PPT1 gene therapy

Abstract

The present invention is characterized by PPT1 polypeptides and coding nucleic acid constructs. The use of the polypeptides and coding nucleic acid constructs includes producing PPT1 polypeptides, increasing PPT1 activity in subjects, and treating PPT1-related disorders such as CLN1 disease in subjects.

Description

[Background technology]

[0001]

[0003] Palmitoyl protein thioesterase 1 (PPT1) is a glycoprotein involved in the removal of thioester-linked fatty acid acyl groups, such as palmitate, from proteins. Full-length PPT1 contains 306 amino acids and a 27-amino acid signal sequence. Removal of the signal sequence results in mature PPT1 (Bellizzi et al. (2000) PNAS 97:9 pp. 4573-4578).

[0002]

[0004] PPT1 is found in lysosomes and plays a role in lipid-modified protein catabolism reactions within lysosomes. PPT1 is also found in other locations, such as synaptosomes, synaptic vesicles, blood, and cerebrospinal fluid (CSF). PPT1 is encoded by the CLN1 gene.

[0003]

[0005] Neuronal ceroid lipofuscinosis 1 (NCL1) is a progressive neurodegenerative disease caused by reduced PPT1 activity. NCL1 can be caused by mutations that result in loss or reduced PPT1 activity or expression, leading to a deficiency of lysosomal PPT1 activity. As the disease progresses, symptoms may include epilepsy, seizures, motor and cognitive decline, visual impairment, behavioral disturbances, sleep disturbances, and death. NCL1 is also known as CLN1, Batten disease, or childhood neuronal ceroid lipofuscinosis (INCL), and symptoms typically begin around 12–18 months of age. In some cases, mutations in PPT1 can lead to a later onset of symptoms, such as late childhood (around 2–4 years), juvenile, or adult onset. NCL1 pathology can occur in various locations, including the brain and spinal cord (Gorenberg et al. (2022) PLos Biol. 20(3):e3001590; Simonati and Williams (2022) Front. Neurol. Mar 11;13:811686; Shyng et al. (2017) PNAS 114(29)E5920~E5929; and Bellizzi et al. (2000) PNAS 97(9):4573~4578).

[0004]

[0006] References mentioning potential enzyme replacement and gene therapy treatments for NCL1 include Nelvagal et al. (2022) J. Clin. Invest. 132(20); e163107; Griffey et al. (2004) Neurobiology of Disease 16: pp. 360-369; Griffey et al. (2006) Molecular Therapy 13(3): pp. 538-547; Shyng et al. (2017) PNAS 114(29) E5920-E5929; International Patent Publication No. 2017 / 219450; and International Patent Publication No. 2020 / 223322. [Overview of the Initiative]

[0005]

[0007] The present invention is characterized by PPT1 polypeptides and coding nucleic acid constructs. The PPT1 polypeptides described herein include polypeptides comprising a PPT1 amino acid sequence, and in different embodiments further comprising a signal sequence, wherein either or both of the mature PPT1 or signal sequence are modified from sequences present in full-length naturally occurring human PPT1. The PPT1 coding constructs include nucleic acid sequences encoding a PPT1 polypeptide having one or more differences from naturally occurring human PPT1 mature or full-length sequences, and / or CpG-reduced sequences. The use of polypeptides and coding nucleic acid constructs includes producing PPT1 polypeptides; increasing PPT1 activity in subjects; and treating PPT1-related disorders such as CLN1 disease in subjects.

[0006]

[0008] A reference to the PPT1 polypeptide indicates the presence of a sequence related to the maturation sequence, and includes either a full-length sequence containing a signal sequence or a mature sequence without a signal sequence, or both. The mature sequence may vary in size depending on the signal sequence and may be subject to further processing. The PPT1 polypeptide sequence may be naturally occurring or a modification of a naturally occurring sequence. A reference to the full-length, naturally occurring human PPT1 is given by Sequence ID No. 29. The mature, naturally occurring human PPT1 sequence is provided by Sequence ID No. 1.

[0007]

[0009] Therefore, a first aspect of the present invention is a polynucleotide comprising a nucleic acid sequence encoding a palmitoyl protein thioesterase-1 (PPT1) polypeptide, wherein the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1. (a) The PPT1 polypeptide further comprises a variant thereof having any of the signal sequences from SEQ ID NOs. 16 to 27 or one amino acid substitution, deletion, or insertion; and / or (b) The PPT1 amino acid sequence includes a glycine (G), valine (V), or leucine (L) substitution of aspartic acid (D) at its amino terminus; and / or (c) The PPT1 sequence contains the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus; and / or (d) The nucleic acid sequence includes a PPT1 coding sequence that has at least 85% identity with any of sequence numbers 61 to 94. List the polynucleotides.

[0008]

[0010] The signal sequences in sequence numbers 16-27 provide signal sequences that are not present in naturally occurring full-length human PPT1.

[0009]

[0011] The reference to glycine (G), valine (V), or leucine (L) substitutions of aspartic acid (D) at its amino terminus indicates that glycine (G), valine (V), or leucine (L) is present at the location corresponding to aspartic acid (D) in the native mature PPT1 sequence (SEQ ID NO: 1).

[0010]

[0012] Another aspect of the present invention is a PPT1 polypeptide comprising a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, (a) The PPT1 polypeptide further comprises a variant thereof having any of the signal sequences from SEQ ID NOs. 16 to 27 or one amino acid substitution, deletion, or insertion; and / or (b) The PPT1 amino acid sequence includes a glycine (G), valine (V), or leucine (L) substitution of aspartic acid (D) at its amino terminus; and / or (c) The PPT1 sequence contains the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus. The PPT1 polypeptide is described.

[0011]

[0013] Another aspect of the present invention relates to an expression cassette comprising a nucleic acid sequence encoding a PPT1 polypeptide and one or more expression regulatory elements operably coupled to the encoding nucleic acid sequence.

[0012]

[0014] A reference to one or more regulatory elements "operably ligated" or "operably coupled" to the nucleic acid encoding the PPT1 polypeptide indicates that the regulatory element(s) affect PPT1 polypeptide expression. PPT1 polypeptide expression can be affected in various ways, including increased production of PPT1 polypeptide mRNA transcripts, increased nuclear transport and stability of mRNA transcripts, and increased mRNA translation.

[0013]

[0015] Another aspect of the present invention relates to a recombinant viral vector nucleic acid comprising (a) an expression cassette comprising a nucleic acid sequence encoding a PPT1 polypeptide and one or more expression control elements operably linked to the encoding nucleic acid sequence, and (b) 5' and / or 3' viral elements resulting in viral packaging and / or replication.

[0014]

[0016] Another aspect of the present invention relates to a delivery vehicle comprising a virus or non-viral vector and a polynucleotide, expression cassette or recombinant viral vector nucleic acid comprising a sequence encoding (a) a PPT1 polypeptide or (b) a PPT1 polypeptide.

[0015]

[0017] Another aspect of the present invention relates to a pharmaceutical composition comprising (a) a PPT1 polypeptide; (b) a polynucleotide, expression cassette, or recombinant viral vector nucleic acid containing a sequence encoding the PPT1 polypeptide; or (c) a delivery vehicle containing (a) or (b); and a pharmaceutically acceptable carrier.

[0016]

[0018] Another aspect of the present invention relates to a method for increasing PPT1 activity in a subject, treating a PPT1 disease or disorder, or treating CLN1, comprising the administration of a pharmaceutical composition comprising (a) a PPT1 polypeptide; (b) a polynucleotide, expression cassette, or recombinant viral vector nucleic acid containing a sequence encoding the PPT1 polypeptide; (c) a delivery vehicle comprising (a) or (b); or (d) (a), (b), or (c) and a pharmaceutically acceptable carrier.

[0017]

[0019] Additional aspects of the present invention include (a) a PPT1 polypeptide; (b) a polynucleotide, expression cassette, or recombinant viral vector nucleic acid comprising a sequence encoding the PPT1 polypeptide; (c) a delivery vehicle comprising (a) or (b); or (d) a pharmaceutical composition comprising (a), (b), or (c) and a pharmaceutically acceptable carrier; (a), (b), (c), or (d) for use in medicine, increasing PPT1 activity in a subject, treating PPT1 disease or (of) disorder, or treating CLN1; and the use of (a), (b), (c), or (d) in the preparation of pharmaceuticals (for example, for use in medicine, increasing PPT1 activity in a subject, treating PPT1-related disorder, or treating CLN1).

[0018]

[0020] Additional aspects of the present invention include a vector genome plasmid containing recombinant viral vector nucleic acid encoding a PPT1 polypeptide, a method for producing recombinant viral vector nucleic acid encoding a PPT1 polypeptide, and a method for obtaining a PPT1 polypeptide.

