cell model.

Example 2. CDS1 and CDIPT Inhibition Rescues Alpha-Synuclein-Dependent Cell Toxicity in Neu ons

Introduction

To assess the relevance of targets in ameliorating alpha-synuclein-induced toxicity, diseaserelevant cellular events were modeled in neurons. Transient transfection of alpha-synuclein is an effective way to acutely increase alpha-synuclein protein expression to model its toxicity in iPSC-derived human neurons.

Materials and Methods

Molecular Biology and Compound Sources

GABAergic cortical neurons (iCELLs) were purchased from Fujifilm Cellular Dynamics.

Expression constructs for alpha-synuclein wild-type and A53T were obtained from the Whitehead Institute (Massachusetts Institute of Technology, Cambridge, MA). The pSF-CAG plasmid was obtained from Oxford Genetics (Oxford, UK). The red fluorescent protein (RFP) reporter plasmid, pSF-MAP2-mApple, was constructed by replacing the CAG promoter with human MAP2 promoter sequence and inserting mApple coding sequence into the multiple cloning site. siRNA for CDS1 or CDIPT were purchased from ThermoFisher Ambion.

Cell Culture

Cell culture was performed essentially as performed in PCT Publication No. WO 2018/129403A1 . Briefly, iCELLs were plated onto a 12-well plate with an approximate density of 80,000 neurons per well. 7 days after plating, iCELLs were co-transfected with a fluorescence reporter plasmid (encoding mApple) and empty or alpha-synuclein-A53T overexpression plasmids together with control scrambled siRNA, siRNA against CDS1 , or siRNA against CDIPT by lipofection at 7 div (days in vitro).

RNA Purification and Quantitative Reverse Transcription-Polymerase Chain Reaction

Transient transfection of siRNA into human neurons cultured in 12-well plate at DIV (days in vitro) 7 was performed using NeuroMAG (Oz Biosciences, Cat# NM51000). For one well of 12-well plate, 5ul of siRNA (CDS1 , CDS2, CDIPT or negative control, 20uM) was added into 195ul of Neurobasal media (Gibco, Cat #21103049). In the separate tube, 5ul of NeuroMAG was diluted in the 195ul of Neurobasal media. The siRNA/Neurobasal media solution was added into NeuroMAG/Neurobasal media solution. This mixture was incubated at room temperature for 40 minutes and then added onto neurons in a drop- by-drop manner. The culture plate was placed on top of magnetic plate (OZ Biosciences, Cat# MF10000) in the CO2 incubator. After 20 minutes, the magnetic plate was removed and the 12-well culture plate was kept in the CO2 incubator for 2 days. At DIV9, neurons were lysed for RNA extraction and qRT-PCR was performed to measure the mRNA level of target genes.

Transfected iCELLs were rinsed with ice-cold PBS (pH 7.4). Total RNA was purified using an RNEasy® Mini Kit following the manufacturer’s instructions (Qiagen). Reverse transcription was performed with 150 ng RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher) in a MASTERCYCLER® Pro thermal cycler (Eppendorf). Real-time PCR analyses of 2 pL cDNA products in a total reaction volume of 20 pL were carried out in duplicates using TaqMan® Fast Advanced Master Mix in a StepOnePlus™ Real-Time PCR System (Thermo Fisher). The primer pairs and probes for real-time amplification of CDS1 , CDIPT, and CDS2 were predesigned TaqMan® gene expression assays (Applied Biosystems Hs00181633_m1 , Hs00197004_m1 , and Hs00197004_m1 , respectively). Human beta-actin was used as an endogenous housekeeping control (Applied Biosystems #4310881 E). The relative quantity of gene transcript abundance was calculated using the AACt method.