[0019]

[0021] Other features and advantages of the present invention will become apparent from the additional descriptions provided herein, including various embodiments. The provided embodiments illustrate different components and methodologies useful in carrying out the present invention. Such embodiments do not limit the claimed invention. Based on this disclosure, those skilled in the art can identify and utilize other components and methodologies useful in carrying out the present invention. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 is a schematic diagram of an rAAV PPT1 expression construct (or cassette) containing various nucleic acid regions: 5'-inverted terminal repeat (ITR), elongation factor-1 alpha (EF-1α) promoter, Kozak sequence (Kozak), signal sequence (SS, also called signal peptide), mature PPT1 sequence (human PPT1), bovine growth hormone polyadenylated sequence (bGH-pA), stuffer (stuffer sequence), synthetic polyadenylated sequence (synthetic pA), and 3'-ITR. [Figure 2A] Figures 2A and 2B are bar graphs illustrating the PPT1 enzyme activity levels in PPT1 knockout HeLa cells transfected with rAAV plasmids containing various manipulated human PPT1 constructs. PPT1 activity is expressed as a percentage of the PPT1 activity obtained from PPT1 with its native signal peptide in total cell lysate (Figure 2A) and cell culture supernatant (secreted PPT1) (Figure 2B). The reference to "Dead" indicates mutant PPT1 lacking enzyme activity. [Figure 2B] Figures 2A and 2B are bar graphs illustrating the PPT1 enzyme activity levels in PPT1 knockout HeLa cells transfected with rAAV plasmids containing various manipulated human PPT1 constructs. PPT1 activity is expressed as a percentage of the PPT1 activity obtained from PPT1 with its native signal peptide in total cell lysate (Figure 2A) and cell culture supernatant (secreted PPT1) (Figure 2B). The reference to "Dead" indicates mutant PPT1 lacking enzyme activity. [Figure 3A] Figures 3A–3C illustrate the results of using the rAAV PPT1 nucleic acid / tdTomato reporter plasmid and the plasmid to evaluate PPT1 secretion from transfected cells. Figure 3A shows various rAAV nucleic acid and tdTomato regions. Figure 3B provides a bar graph showing the ratio of the number of cells with PPT1 to the number of cells transfected with the PPT1 / tdTomato reporter plasmid. Figure 3C provides a bar graph showing the results of Figure 3B normalized to the native construct. For both Figures 3B and 3C, each point (transparent circle; mean of 30 fields of view) is an independent transfection (obtained from 2–3 experiments), and the value is mean ± SD. P values ​​by one-way ANOVA: ****p<0.0001, ***p<0.001, **p<0.01. [Figure 3B]Figures 3A–3C illustrate the results of using the rAAV PPT1 nucleic acid / tdTomato reporter plasmid and the plasmid to evaluate PPT1 secretion from transfected cells. Figure 3A shows various rAAV nucleic acid and tdTomato regions. Figure 3B provides a bar graph showing the ratio of the number of cells with PPT1 to the number of cells transfected with the PPT1 / tdTomato reporter plasmid. Figure 3C provides a bar graph showing the results of Figure 3B normalized to the native construct. For both Figures 3B and 3C, each point (transparent circle; mean of 30 fields of view) is an independent transfection (obtained from 2–3 experiments), and the value is mean ± SD. P values ​​by one-way ANOVA: ****p<0.0001, ***p<0.001, **p<0.01. [Figure 3C] Figures 3A–3C illustrate the results of using the rAAV PPT1 nucleic acid / tdTomato reporter plasmid and the plasmid to evaluate PPT1 secretion from transfected cells. Figure 3A shows various rAAV nucleic acid and tdTomato regions. Figure 3B provides a bar graph showing the ratio of the number of cells with PPT1 to the number of cells transfected with the PPT1 / tdTomato reporter plasmid. Figure 3C provides a bar graph showing the results of Figure 3B normalized to the native construct. For both Figures 3B and 3C, each point (transparent circle; mean of 30 fields of view) is an independent transfection (obtained from 2–3 experiments), and the value is mean ± SD. P values ​​by one-way ANOVA: ****p<0.0001, ***p<0.001, **p<0.01. [Figure 4]Figure 4 illustrates the dose- and time-dependent increase in PPT1 activity detected in culture medium of rat primary cortical neurons transduced with rAAV containing Sp7-F.PPT1. Cultured neurons were transduced at three different MOIs (low, 1E+5; medium, 5E+5; and high, 1E+6), and PPT1 activity in culture medium was analyzed at 3, 4, or 6 days post-transduction. "Dead" PPT1 refers to catalytically inactive PPT1. Untreated or diluent-treated cells were used as negative controls. Purified recombinant PPT1 was used as a positive control for the assay. Each circle represents a value obtained from independently transduced cells in a single well. Data are mean ± SD. [Figure 5A] Figures 5A, 5B, and 5C illustrate serum PPT1 expression and activity at various time points obtained from mice administered rAAV containing Sp7-F.PPT1 viral vector nucleic acid. Figure 5A illustrates the production of glycosylated PPT1 activity in the serum of intravenously (IV) administered mice. Figure 5B shows the serum PPT1 activity of IV-administered mice. Figure 5C shows the serum activity of IPA-administered mice (delivered intraparenchymally to the hippocampus by stereotactic injection). ND indicates not detected. Data are mean ± SD. P values ​​by ANOVA: *P<0.05, ***P<0.001, ****P<0.0001. [Figure 5B] Figures 5A, 5B, and 5C illustrate serum PPT1 expression and activity at various time points obtained from mice administered rAAV containing Sp7-F.PPT1 viral vector nucleic acid. Figure 5A illustrates the production of glycosylated PPT1 activity in the serum of intravenously (IV) administered mice. Figure 5B shows the serum PPT1 activity of IV-administered mice. Figure 5C shows the serum activity of IPA-administered mice (delivered intraparenchymally to the hippocampus by stereotactic injection). ND indicates not detected. Data are mean ± SD. P values ​​by ANOVA: *P<0.05, ***P<0.001, ****P<0.0001. [Figure 5C]Figures 5A, 5B, and 5C illustrate serum PPT1 expression and activity at various time points obtained from mice administered rAAV containing Sp7-F.PPT1 viral vector nucleic acid. Figure 5A illustrates the production of glycosylated PPT1 activity in the serum of intravenously (IV) administered mice. Figure 5B shows the serum PPT1 activity of IV-administered mice. Figure 5C shows the serum activity of IPA-administered mice (delivered intraparenchymally to the hippocampus by stereotactic injection). ND indicates not detected. Data are mean ± SD. P values ​​by ANOVA: *P<0.05, ***P<0.001, ****P<0.0001. [Figure 6] Figure 6 illustrates the liver PPT1 activity obtained from mice IV-administered with rAAV containing the Sp7-F.PPT1 viral vector nucleic acid. Mice injected with a diluent were used as negative controls. [Figure 7] Figure 7 illustrates immunohistochemical analysis showing increased PPT1 staining (indicated by black asterisks) in the mouse hippocampus after administration of rAAV containing Sp7-F.PPT1 viral vector nucleic acid to the hippocampus. [Figure 8] Figure 8 shows an automated capillary-based immunoassay (JESS, ProteinSimple) analysis demonstrating glycosylation of overexpressed PPT1 in mouse brains. Recombinant AAV or diluent containing Sp7-F.PPT1 viral vector nucleic acid was administered to the hippocampus (4 mice per group). [Figure 9] Figure 9 shows automated capillary-based immunoassay (JESS, ProteinSimple) analysis of hippocampal protein extracts from two different mice, untreated (-) or deglycosylase-treated (+), after administration of rAAV containing the Sp7-F.PPT1 viral vector to the hippocampus. The reduced molecular weight after deglycosylase treatment (lanes marked with +) suggested that PPT1 was glycosylated. [Figure 10]Figure 10 illustrates a PPT1 activity assay in mouse hippocampal lysate, showing PPT1 expression in the mouse brain. Recombinant AAV containing Sp7-F.PPT1 viral vector nucleic acid was administered to the hippocampus. [Figure 11] Figure 11 is a bar chart illustrating vector genome copy number (VGCN) as an indicator of viral transduction in various brain regions. Total DNA was isolated from frozen tissue, and VGCN was quantified by quantitative PCR (qPCR) using a standard curve. Each circle represents data obtained from one mouse. The height of each bar indicates the average. Hpc: hippocampus; Br.Stem: brainstem; crblm: cerebellum; Cerv: cervical spinal cord; Thor: thoracic spinal cord; lumb: lumbar spinal cord. Mice treated with diluents were used as negative controls. [Figure 12] Figure 12 is a bar chart showing the multiplicative change (FC) of PPT1 activity in various brain and spinal cord regions of rAAV-injected animals compared to diluent-injected mice. PPT1 activity in tissue lysates was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as the substrate. PPT1 activity was determined using a standard curve (created by using 4 MU of known concentration). FC activity was calculated compared to the mean activity of diluent-injected animals. Each circle represents the result for one mouse. Data are mean ± SD. One-way ANOVA, Tukey's test, *P<0.05, ***P<0.001, ****P<0.0001. [Figure 13] Figure 13 is a bar chart illustrating the FC of PPT1 activity in the cerebrospinal fluid (CSF) of mice injected with rAAV compared to mice injected with a diluent. FC is the relative mean activity level of the diluent group. Each data point on the graph represents data obtained from a single mouse. Data are expressed as mean ± SD. Statistical analysis involved one-way ANOVA followed by Tukey's test. *P<0.05. [Figure 14]Figure 14 is a scatter plot illustrating the correlation between VGCN and PPT1 enzyme activity in the brain. The Spearman correlation coefficient was r=0.066, p<0.0001. Each circle represents data obtained from a brain region derived from mice administered rAAV. [Figure 15] Figure 15 shows the detection of glycosylated and deglycosylated PPT1 protein in brain lysates, as analyzed by JESS. Protein extracts from brain lysates derived from the cortex of two independent mice (Mouse 1: lanes 1 and 3; Mouse 2: lanes 2 and 4) administered rAAV containing Sp7-F.PPT1 were treated with deglycosylase and analyzed by the JESS assay. The double lines seen in lanes 1-2 indicate two glycosylated forms of PPT1. The reduced molecular weight after deglycosylase treatment (lanes 3-4) suggests that PPT1 was glycosylated. [Figure 16A]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 16B]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 16C]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 16D]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 16E]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 16F]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 16G]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 16H]Figures 16A–16H are bar charts depicting vector genome copy number (VGCN) as a measure of viral transduction across various brain regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); additional brain regions shown (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN obtained from frozen tissue DNA was quantified using quantitative PCR (qPCR) with standard curves. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). White circles correspond to sheep administered with rAAV containing Sp7-F.PPT1, while black circles depict results from sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), ectolateral gyrus (EcG), entolateral gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaquaductal gray (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord (Cer), spinal cord (Tho), and spinal cord (Lum). [Figure 17A]Figures 17A–17D are bar charts showing functional PPT1 expression and secretion in the brain and CSF of sheep injected with rAAV containing the Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep) transgene. Figure 17A is a bar chart illustrating PPT1 activity in the cortex. Figure 17B is a bar chart illustrating PPT1 activity in the thalamus. Figure 17C is a bar chart illustrating PPT1 activity in the cerebellar cortex. Figure 17D is a bar chart illustrating PPT1 activity in the caudate nucleus. PPT1 activity in tissue lysates was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate. Each circle represents the result of one tissue punch from a brain region. N indicates the number of regions from 2 GFP-treated animals and 4 PPT1-treated animals. Data are mean ± SEM. Statistical analysis by Mann-Whitney U test, *P<0.05, ***P<0.001. [Figure 17B] Figures 17A–17D are bar charts showing functional PPT1 expression and secretion in the brain and CSF of sheep injected with rAAV containing the Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep) transgene. Figure 17A is a bar chart illustrating PPT1 activity in the cortex. Figure 17B is a bar chart illustrating PPT1 activity in the thalamus. Figure 17C is a bar chart illustrating PPT1 activity in the cerebellar cortex. Figure 17D is a bar chart illustrating PPT1 activity in the caudate nucleus. PPT1 activity in tissue lysates was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate. Each circle represents the result of one tissue punch from a brain region. N indicates the number of regions from 2 GFP-treated animals and 4 PPT1-treated animals. Data are mean ± SEM. Statistical analysis by Mann-Whitney U test, *P<0.05, ***P<0.001. [Figure 17C]Figures 17A–17D are bar charts showing functional PPT1 expression and secretion in the brain and CSF of sheep injected with rAAV containing the Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep) transgene. Figure 17A is a bar chart illustrating PPT1 activity in the cortex. Figure 17B is a bar chart illustrating PPT1 activity in the thalamus. Figure 17C is a bar chart illustrating PPT1 activity in the cerebellar cortex. Figure 17D is a bar chart illustrating PPT1 activity in the caudate nucleus. PPT1 activity in tissue lysates was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate. Each circle represents the result of one tissue punch from a brain region. N indicates the number of regions from 2 GFP-treated animals and 4 PPT1-treated animals. Data are mean ± SEM. Statistical analysis by Mann-Whitney U test, *P<0.05, ***P<0.001. [Figure 17D] Figures 17A–17D are bar charts showing functional PPT1 expression and secretion in the brain and CSF of sheep injected with rAAV containing the Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep) transgene. Figure 17A is a bar chart illustrating PPT1 activity in the cortex. Figure 17B is a bar chart illustrating PPT1 activity in the thalamus. Figure 17C is a bar chart illustrating PPT1 activity in the cerebellar cortex. Figure 17D is a bar chart illustrating PPT1 activity in the caudate nucleus. PPT1 activity in tissue lysates was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate. Each circle represents the result of one tissue punch from a brain region. N indicates the number of regions from 2 GFP-treated animals and 4 PPT1-treated animals. Data are mean ± SEM. Statistical analysis by Mann-Whitney U test, *P<0.05, ***P<0.001. [Figure 18]Figure 18 shows the 95% confidence interval for mean PPT1 activity across the overall treatment groups. Logarithmically transformed activity results for all data in Figures 17A–17D were obtained, and after explaining the differences in brain regions in punch count and mean values, a hypothesis test for mean treatment-type differences was performed at the 0.05 alpha level. The mean estimated multiplier change of PPT1 over GFP was 3.9, with a median of 73 nmol / mg / hour for PPT1 versus 19 nmol / mg / hour for GFP. ****P<0.0001, weighted two-way ANOVA. [Figure 19] Figure 19 is a bar chart showing the percentage change in PPT1 activity in cerebrospinal fluid (CSF) of sheep administered rAAV carrying Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep). The percentage change is relative to the mean activity of the control (GFP animals). Each circle represents one animal. N indicates the number of animals. Data are mean ± SEM. [Figure 20] Figure 20 shows the results of a JESS assay detecting recombinant PPT1 expression in tissue lysates of sheep spinal cord injected with rAAV vectors containing either PPT1 (sheep 1-4) or GFP (sheep 1-2). The first lane indicates the position of the molecular weight standard (std). Protein bands potentially representing sheep PPT1 were observed in all subjects, but the human PPT1 band was only identifiable in animals treated with vectors encoding human PPT1. The abbreviations C, T, and L represent the cervical, thoracic, and lumbar segments of the spinal cord, respectively. KDa stands for kilodalton. [Figure 21] Figure 21 illustrates the results showing the increased mean levels of PPT1 activity in the thoracic and lumbar segments of the spinal cord of sheep administered with a PPT1-containing vector. Each circle represents the results for a spinal cord segment of a particular animal. Data are mean ± SEM. [Figure 22]Figure 22 is a bar chart illustrating the rotarod evaluation of PPT1 knockout (KO) mice administered with rAAV (1E+11vg / animal) containing Sp7-F.PPT1(1) or SPARC.PPT1(2). KO mice were administered rAAV by bilateral ICV injection on postnatal day 1 and evaluated at 7 months of age. Untreated (Un) or vehicle-treated (Veh) KO mice were used as negative controls. "Natural type" refers to the rAAV vector encoding natural human PPT1. Each circle represents one mouse. Bars represent the mean ± SEM. One-way ANOVA, Tukey's post-hoc test, *P<0.05, ***P<0.001, ****P<0.0001. [Figure 23] Figure 23 illustrates the ability of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 to improve motor coordination and balance, as assessed by the waiting time from falling from an accelerating rod in Ppt1- / - mice (KO). Values ​​are mean ± SEM. One-way ANOVA, Tukey's post-hoc test. ####p<0.0001, WT, 7mo vs. KO, Un, 7mo; KO, Veh, 7mo. ****p<0.0001, KO, Veh, 7mo vs. rAAV-Sp7-F.PPT1, hi, all treatment groups except 9mo. **p<0.005, KO, Veh, 7mo vs. rAAV-Sp7-F.PPT1, hi, 9 months. AAV = Adeno-associated virus; ANOVA = Analysis of variance; CNS = Central nervous system; lo = Low dose (1 × 10¹¹ vg / animal); hi = High dose (3.82 × 10¹¹ vg / animal); KO = Ppt1- / -; mo = Months; PND = Days after birth; sec = Seconds; SEM = Standard error of mean; Un = Untreated; Veh = Vehicle; WT = Wild type. [Figure 24]Figure 24 illustrates the effects of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 on grip strength in Ppt1- / - mice (KO). Values ​​are mean ± SEM. One-way ANOVA, Tukey's post-hoc test. ####p<0.0001, WT, 7mo vs KO, Un, 7mo; KO, Veh, 7mo. ****p<0.0001, KO, Veh, 7mo vs all treatment groups. AAV = adeno-associated virus; ANOVA = analysis of variance; CNS = central nervous system; lo = low dose (1 × 10¹¹ vg / animal); hi = high dose (3.82 × 10¹¹ vg / animal); KO = Ppt1- / -; mo = months; PND = days after birth; sec = seconds; SEM = standard error of mean; Un = untreated; Veh = vehicle; WT = wild type. [Figure 25A] Figures 25A and 25B illustrate serum PPT1 activity in mice administered rAAV-Sp7-F.PPT1 or rAAV-SPARC.PPT1. Figure 25A shows activity at various time points. Figure 25B shows activity at 8 months. The number of mice in each group varied from 11 to 18. The sex distribution was generally balanced within each group. Each circle represents the result for one mouse. Each point in Figure 25A and each bar in Figure 25B represent the mean ± SEM. One-way ANOVA and Tukey's post-hoc test were performed using Log10 transformed values. ####p<0.0001, WT vs KO, Un;WT vs KO Veh;**p<0.01, KO, Veh vs wild type, lo;***p<0.001, KO, Veh vs rAAV-SPARC.PPT1, lo;***p<0.0001, KO, Veh vs rAAV-Sp7-F.PPT1, lo; wild type, hi;rAAV-Sp7-F.PPT1, hi;rAAV-SPARC.PPT1, hi. AAV = adeno-associated virus; ANOVA = analysis of variance; lo = low dose (1 × 10¹¹ vg / animal); hi = high dose (3.82 × 10¹¹ vg / animal); KO = Ppt1- / -; mo = months; LLOQ = lower limit of quantification; PND = days after birth; sec = seconds; SD = standard deviation; Un = untreated; Veh = vehicle; WT = wild type. [Figure 25B]Figures 25A and 25B illustrate serum PPT1 activity in mice administered rAAV-Sp7-F.PPT1 or rAAV-SPARC.PPT1. Figure 25A shows activity at various time points. Figure 25B shows activity at 8 months. The number of mice in each group varied from 11 to 18. The sex distribution was generally balanced within each group. Each circle represents the result for one mouse. Each point in Figure 25A and each bar in Figure 25B represent the mean ± SEM. One-way ANOVA and Tukey's post-hoc test were performed using Log10 transformed values. ####p<0.0001, WT vs KO, Un;WT vs KO Veh;**p<0.01, KO, Veh vs wild type, lo;***p<0.001, KO, Veh vs rAAV-SPARC.PPT1, lo;***p<0.0001, KO, Veh vs rAAV-Sp7-F.PPT1, lo; wild type, hi;rAAV-Sp7-F.PPT1, hi;rAAV-SPARC.PPT1, hi. AAV = adeno-associated virus; ANOVA = analysis of variance; lo = low dose (1 × 10¹¹ vg / animal); hi = high dose (3.82 × 10¹¹ vg / animal); KO = Ppt1- / -; mo = months; LLOQ = lower limit of quantification; PND = days after birth; sec = seconds; SD = standard deviation; Un = untreated; Veh = vehicle; WT = wild type. [Figure 26A] Figures 26A–26C provide bar charts showing PPT1 activity in the cortex (Figure 26A), brainstem (Figure 26B), and cerebellum (Figure 26C) of Ppt1- / - mice injected with rAAV-Sp7-F.PPT1 or rAAV-SPARC.PPT1. Mice were administered PND1 by bilateral intraventricular injection. "Natural" refers to an AAV vector containing the unmodified human PPT1 gene. Bars are mean ± SEM. Each circle represents a mouse. [Figure 26B]Figures 26A–26C provide bar charts showing PPT1 activity in the cortex (Figure 26A), brainstem (Figure 26B), and cerebellum (Figure 26C) of Ppt1- / - mice injected with rAAV-Sp7-F.PPT1 or rAAV-SPARC.PPT1. Mice were administered PND1 by bilateral intraventricular injection. "Natural" refers to an AAV vector containing the unmodified human PPT1 gene. Bars are mean ± SEM. Each circle represents a mouse. [Figure 26C] Figures 26A–26C provide bar charts showing PPT1 activity in the cortex (Figure 26A), brainstem (Figure 26B), and cerebellum (Figure 26C) of Ppt1- / - mice injected with rAAV-Sp7-F.PPT1 or rAAV-SPARC.PPT1. Mice were administered PND1 by bilateral intraventricular injection. "Natural" refers to an AAV vector containing the unmodified human PPT1 gene. Bars are mean ± SEM. Each circle represents a mouse. [Figure 27] Figure 27 is a schematic diagram showing the design of the rAAV nucleic acid present in the plasmid. Expression of the human PPT1 sequence is driven by a longer version of the EF1a promoter (SEQ ID NO: 173). SS = signal sequence. [Figure 28A] Figures 28A and 28B are bar graphs depicting PPT1 expressed in PPT1 knockout HeLa cells transfected with various AAV plasmids containing codon-optimized PPT1 sequences. Different codon-optimized PPT1 cDNA constructs (without the signal sequence) are labeled CO followed by a number on the x-axis and paired with the signal sequence. Figure 28A depicts Sp7F or SP7F (codon-optimized). Figure 28B depicts SpSPARC or SpSPARC (codon-optimized). [Figure 28B]Figures 28A and 28B are bar graphs depicting PPT1 expressed in PPT1 knockout HeLa cells transfected with various AAV plasmids containing codon-optimized PPT1 sequences. Different codon-optimized PPT1 cDNA constructs (without the signal sequence) are labeled CO followed by a number on the x-axis and paired with the signal sequence. Figure 28A depicts Sp7F or SP7F (codon-optimized). Figure 28B depicts SpSPARC or SpSPARC (codon-optimized). [Figure 29] Figure 29 is a diagram providing survival curves illustrating the ability of CNS-targeted AAV gene therapy to deliver functional human PPT1 in Ppt1- / - mice to extend survival time. [Figure 30] Figure 30 is a bar chart illustrating the effect of rAAV containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 on brain weight in Ppt1- / - mice. Mean + SD. ***P<0.0001, one-way ANOVA, Tukey's post-hoc. AAV = adeno-associated virus; CNS = central nervous system; lo = low dose (1 × 10¹¹ vg / animal); hi = high dose (3.82 × 10¹¹ vg / animal); KO = Ppt1- / -; PND = days after birth; Un = untreated; Veh = vehicle; WT = wild type. [Modes for carrying out the invention]