Neuron Survival Assay

Transient transfection of plasmids and siRNA into human neurons at DIV (days in vitro) 7 was performed using NeuroMAG (Oz Biosciences, Cat# NM51000). For one well of 96-well plate, red fluorescence tracer plasmid (pSF-MAP2-mApple, 100ng) and empty or a-synuclein-A53T mutant plasmid (50ng) were diluted in the Neurobasal media (Gibco, Cat #21103049) as the total amount of 20ul. Then siRNA (CDS1 , CDS2, CDIPT or negative control, final 50 nM concentration) was added into this plasmids/Neurobasal media solution. In the separate tube, 0.5ul of NeuroMAG was diluted in the 19.5ul of

Neurobasal media. The DNA & siRNA/Neurobasal media solution described above was added into NeuroMAG/Neurobasal media solution. This mixture was incubated at room temperature for 40 minutes and then added onto neurons in a drop-by-drop manner. The culture plate was placed on top oi 96-weil magnetic plate (OZ Biosciences, Cat# MFI 0096) in the CO2 incubator and incubated for 20 minutes. Then the magnetic plate was removed and the culture plate was inserted into BioStation CT (Nikon) tor automated imaging. Neurons which express red fluorescence were automatically imaged every 12 hours for 2 weeks and the images were analyzed by CL-Quant software with automated algorithms developed by Nikon. Individual neurons were tracked by monitoring red fluorescence with the algorithms and the risk of neuron death was determined over time by determining neuronal death based on the loss of red fluorescent tracer signal. Cox proportional hazard analysis was then used to estimate relative risk of death, or hazard ratio (HR) and to generate cumulative hazard plots. Log-rank test was used to determine statistical significance of survival curve divergence between neuron cohorts.

Results

To evaluate the effects of CDS1 or CDIPT inhibition on alpha-synuclein-induced toxicity in human neurons, iCELLs harboring the alpha-synuclein A53T mutation or an isogenic control line in which the A53T mutation was corrected to wild-type, were trans-differentiated into neurons, and cell survival was monitored over the course of 14 d. iCELLs were transfected with control scrambled siRNA, siCDSI , siRNA against the closely related CDS enzyme, CDS2 (siCDS2), or CDIPT (siCDIPT). Quantitative RT- PCR was performed to measure mRNA levels of CDS1 , CDS2 and CDIPT. siCDSI , siCDS2, and siCDIPT reduced the mRNA levels of the target genes compared to the scrambled siRNA control (FIG. 5).

Analysis of cumulative single cell survival data indicated that the risk of neuron death was significantly reduced by treatment with siCDSI or siCDIPT (FIGs. 6 and 7) relative to scrambled siRNA controls in isogenic control neurons. Interestingly, siRNA against CDS2, did not produce the same cell survival rescue effect, suggesting that the amelioration of toxicity of alpha-synuclein A53T on cell viability was CDS1 inhibition-specific. siCDSI showed more consistent and robust protection compared to siCDIPT (FIG. 7). Taken together, these data demonstrate that CDS1 or CDIPT inhibition rescues cell death associated with alpha-synuclein-induced toxicity in a relevant disease model, providing further evidence that phosphatidic acid (PA) to phosphoinositide (PI) biosynthesis pathway inhibition as a therapeutic approach for reducing alpha-synuclein-induced toxicity and treating a neurological disorder including Alzheimer’s disease and Parkinson’s disease.

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

Claims

What is claimed is:

1 . A method of reducing alpha-synuclein-induced toxicity in a subject, the method comprising administering an effective amount of a phosphatidic acid (PA) to phosphoinositide (PI) biosynthesis pathway inhibitor to the subject.

2. A method of treating a neurological disorder in a subject, the method comprising administering an effective amount of a PA to PI biosynthesis pathway inhibitor to the subject.

3. A method of suppressing toxicity in a cell related to protein misfolding and/or aggregation in a subject, the method comprising contacting a cell with a PA to PI biosynthesis pathway inhibitor.