[0021]

[0052] The present invention is characterized by PPT1 polypeptides and nucleic acid constructs encoding PPT1 polypeptides. Using the polypeptides and encoding nucleic acid constructs, for example, PPT1 polypeptides can be produced, PPT1 activity in a subject can be increased, and / or PPT1-related diseases or disorders such as CLN1 can be treated.

[0022]

[0053] Polynucleotides encoding the PPT1 polypeptide can be delivered to the target by non-viral or viral delivery. Viral vectors that can be used include retroviral vectors, adenovirus vectors, AAV vectors, and herpes simplex virus vectors. Non-viral delivery methods include the use of naked DNA and nanoparticles.

[0023]

[0054] The term "subject" refers to mammals, including humans; non-human primates such as apes, gibbons, gorillas, chimpanzees, orangutans, and macaques; pet animals such as dogs and cats; domesticated animals such as poultry, ducks, horses, cattle, goats, sheep, and pigs; and laboratory animals such as mice, rats, rabbits, sheep, and guinea pigs. The preferred subject is humans.

[0024]

[0055] In certain embodiments, the PPT1 polypeptide or nucleic acid construct encoding the PPT1 polypeptide is evaluated for PPT1 expression and efficacy using a sheep model (see, for example, Nelvagal et al. (2022) J. Clin. Invest. 132(20); e163107; and the Examples section provided below, which are incorporated herein in their entirety by reference).

[0025]

[0056] The indicated percent identity for one or more reference sequences, and the references to similar language throughout this specification that provide indicated percent identity for one or more reference sequences, independently provide indicated percent identity or percent identity range for each of the referenced sequences. In determining the percent identity of polynucleotides, RNA and corresponding DNA are considered the same unless otherwise provided by the context in which they are used, for example, by providing a reference to a polynucleotide that is RNA or DNA. Examples of corresponding RNA and DNA include the substitution of uracil for thymine and the substitution of a ribose skeleton for a deoxyribose skeleton.

[0026]

[0057] Percent "identical," "identical," and similar terms refer to two sequences having the maximum alignment within a particular region. The region provided refers to the indicated reference sequence. For example, the "identical" or "identical" nature of a sequence to the PPT1 polypeptide of SEQ ID NO: 1 can be calculated by determining the number of identical amino acids in the aligned sequence, dividing by the total number of amino acids in SEQ ID NO: 1 (279 amino acids), and multiplying by 100. The percentage "identical" or "identical" nature of a nucleic acid sequence can be determined in a similar manner by aligning nucleotides to a reference sequence to achieve the maximum alignment taking into account nucleotide differences and gaps, dividing by the total number of nucleotides in the reference sequence, and multiplying by 100.

[0027]

[0058] The percentage identical or identical status of PPT1 code sequences shown to contain two or more exons is determined independently of any introns. For calculation purposes, introns are removed prior to alignment.

[0028]

[0059] In determining sequence identity for a reference sequence having one or more indicated variants, the specific variant chosen to determine sequence identity is the variant that provides the greatest sequence identity. For example, sequence number 2 provides X which is G, V, or L; for the purpose of determining sequence identity for sequence number 2, the X chosen to determine sequence identity is the X that provides the greatest sequence identity.

[0029]

[0060] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to mean all forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of providing the sequence in the 5' to 3' direction.

[0030]

[0061] In certain embodiments, nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA (poly and oligonucleotides), chromosomal DNA, splicing or unsplicing mRNA, rRNA, tRNA inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, transsplicing RNA, or antisense RNA), locked nucleic acid analogs (LNA), single-stranded and double-stranded oligonucleotide DNA (ODN), immunostimulatory sequences (ISS), riboswitches, and ribozymes.

[0031]

[0062] In certain embodiments, nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids may be single-stranded, double-stranded, or triple-stranded, linear or cyclic, and may have varying lengths.

[0032]

[0063] In certain embodiments, a polynucleotide is a single-stranded (ssDNA) or double-stranded (dsDNA) molecule. In certain embodiments, a dsDNA molecule is a minicircle, nanoplasmid, open-ring linear double-stranded DNA, or closed-ring linear double-stranded DNA (CELiD / ceDNA / doggyboneDNA). In certain embodiments, an ssDNA molecule is a closed-ring or open-ring linear DNA.

[0033]

[0064] A "transgene" refers to a nucleic acid that is intended to be introduced into a cell and operably ligated to a promoter, or that has been introduced and ligated. Examples of transgenes include heterologous polynucleotide sequences, such as the PPT1 polypeptide and nucleic acids encoding heterologous promoters.

[0034]

[0065] Certain embodiments focus on "CpG-reduced" or "CpG-depleted" nucleotide sequences, meaning (i) a nucleotide sequence in which one or more CpG dinucleotides (or motifs) are removed from a reference nucleic acid sequence; and / or (ii) a nucleotide sequence in which the percentage of CpG in the referenced polynucleotide is between 0% and 15%. In different embodiments, the CpG percentage is 0%, approximately 0.5%, approximately 1.0%, approximately 2.0%, approximately 3.0%, approximately 4.0%, approximately 5.0%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, or approximately 15% CpG; and / or up to approximately 0.5%, up to approximately 1.0%, up to approximately 2.0%, up to approximately 3.0%, up to approximately 4.0%, up to approximately 5.0%, up to approximately 6%, up to approximately 7%, up to approximately 8%, up to approximately 9%, up to approximately 10%, up to approximately 11%, up to approximately 12%, up to approximately 13%, up to approximately 14%, or up to approximately 15% CpG.

[0035]

[0066] The CpG motif can be suitably reduced or removed in the nucleotide sequence encoding the PPT1 protein and in other sequences that may be present in certain constructs (e.g., expression cassettes and viral vectors). Other sequences that may be present include the 5' and 3' untranslated regions (UTRs), stuffer sequences, promoters, enhancers; polyadenylation signals, ITRs, and non-coding sequences such as introns.

[0036]

[0067] The singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise.

[0037]

[0068] The conjunction "and / or" between multiple specified elements encompasses both individual options and combined options. For example, when two elements are joined by "and / or," the first option means the applicability of the first option without the second option, the second option means the applicability of the second option without the first option, and the third option means the applicability of the first and second options together. It is understood that any one of the options falls within the scope of meaning and thus satisfies the requirements of the term "and / or." The simultaneous applicability of two or more of the options is also understood to fall within the scope of meaning of the term "and / or."

[0038]

[0069] Unless clearly indicated otherwise by the context in which they are used, the terms "or" and "and" have the same meaning as "and / or".

[0039]

[0070] References to terms such as “beginning,” “for example,” “e.g.,” and “etc.” that follow different components or examples are open-ended, the listed components or examples are illustrative, and other components or examples may be provided or used.

[0040]

[0071] The terms “polypeptide,” “protein,” and “peptide” can be used interchangeably to refer to amino acid sequences, regardless of function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about ten amino acids. Amino acids include naturally occurring amino acids and those provided by cellular modifications.

[0041]

[0072] References to “comprise,” as used in reference to an element or group of elements, and variations such as “comprises” and “comprises,” are open-ended and do not exclude additional unspecified elements or method steps. Terms such as “begin,” “contain,” and “characterized by” are synonymous with “comprise.” In different embodiments and examples described herein, references to open-ended terms such as “comprises” may be replaced with “consisting of” or “essentially consisting of.”

[0042]

[0073] The reference to “consisting of” excludes any element, step, or component not specified in the enumerated claim elements, where such element, step, or component is related to the claimed invention.

[0043]

[0074] A reference to "essentially consisting of" limits the scope of the claim to those that do not substantially affect the specified material or step and the fundamental and novel features of the claimed invention.

[0044]

[0075] The term "approximately" means a value within 10% of the underlying parameter (i.e., plus or minus 10%). For example, "approximately 1:10" includes 1.1:10.1 or 0.9:9.9, and "approximately 5 hours" includes 4.5 hours or 5.5 hours. The term "approximately" at the beginning of a range of values ​​modifies each of the values ​​up to 10%.

[0045]

[0076] Every number or range of numbers includes integers within that range and any values ​​or integers within that range unless the context clearly indicates otherwise. For example, a reference to a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, and 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., as well as 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. A reference to a range of numbers such as "1-4" includes 1, 2, 3, 4, and 1.1, 1.2, 1.3, 1.4, etc. As a further example, “1 to 4 weeks” includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0046]

[0077] Furthermore, references to numerical ranges such as "0.01 to 10" include 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, and others. For example, dosages for a target body weight of approximately "0.01 mg / kg to approximately 10 mg / kg" include 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, and others.

[0047]

[0078] References to integers with "greater than" or "less than" each include digits greater than or less than the reference number. For example, a reference to greater than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more; and a “two or more” dose includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0048]

[0079] Various references, including papers and patent publications, are cited or mentioned throughout the background and this specification. Each of these references is incorporated in its entirety by reference within this specification. None of the references are recognized as prior art with respect to any of the disclosed or claimed inventions. In some cases, a particular reference is indicated as being incorporated by reference within this specification to emphasize its incorporation.

[0049]

[0080] The definitions provided herein, including those in this section and other sections of this application, shall apply throughout this application.

[0050]

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0051]

[0082] The description is separated into various sections and paragraphs and provides examples of various embodiments. These separations should not be considered to decouple the content of a paragraph or section or embodiment from the content of another paragraph or section or embodiment. The description provided has broad applicability and encompasses all possible combinations of sections, paragraphs and sentences that can be considered. Any discussion of any embodiment is illustrative only and is not intended to imply that the scope of this disclosure, including the claims, is limited to these examples (unless otherwise given in the claims).

[0052]

[0083] To describe numerous embodiments of the present invention, the invention is generally disclosed herein using definitive language. The invention also specifically includes embodiments in which certain subjects, such as substances or materials, method steps and conditions, protocols, or procedures, are completely or partially excluded. For example, in certain embodiments of the invention, materials and / or method steps are excluded. Thus, embodiments that are not expressly excluded in the invention are still disclosed herein, even if the invention is not generally expressed herein with respect to what is not included in the invention.

[0053] I.PPT1 polypeptide

[0084] The PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1. In certain embodiments, the polypeptide comprises a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, and (a) the PPT1 polypeptide further comprises a variant thereof having one of the signal sequences from SEQ ID NOs: 16-27 or one amino acid substitution, deletion or insertion; and / or (b) the PPT1 amino acid sequence comprises a G, V or L substitution of D at its amino terminus; and / or (c) the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N terminus. SEQ ID NO: 38 is an example of a full-length polypeptide in which the region corresponding to the mature human sequence has leucine-glutamine-histidine-leucine added to the N terminus of a naturally occurring sequence.

[0054]

[0085] In certain embodiments, the mature sequence contains a deletion at its N-terminus. Sequence ID No. 37 is a full-length example in which the mature sequence has an aspartate-proline-proline-alanine deletion at the N-terminus of the naturally occurring sequence.

[0055]

[0086] In certain embodiments, the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with respect to the sequence of SEQ ID NO: 1. In further embodiments, the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with respect to the sequence of SEQ ID NO: 2, and X is glycine; the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with respect to the sequence of SEQ ID NO: 2, and X is valine; the PPT1 polypeptide comprises at least 96%, at least 97%, at least 98%, at least 98%, or less The PPT1 polypeptide contains a PPT1 amino acid sequence having at least 99% or 100% identity, and X is leucine; the PPT1 polypeptide contains a PPT1 amino acid sequence having at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with respect to the sequence of SEQ ID NO: 3; or the PPT1 polypeptide contains the amino acid sequence leucine-glutamine-histidine-leucine in its N-terminal sequence, and contains a PPT1 amino acid having at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with respect to the sequence of SEQ ID NO: 4.

[0056]

[0087] PPT1 is a well-characterized enzyme with a wide range of amino acids that contribute to its activity and various mutations that result in reduced activity (for example, each of these is incorporated herein by reference as follows: Kumar et al., Advances in Protein Chemistry and Structural Biology (2022) 132: pp. 89-109; Bellizzi et al., PNAS (2000) 97(9): pp. 4573-4578; and hyperlink / / www.uniprot.org / uniprotkb / P50897 / entry (December 3, 2022)).

[0057]

[0088] The signal sequence provides the amino acid sequence of the signal peptide, which is a short N-terminal amino acid sequence that leads to protein secretion. The terms signal sequence and sequence peptide are used interchangeably herein. The signal sequence directs proteins to or through the endoplasmic reticulum secretory pathway and is generally cleaved within the endoplasmic reticulum prior to secretion. Thus, the signal peptide enhances the secretion of polypeptides from cells compared to the secretion levels of the corresponding polypeptide lacking the signal peptide.

[0058]

[0089] The presence of a signaling sequence in the PPT1 polypeptide facilitates the extracellular secretion of mature PPT1 polypeptide. The secreted polypeptide may then be taken up by other cells, providing cross-correction.

[0059]

[0090] In certain embodiments, the signal peptide contains any of the amino acid sequences from SEQ ID NOs: 16 to 27, or contains an amino acid sequence in which one amino acid is different from any of SEQ ID NOs: 16 to 27. In further embodiments, the signal peptide contains the amino acid sequence of SEQ ID NO: 16; the amino acid sequence of SEQ ID NO: 19; or the amino acid sequence of SEQ ID NO: 23.

[0060]

[0091] In certain embodiments, the PPT1 polypeptide comprises a signal sequence and a PPT1 sequence. a) The signal sequence includes one of the amino acid sequences from SEQ ID NOs. 16-21 and 24-27, or an amino acid sequence different from one of SEQ ID NOs. 16-21 and 24-27 due to the addition, deletion, or substitution of one amino acid; the PPT1 polypeptide sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO. 1; b) The signal sequence comprises one of the amino acid sequences from SEQ ID NOs. 16-21 and 24-27, and the PPT1 polypeptide sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to SEQ ID NO. 2, where X is G, V, or L; c) The signal sequence includes the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence different from SEQ ID NO: 16 due to the addition, deletion, or substitution of one amino acid; the PPT1 polypeptide sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1; d) The signal sequence comprises the amino acid sequence of SEQ ID NO: 16, and the PPT1 polypeptide sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 2, where X is G, V, or L; e) The signal sequence contains the amino acid sequence of SEQ ID NO: 16, the PPT1 polypeptide sequence contains the sequence of SEQ ID NO: 2, and X is G. f) The signal sequence includes the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence different from SEQ ID NO: 19 due to the addition, deletion, or substitution of one amino acid; the PPT1 polypeptide sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1; h) The signal sequence comprises the amino acid sequence of SEQ ID NO: 19, and the PPT1 polypeptide sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 2, where X is G, V, or L; i) The signal sequence contains the amino acid sequence of SEQ ID NO: 19, the PPT1 polypeptide sequence contains the sequence of SEQ ID NO: 2, and X is G.

[0061]

[0092] In certain embodiments, the PPT1 polypeptide comprises a signal sequence and a PPT1 sequence, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence different from SEQ ID NO: 23 due to the addition, deletion, or substitution of one amino acid; and the PPT1 polypeptide sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus and has a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4.

[0062]

[0093] In certain embodiments relating to a PPT1 polypeptide including a signal sequence and a PPT1 sequence, the polypeptide contains an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NOs: 31-42, or differs from any of SEQ ID NOs: 31-42 by differences of 1 to 10 amino acids, or by differences of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The reference to the amino acid difference indicates any combination of addition, substitution, and / or deletion.