4. The method of any one of claims 1 -3, wherein the PA to PI biosynthesis pathway inhibitor is selected from the group consisting of a CDP-Diacylglycerol Synthase 1 (CDS1 ) inhibitor, a CDP-DAG inositol 3-phosphatidyltransferase (CDIPT) inhibitor, a diacylglycerol kinase (DGK) activator, a phosphatidate phosphatase lipin (LPIN) inhibitor, and a phospholipase D (PLD) inhibitor.

5. The method of claim 4, wherein the PA to PI biosynthesis pathway inhibitor is a CDS1 inhibitor or a CDIPT inhibitor.

6. The method of claim 5, wherein the PA to PI biosynthesis pathway inhibitor is a CDS1 inhibitor.

7. The method of claim 5, wherein the PA to PI biosynthesis pathway inhibitor is a CDIPT inhibitor.

8. The method of any one of claims 1 -7, wherein the PA to PI biosynthesis pathway inhibitor is selected from the group consisting of a nucleic acid molecule and a nuclease.

9. The method of claim 8, wherein the nucleic acid molecule is selected from the group consisting of small interfering RNA, short hairpin RNA, micro RNA, antisense oligonucleotide, and guide RNA.

10. The method of claim 8 or 9, wherein the nucleic acid molecule is complementary to a portion of a full-length CDS1 , CDIPT, DGK, LPIN, or PLD nucleic acid.

11 . The method of claim 10, wherein the nucleic acid molecule is a CDS1 inhibitor, and the nucleic acid molecule is complementary to a portion of SEQ ID NO: 11 .

12. The method of claim 10, wherein the nucleic acid molecule is a CDIPT inhibitor, and the nucleic acid molecule is complementary to a portion of SEQ ID NO: 13.

13. The method of claim 10, wherein the nucleic acid molecule is a DGK activator, and the nucleic acid molecule is complementary to a portion of SEQ ID NOs: 15, 17, 19, 21 , 23, 25, 27, 29, or 31 .

43

14. The method of claim 10, wherein the nucleic acid molecule is a LPIN inhibitor, and the nucleic acid molecule is complementary to a portion of SEQ ID NOs: 35, 37, or 39.

15. The method of claim 10, wherein the nucleic acid molecule is a PLD inhibitor, and the nucleic acid molecule is complementary to a portion of SEQ ID NOs: 41 , 43, 45, 47, 49, or 51 .

16. The method of claim 8, wherein the nuclease is selected from the group consisting of a meganuclease, a transcription activator-like effector nuclease, a zinc-finger nuclease, a CRISPR associated protein 9, and a CRISPR-associated protein 12a.

17. The method of claim 16, wherein the nuclease disrupts endogenous CDS1 , CDIPT, LPIN, or PLD in cells of the subject.

18. The method of any one of claims 1 -17, wherein the PA to PI biosynthesis pathway inhibitor is administered to cells of the subject by transduction with a viral vector.

19. The method of claim 18, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpes virus, a poxvirus, and a Retroviridae family virus.

20. The method of any one of claims 1 -19, wherein the PA to PI biosynthesis pathway inhibitor is administered to the subject by way of systemic administration, by way of direct administration to the central nervous system of the subject.

21 . The method of any one of claims 1 -20, wherein cells of the subject are transfected or transduced ex vivo to express the PA to PI biosynthesis pathway inhibitor.

22. The method of any one of claims 1 -21 , wherein cells of the subject are transfected with a PA to PI biosynthesis pathway inhibitor using: a) an agent selected from the group consisting of a cationic polymer, diethylaminoethyldextran, polyethylenimine, a cationic lipid, a liposome, calcium phosphate, an activated dendrimer, and a magnetic bead; or b) a technique selected from the group consisting of electroporation, NUCLEOFECTION™, squeeze-poration, sonoporation, optical transfection, MAGNETOFECTION ™, and impalefection.

23. The method of any one of claims 1 and 3-22, wherein the toxicity is related to misfolding and/or aggregation of a protein.

24. The method of any one of claims 1 and 3-23, wherein the toxicity is related to misfolding and/or aggregation of alpha-synuclein.