[0063]

[0094] In further embodiments, the polypeptide comprises an amino acid sequence having at least 99% or 100% sequence identity with SEQ ID NO: 31, or 1 to 5 amino acids different from SEQ ID NO: 31; the polypeptide comprises the amino acids of SEQ ID NO: 31, and X is D or G; the polypeptide comprises an amino acid sequence having at least 99% or 100% sequence identity with SEQ ID NO: 34, or 1 to 5 amino acids different from SEQ ID NO: 34; the polypeptide comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, and has at least 99% or 100% sequence identity with SEQ ID NO: 38, or 1 to 5 amino acids different from SEQ ID NO: 38; or the polypeptide comprises SEQ ID NO: 30.

[0064] II. Polynucleotides containing the PPT1 coding sequence

[0095] The production and intracellular delivery of the PPT1 polypeptide can be facilitated by using a polynucleotide containing a nucleic acid sequence encoding the PPT1 polypeptide. In certain embodiments, the polynucleotide comprises a nucleic acid sequence encoding the PPT1 polypeptide, and the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 1. (a) The PPT1 polypeptide further comprises a variant thereof having any of the signal sequences from SEQ ID NOs. 16 to 27 or one amino acid substitution, deletion or insertion; and / or (b) The PPT1 amino acid sequence includes a glycine (G), valine (V), or leucine (L) substitution of aspartic acid (D) at its amino terminus; and / or (c) The PPT1 sequence contains the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus; and / or (d) The nucleic acid sequence includes a PPT1 coding sequence that has at least 85% identity with any of sequence numbers 61 to 94.

[0065]

[0096] In certain embodiments, (a) the polynucleotide encodes a PPT1 polypeptide comprising a PPT1 amino acid sequence having at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity with respect to the sequence of SEQ ID NO: 1; (b) the encoded PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity with respect to the sequence of SEQ ID NO: 2, where X is glycine (c) The encoded PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity with respect to the sequence of SEQ ID NO: 2, where X is valine; or (d) The encoded PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity with respect to the sequence of SEQ ID NO: 2, where X is leucine. In further embodiments, a nucleotide acid sequence encoding any of (a), (b), (c), or (d) comprises a nucleic sequence or variant thereof having at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with any of sequence numbers 61-94, wherein the first three nucleotides encode G, D, V, or L, or encode G. Nucleotides encoding G, V, D, or L are shown in Table 1.

[0066]

[0097] [Table 1]

[0067]

[0098] In certain embodiments, the nucleic acid encoding the mature PPT1 amino acid comprises the amino acid sequence leucine-glutamine-histidine-leucine in its N-terminal sequence and has at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with respect to the sequence of Sequence ID No. 4.

[0068]

[0099] In certain embodiments, the nucleic acid encoding the mature PPT1 polypeptide further comprises a signal sequence containing any of the amino acid sequences of SEQ ID NOs. 16-27, or an amino acid sequence in which one amino acid is different from any of SEQ ID NOs. 16-27. In further embodiments, the signal coding sequence is any of the sequences of SEQ ID NOs. 43-58. A reference to “signal coding sequence” indicates a nucleotide sequence encoding a signal sequence or signal peptide.

[0069]

[0100] In certain embodiments, the polynucleotide comprises one of the sequence numbers 16-21 and 24-27; sequence number 16; or the signal coding sequence of sequence number 19.

[0070]

[0101] In certain embodiments, the signal coding sequence includes a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any of SEQ ID NOs: 43-47, and the signal sequence includes the sequence of SEQ ID NO: 16, or a sequence in which one amino acid differs from SEQ ID NO: 16. In further embodiments, the nucleic acid sequence includes a signal coding sequence with any of SEQ ID NOs: 43, 44, 45, 46, or 47.

[0071]

[0102] In certain embodiments, the signal coding sequence includes a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 43, and the signal sequence includes the sequence of SEQ ID NO: 16, or a sequence in which one amino acid differs from SEQ ID NO: 16. In further embodiments, the signal coding sequence includes the sequence of SEQ ID NO: 43.

[0072]

[0103] In certain embodiments, the signal coding sequence includes a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 50, and the signal coding sequence includes the sequence of SEQ ID NO: 19, or a sequence in which one amino acid differs from SEQ ID NO: 19. In different embodiments, the nucleic acid sequence includes the sequence of SEQ ID NO: 50.

[0073]

[0104] In certain embodiments, the signal coding sequence includes a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 54, and the signal coding sequence includes the sequence of SEQ ID NO: 23, or a sequence in which one amino acid differs from SEQ ID NO: 23. In different embodiments, the nucleic acid sequence includes the sequence of SEQ ID NO: 54.

[0074]

[0105] In certain embodiments, the polynucleotide encodes a PPT1 polypeptide comprising a signal sequence and a PPT1 sequence, a) The signal sequence includes sequence number 16, the signal code sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 43, and the PPT1 sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 1 or sequence number 2; b) The signal code sequence includes the sequence of sequence number 43, and the PPT1 sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 1 or 2; c) The signal code sequence includes the sequence of sequence number 43, and the PPT1 sequence includes a sequence having at least 97% sequence identity with sequence number 1; d) The signal code sequence includes the sequence of sequence number 43, the PPT1 sequence includes a sequence having at least 97% sequence identity with sequence number 2, and X is G, V, or L; e) The signal code sequence includes the sequence of sequence number 43, the PPT1 sequence includes a sequence having at least 99% sequence identity with sequence number 2, and X is G; f) The signal code sequence includes the sequence of sequence number 43; the PPT1 sequence includes the sequence of sequence number 1; g) The signal code sequence includes the sequence of sequence number 43; the PPT1 sequence includes the sequence of sequence number 2, where X is G; h) In a different embodiment, the sequence encoding the polypeptide sequence for any of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs. 61 to 79 and 80 to 94, wherein the first three nucleotides are codons from Table 1. In further embodiments, a sequence encoding a polypeptide sequence for any of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs. 62-64, 71, 74, 78, 79, and 83, wherein the first three nucleotides are codons from Table 1; a sequence encoding a polypeptide sequence for any of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs. 64, wherein the first three nucleotides are codons from Table 1; or a sequence encoding a polypeptide sequence for any of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NOs. 79, wherein the first three nucleotides are codons from Table 1.

[0075]

[0106] In certain embodiments, the polynucleotide encodes a PPT1 polypeptide comprising a signal sequence and a PPT1 sequence, a) The signal sequence includes sequence number 19, the signal code sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 50, and the PPT1 sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 1 or sequence number 2; b) The signal code sequence includes the sequence of sequence number 50, and the PPT1 sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with sequence number 1 or 2; c) The signal code sequence includes the sequence of sequence number 50, and the PPT1 sequence includes a sequence having at least 97% sequence identity with sequence number 1; d) The signal code sequence includes the sequence of sequence number 50, the PPT1 sequence includes a sequence having at least 97% sequence identity with sequence number 2, and X is G, V, or L; e) The signal code sequence includes the sequence of sequence number 50, the PPT1 sequence includes a sequence having at least 99% sequence identity with respect to sequence number 2, and X is G; f) The signal code sequence includes the sequence with sequence number 50, and the PPT1 sequence includes the sequence with sequence number 1; g) The signal code sequence includes the sequence of sequence number 50, the PPT1 sequence includes the sequence of sequence number 2, and X is G; h) In a different embodiment, the nucleic acid encoding the polypeptide sequence for any of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs. 61 to 79 and 80 to 94, wherein the first three nucleotides are codons from Table 1.

[0107] In certain embodiments, the polynucleotide encodes a PPT1 polypeptide comprising a signal sequence and a PPT1 sequence: a) The signal sequence includes SEQ ID NO: 23, the signal coding sequence includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54, and the PPT1 sequence includes leucine-glutamine-histidine-leucine at its N-terminus and at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; b) The signal coding sequence includes the sequence of SEQ ID NO: 54, and the PPT1 sequence includes leucine-glutamine histidine-leucine at its N-terminus and at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; c) The signal coding sequence includes the sequence of SEQ ID NO: 54, and the PPT1 sequence includes leucine-glutamine histidine-leucine at its N-terminus and at least 97% sequence identity to SEQ ID NO: 1; d) The signal coding sequence includes the sequence of SEQ ID NO: 54, and the PPT1 sequence includes leucine-glutamine-histidine-leucine at its N-terminus and at least 98% sequence identity to SEQ ID NO: 4; e) The signal coding sequence includes the sequence of SEQ ID NO: 54, and the PPT1 sequence includes leucine-glutamine-histidine-leucine at its N-terminus and at least 99% sequence identity to SEQ ID NO: 4; f) The signal code sequence includes the sequence with sequence number 54, and the PPT1 sequence includes the sequence with sequence number 4.

[0076]

[0108] In certain embodiments, the nucleic acid encodes a PPT1 polypeptide containing a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NOs: 29-42, or 1 to 10 amino acids, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that are different from any of SEQ ID NOs: 29-42; (b) a PPT1 polypeptide containing an amino acid sequence having at least 99% or 100% sequence identity with SEQ ID NO: 31, or 1 to 5 amino acids that are different from SEQ ID NO: 31; (c) a PPT1 polypeptide containing the amino acids of SEQ ID NO: 31, where X is D or G; (d) a PPT1 polypeptide containing an amino acid sequence having at least 99% or 100% sequence identity with SEQ ID NO: 34, or 1 to 5 amino acids that are different from SEQ ID NO: 34; (e) a PPT1 polypeptide containing the amino acids of SEQ ID NO: 34, where X is D or G; (f) SEQ ID NO: 38 (g) Encodes a PPT1 polypeptide containing the amino acid sequence of (b), wherein the nucleic acid contains a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 43; (h) Encodes (b), wherein the nucleic acid contains a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of SEQ ID NOs: 107-125 and 168; (i) Encodes (c), wherein the nucleic acid contains at least (h)(c) Encodes (h)(c) and the nucleic acid contains a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of sequence numbers 126-140 and 161-167; or (i)(f) Encodes (h)(c) and the nucleic acid contains a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to sequence number 103.

[0077]

[0109] In certain embodiments, the polynucleotide comprises a nucleic acid sequence encoding the PPT1 polypeptide described in Section I, above reference.

[0078]

[0110] In certain embodiments, the polynucleotide sequence encoding the PPT1 polypeptide comprises two or more exons encoding the PPT1 polypeptide, and one or more introns.

[0079]

[0111] In certain embodiments, the polynucleotide comprises a PPT1 coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any of the sequences 95 to 106.

[0080]

[0112] References to sequences provided in this application, such as Table 2 below which include stop codons, include embodiments in which there are no stop codons, multiple stop codons, and different stop codons.

[0081]

[0113] References to sequences provided in this application, such as Table 2 below, which encode proteins that provide stop codons, include embodiments in which there is no stop codon immediately following the provided sequence, a stop codon is present, multiple stop codons are present, and different stop codons are present.

[0082]

[0114] In certain embodiments, the PPT1 coding nucleotide sequence contains 0 to 5, 0 to 10, or 0 to 15 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% CpG; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, or up to about 5.0% CpG.

[0083] III. Expression Cassette

[0115] A polynucleotide expression cassette comprises a nucleic acid encoding a PPT1 polypeptide operably linked to one or more regulatory elements. Expression regulation can affect, for example, transcription, translation, splicing, and message stability. Regulatory elements are typically located at the 5' ("upstream") or 3' ("downstream") end of the nucleic acid being transcribed. Regulatory elements may be located within the transcript (e.g., in an intron) at a distance adjacent to or away from the sequence being transcribed. One or more regulatory elements of the same or different types may be present. Examples of regulatory elements include promoters, enhancers, introns, polyadenylation signals, Kozak sequences, post-transcriptional regulatory elements, and termination sequences.

[0084]

[0116] The promoter is the DNA region from which transcription begins. Generally, the nucleic acid to be transcribed is located 3' of the promoter sequence. In certain embodiments, the promoter sequence is coupled to an enhancer. An enhancer is a DNA region that increases promoter transcription. The enhancer may be adjacent to the promoter, within the promoter, or distal to it. Typically, the enhancer is located upstream of the promoter, but it may be located downstream of or within the promoter sequence.

[0085]

[0117] Expression regulatory elements, such as promoters and enhancers, can be selected to preferentially drive expression in specific cell or tissue types. Since expression regulatory elements are recognized by transcription activator proteins or other transcription regulators specific to particular cell, tissue, or organ types, they are typically active in those specific cells, tissues, or organs (see, for example, Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, 4th edition, Vol. II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al., (2010) Current protocols in molecular biology, John Wiley & Sons, New York).

[0086]

[0118] The incorporation of tissue-specific regulatory elements in the expression construct provides at least partial tissue-specificity for PPT1 protein expression. Reference to a promoter or enhancer specific to a particular cell type of tissue indicates that the promoter or enhancer provides relatively high levels of expression and / or secretion in the indicated cell or tissue type. Examples of liver-specific promoters include the transthyretin (TTR) gene promoter; the human α-1 antitrypsin (hAAT) promoter; the apolipoprotein AI promoter; albumin, Miyatake et al., J. Virol., 71:51 pp. 24-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther. 3:10 pp. 2-9 (1996); α-fetoprotein (AFP), Arbuthnot et al., Hum. Gene. Ther., 7:15 pp. 3-14 (1996); the human factor IX promoter; thyroxine-binding globulin (TBG) promoter; the TTR minimal enhancer / promoter; the α-antitrypsin promoter; LSP (845nt) (requiring intron-less scAAV); and the LSP1 promoter. Examples of active enhancers in the liver include apolipoprotein E (apoE)HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0087]

[0119] Expression regulatory elements also include ubiquitous or indiscriminate promoters and promoters / enhancers capable of driving polynucleotide expression in a number of different cell types. Such elements include the EF1-α promoter, the cytomegalovirus (CMV) early promoter / enhancer sequence, the Roussarcoma virus (RSV) promoter / enhancer sequence, the phosphoglycerate kinase (PKG) promoter, CAG (a compound of the CMV enhancer, chicken β-actin promoter (CBA), and rabbit β-globin intron) (see, e.g., Boshart et al., (1985) Cell, 41: pp. 521-530), the SV40 promoter, the dihydrofolate reductase promoter, and the cytoplasmic β-actin promoter.

[0088]

[0120] Examples of CNS-specific promoters include NSE (neuron-specific enolase), synapsin or NeuN, platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), methyl-CpG-binding protein 2 (MeCP2), and Ca 2Examples of neuron-specific promoters include calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin minigene nβ2, preproenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters; astrocyte-specific promoters such as glial fibrillary acid protein (GFAP) and EAAT2 promoters; oligodendrocyte-specific promoters such as myelin basic protein (MBP) / myelin-associated glycoprotein and oligodendrocyte transcription factor 2 promoters; neuron / hypothalamic-specific promoters such as proopiomelanocortin (POMC) promoter; and neuron / spinal cord-specific promoters such as superoxide dismutase 1 (SOD1). (For example, see U.S. Patent Application Publication No. 2021 / 214749 and Adeno-Associated Virus Vectors (2019), Castle, ed., 1st edition, Springer New York, New York, NY., both of which are incorporated herein in their entirety by reference.)

[0089]

[0121] Additional promoters include the SV40 early promoter, mouse mammary cancer virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, SFFV promoter, rat insulin promoter, TBG promoter, desmin promoter and similar muscle-specific promoters, synthetic promoters, hybrid promoters, and promoters with multi-tissue specificity.

[0090]

[0122] Expression regulatory elements can also influence expression in a manner modulated by signals or stimuli that increase or decrease expression. Regulatory elements that increase the expression of a target nucleic acid in response to a signal or stimulus are also referred to as “inducible elements” (i.e., signal-induced). Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present. Specific examples include the zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary cancer virus (MMTV) promoter; the tetracycline-inhibiting system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89: pp. 5547-5551 (1992)); the tetracycline-inducible system (Gossen et al., Science 268: pp. 1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2: pp. 512-518 (1998)); and the RU486-inducible system (Wang et al., Nat. Biotech. 15: pp. 239-243 (1997) and Wang et al., Gene Examples include Ther. 4: pp. 432-441 (1997); and rapamycin-inducible systems (Magari et al., J. Clin. Invest. 100: pp. 2865-2872 (1997); and Rivera et al., Nat. Medicine. 2: pp. 1028-1032 (1996)). Other examples of adjustable control elements include those regulated by specific physiological conditions such as temperature, acute phase, or development.

[0091]

[0123] In certain embodiments, the expression cassette further comprises one or more introns independent of the PPT1 coding nucleotide acid. Various different introns can be used to enhance gene expression. Examples of introns that can be used include rabbit β-globin introns containing splicing donor / splicing acceptor, SV40 introns containing splicing donor / splicing acceptor, human β-globin intron, intron 2 of the human hemoglobin β gene, hFIX int1 (intron 1 of the human coagulation factor IX gene), CBA-rHHB (synthetic intron derived from a fusion of intron 1 of the chicken β-actin gene and intron 2 of rabbit hemoglobin β), CBA (intron 1 of the chicken β-actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (derived from different parts of the human coagulation factor IX gene, and also used in pLIVE vectors, Mirus Examples include synthetic introns present in Bio, Madison, and WI; human hemoglobin subunit β (HBB2) synthetic introns and optimized HBB2; and chimeric introns such as the 5' splicing donor of the first human β-globin intron and the intron-derived branching site and 3' acceptor site between the leader and body of the immunoglobulin gene heavy chain variable region (Buck et al., Int. J. Mol. Sci. (2020), 21, p. 4197; Ronzitti et al., Mol. Ther. Methods Clin Dev. (2016) July 20; 3: 16049; and HBB-IG introns provided by pCMVNT® vectors).

[0092]

[0124] In certain embodiments, the expression cassette includes post-transcriptional regulatory elements. Post-transcriptional regulatory elements such as the Woodchuck post-transcriptional regulatory element (WPRE) and the hepatitis B regulatory element can increase gene expression (Buck et al., Int. J. Mol. Sci. (2020), 21, pp. 4197).

[0093]

[0125] Polyadenylation signal sequences contribute to the formation of the poly(A) tail, which promotes nuclear export, translation, and / or mRNA stability, and may also be involved in transcription termination. Examples of polyadenylation signal sequences include the SV40 late polyadenylation signal, the bovine growth hormone poly(A) (bGHpA) signal sequence, synthetic poly(A), mouse β-globin pA, rabbit β-globin pA, and H4-based pA (Buck et al., Int. J. Mol. Sci. (2020), 21, pp. 4197).

[0094]

[0126] In certain embodiments, the expression cassette includes the Kozak consensus sequence or a variation thereof. The Kozak consensus sequence plays a role in translation initiation. The Kozak consensus sequence and its variants are provided, for example, in McClements et al. (2021), Molecular Vision, 27, pp. 233-242, which are incorporated herein by reference.

[0095]

[0127] In certain embodiments, the expression cassette is operably coupled from 5' to 3' to a PPT1 coding sequence and includes a promoter, promoter / enhancer, intron, Kozak sequence, PPT1 coding sequence, and polyadenylation signal. In certain embodiments, the intron includes the amino acid sequence of SEQ ID NO: 11.

[0096]

[0128] In certain embodiments, the expression cassette further comprises a miRNA target sequence, which in further embodiments is incorporated into the 3'UTR of the expression cassette. The miRNA target sequence is recognized by miRNAs present in specific cells or tissues, leading to degradation of the mRNA transcript. Incorporating a miRNA target sequence(s) based on the presence of certain miRNAs in specific cells can be used to reduce expression in specific cell or tissue types. Multiple tandem repeats of the miRNA target sequence can be used to increase degradation (Geisle et al., (2016) World Journal of Experimental Medicine 6(2): pp. 37-54).