25. The method of any one of claims 3-24, wherein cells of the subject are neural cells.

44

26. The method of claim 25, wherein the neural cells are neurons or glial cells.

27. The method of any one of claims 3-26, wherein cells of the subject are non-neural cells.

28. The method of any one of claims 1 -15 and 18-27, wherein the PA to PI biosynthesis pathway inhibitor is a nucleic acid molecule comprising a region having at least 80% sequence identity to any one of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10.

29. The method of claim 1 or 3, wherein the subject is suffering from a neurological disorder.

30. The method of any one of claims 2 and 4-29, wherein the neurological disorder is Alzheimer’s disease (AD), mild cognitive impairment (MCI), cerebral amyloid angiopathy, dementia associated with Down syndrome, or other neurodegenerative diseases characterized by the formation or accumulation of amyloid plaques comprising Ap42.

31 . The method of any one of claims 2 and 4-30, wherein the neurological disorder is AD, Parkinson's disease (PD), dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease, Alpers’ disease, Leigh's disease, Pelizaeus-Merzbacher disease, Olivopontocerebellar atrophy, Friedreich's ataxia, leukodystrophies, Rett syndrome, Ramsay Hunt syndrome type II, Down's syndrome, multiple sclerosis, and MCI.

32. The method of any one of claims 2 and 4-31 , wherein the neurological disorder is a proteinopathy.

33. The method of claim 32, wherein the proteinopathy is a synucleinopathy.

34. The method of claim 33, wherein the synucleinopathy is PD, dementia with Lewy bodies, pure autonomic failure, multiple system atrophy, incidental Lewy body disease, pantothenate kinase- associated neurodegeneration, AD, Down's Syndrome, Gaucher disease, or the Parkinsonism-dementia complex of Guam.

35. The method of claim 32, wherein the proteinopathy is AD, Alexander disease, ALS, a prion disease, Huntington’s disease, Machado-Joseph disease, Pick's disease, or frontotemporal dementia.

36. The method of claim 35, wherein the prion disease is Creutzfeldt-Jakob disease.

37. The method of any one of claims 2 and 4-36, wherein the neurological disorder is a neurodegenerative disorder.

38. The method of claim 37, wherein the neurodegenerative disorder is Alpers’ disease, ataxia telangectsia, Canavan disease, Cockayne syndrome, corticobasal degeneration, Kennedy’s disease, Krabbe disease, Pelizaeus-Merzbacher disease, primary lateral sclerosis, Refsum’s disease, Sandhoff

45 disease, Schilder' s disease, Steele-Richardson-Olszewski disease, tabes dorsalis, vascular dementia, or Guillain-Barre Syndrome.

39. The method of any one of claims 2 and 4-38, wherein the neurological disorder is an Apolipoprotein E4 (ApoE)-associated neurodegenerative disorder.

40. The method of claim 39, wherein the ApoE-associated neurodegenerative disorder is AD, vascular cognitive impairment, cerebral amyloid angiopathy, traumatic brain injury, or multiple sclerosis.

41 . The method of claim 39 or 40, wherein the ApoE-associated disorder is AD.

42. The method of any one of claims 1 -41 , wherein the subject has an elevated level, or is predicted to have an elevated level of alpha-synuclein, ApoE4, or an undesired form thereof.

43. The method of claim 42, wherein the subject is predicted to have an elevated level of alpha- synuclein, ApoE4, and/or an undesired form thereof based on genetic markers.

44. The method of any one of claims 1 -43, wherein the method further comprises administering an additional therapeutic agent to the subject.

45. The method of claim 44, wherein the additional therapeutic agent is a small molecule, an antibody or fragment thereof, or a nucleic acid.

46. The method of claim 44 or 45, wherein the additional therapeutic agent is a cognitionenhancing agent, an antidepressant agent, an anxiolytic agent, an antipsychotic agent, a sedative, a dopamine promoter, or an anti-tremor agent.