[0097]

[0129] In certain embodiments, the expression cassette comprises a nucleic acid sequence encoding a PPT1 polypeptide provided in Section I, above reference, and a nucleic acid sequence encoding a PPT1 polynucleotide provided in Section II, above reference; and / or comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any of SEQ ID NOs. 141-143, 169, and 170.

[0098]

[0130] In certain embodiments, the expression cassette nucleotide sequence includes 0-5, 0-10, 0-15, 0-50, or 0-100 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpGs; 0%, approximately 0.5%, approximately 1.0%, approximately 2.0%, approximately 3.0%, approximately 4.0%, approximately 5.0%, Approximately 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of CpG; and / or any of the following CpGs: up to approximately 0.5%, up to approximately 1.0%, up to approximately 2.0%, up to approximately 3.0%, up to approximately 4.0%, up to approximately 5.0%, up to approximately 6%, up to approximately 7%, up to approximately 8%, up to approximately 9%, up to approximately 10%, up to approximately 11%, up to approximately 12%, up to approximately 13%, up to approximately 14%, or up to approximately 15%.

[0099] IV. Recombinant viral vector nucleic acids

[0131] Polynucleotide recombinant viral vector nucleic acids contain 5' and / or 3' viral elements that provide viral packaging and can confer additional activities such as self-priming, DNA replication, promoter activity, genomic integration, or episomal concatemerization. The 5' and 3' elements are typically located at or near the 5' and 3' ends of the recombinant viral vector nucleic acid and may be naturally occurring or modified versions of naturally occurring sequences. Examples of 5' and 3' elements include adenovirus ITRs, adeno-associated virus ITRs and packaging sequences; retrovirus 5' and 3' long-chain terminal repeats (LTRs) and packaging sequences (Naso et al., (2017) BioDrugs, 31(4), pp. 317-334; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: p. 53 (2021); and Liu and Seol (2020) BMB Reports; 53(11): pp. 565-575).

[0100]

[0132] The term "recombinant," as a modifier for nucleic acids or vectors, indicates a combination of elements not found in nature. For example, recombinant viral vector nucleic acids provide 5' and / or 3' viral elements along with an expression cassette containing one or more elements not found in nature, including the 5' and / or 3' elements. Similarly, viral vectors such as rAAV vectors may contain naturally occurring or modified capsids that capsidize the recombinant viral vector nucleic acid.

[0101]

[0133] In certain embodiments, the viral vector nucleic acid sequence comprises a 5'UTR and a 3'UTR, or a 5'ITR and a 3'ITR, and includes (1) a sequence encoding a polypeptide as referenced in Section I above; (2) a nucleic acid sequence encoding a PPT1 polynucleotide provided as referenced in Section II above; and / or (3) an expression cassette encoding a PPT1 polynucleotide provided as referenced in Section III above.

[0102]

[0134] In certain embodiments, the viral vector includes a polyA signal operably ligated to the 3'ITR, the polyA signal antagonizing potential transcriptions that begin at the 3'ITR. The operably ligated polyA signal is located upstream of the 3'ITR.

[0103]

[0135] In certain embodiments, the viral vector nucleic acid contains 0-5, 0-10, 0-15, 0-50, 0-100, or 0-150 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 44, 46, 47, 48, 49, or 50 CpGs; 0%, approximately 0.5 CpGs of approximately 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%; and / or any of the following CpGs: up to approximately 0.5%, up to approximately 1.0%, up to approximately 2.0%, up to approximately 3.0%, up to approximately 4.0%, up to approximately 5.0%, up to approximately 6%, up to approximately 7%, up to approximately 8%, up to approximately 9%, up to approximately 10%, up to approximately 11%, up to approximately 12%, up to approximately 13%, up to approximately 14%, or up to approximately 15%.

[0104]

[0136] In certain embodiments, the recombinant viral vector nucleic acid includes a sequence having at least 95% identity, at least 97% identity, at least 99% identity, or 100% identity with any of the sequences SEQ ID NOs. 144-154, 171, and 172.

[0105] V. Virus vector

[0137] In certain embodiments, the gene delivery vehicle is a viral vector containing a protein capsid that capsidates recombinant viral vector nucleic acid. The viral vector can deliver the viral vector nucleic acid to cells or tissues. Depending on the specific vector, the viral vector may further include a viral envelope. Examples of viral vectors that can be used for gene delivery include adenovirus vectors, rAAV, retroviral vectors, and herpes simplex vectors.

[0106]

[0138] Various serotypes exist within various types of viruses. Different serotypes can result in different activities, such as cell or tissue targeting and the potential to elicit host immune responses. The term "serotype" broadly refers to both serologically distinct viruses and serologically non-distinguishable viruses that may exist within a given serotype subgroup or variant. Serological differences can be determined based on the lack of cross-reactivity between antibodies against a particular capsid when compared to another capsid. Such differences in cross-reactivity usually result from differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0107]

[0139] As more naturally occurring virus isolates are discovered or capsid variants are generated, there may or may not be serological differences from any of the currently existing serotypes. Therefore, if a new virus does not exhibit serological differences, it is likely a subgroup or variant of the corresponding serotype.

[0108] VA adenovirus vector

[0140] Adenoviruses are non-enveloped double-stranded DNA viruses. Recombinant adenovirus vectors contain recombinant adenovirus nucleic acid lacking one or more proteins involved in viral replication, and further contain an adenovirus capsid. Recombinant adenovirus vectors containing different amounts of adenovirus DNA can be constructed. The adenovirus (Ad) genome is flanked at its ends by hairpin-like inverted terminal repeats (ITRs) whose length varies from 30 to 371 bp. ITRs function as self-priming structures that promote primase-independent DNA replication. Packaging signals located on the left arm of the genome are required for viral genome packaging (Liu and Seol (2020) BMB Reports; 53(11): pp. 565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: p. 53).

[0109]

[0141] In certain embodiments, the recombinant adenovirus vector is a third-generation vector also referred to as "gutless" or "helper-independent." Gutless vectors can be prepared from recombinant adenovirus nucleic acids that are entirely, or substantially, devoid of the viral sequence except for the ITR and packaging signal. Gutless adenovirus vectors are high-capacity vectors capable of accommodating up to approximately 36 kb of DNA inserts. Preferred recombinant adenovirus nucleic acids are approximately 27 kb to 37 kb. Stuffer sequences can be added to the recombinant adenovirus nucleic acids to increase nucleic acid size and capsid integration. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences (each of which is incorporated in whole herein by reference: Liu and Seol (2020) BMB Reports, 53(11): pp. 565-575; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: p. 53; and Sandig et al., PNAS (2000) 97(3): pp. 1002-1007).

[0110]

[0142] In certain embodiments, recombinant adenovirus vectors can be constructed based on rare human or chimpanzee serotypes. The use of chimpanzee and rare human serotypes may be useful in reducing the host immune response to recombinant adenovirus vectors due to pre-existing immunity (Guo et al., (2018) Human vaccines & immunotherapeutics, 14(7): pp. 1679-1685 and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: p. 53).

[0111]

[0143] Adenovirus vectors can be generated, for example, by supplying the viral proteins necessary for vector production in trans using a suitable helper virus or plasmid and cell line (Liu and Seol (2020) BMB Reports; 53(11): pp. 565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: p. 53).

[0112] VB Recombination AAV Vector

[0144] Recombinant adeno-associated virus (also referred to herein as "rAAV") vectors are based on adeno-associated virus. Adeno-associated virus is a single-stranded DNA virus containing a 4.7kb genome flanked by 145nt ITRs at both ends of the genome. ITR activity is important for self-priming and packaging and can also provide additional activity such as promoter activity. The AAV 5' and 3' ITRs can vary in size, and the 5' and 3' inverted repeats do not need to be exact inverted repeats.

[0113]

[0145] The rAAV vector comprises AAV recombinant nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV proteins involved in viral replication. In certain embodiments, the rAAV vector contains AAV 5' and / or 3' ITRs along with the DNA insert. In certain embodiments, the rAAV nucleic acid comprises a 5' ITR and / or 3' ITR independently selected from the 5' and 3' ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B ITRs. In further embodiments, both 5' and 3' ITRs are present, and both ITRs originate from the same serotype genome.

[0114]

[0146] In a particular embodiment, the 5'ITR includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 8; the 3'ITR independently includes (a) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 9; (b) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 158; (c) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 159; or (d) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 160.

[0115]

[0147] In a particular embodiment, the 3'ITR includes a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 9; the 5'ITR independently includes (a) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 10; (b) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 156; or (c) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to sequence number 157.

[0116]

[0148] Recombinant adeno-associated virus vectors typically accept DNA insertions having a size range of approximately 4 kb to 5.2 kb. If necessary, stuffer sequences can be used to increase rAAV nucleic acid size and packaging efficiency. In different embodiments, rAAV nucleic acids containing stuffers are 4–5.2 kb, 3.0–5.5 kb, 4.0–5.0 kb, 4.3–4.8 kb, approximately 4.2 kb, approximately 4.3 kb, approximately 4.4 kb, approximately 4.5 kb, approximately 4.6 kb, or approximately 4.7 kb. Preferred stuffer sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences.

[0117]

[0149] In certain embodiments, rAAV is a self-complementary adeno-associated virus vector (scAAV) or a short-chain hairpin adeno-associated virus vector (shAAV). scAAV and shAAV provide double-stranded rAAV nucleic acid that can be incorporated into an AAV capsid. scAAV and shAAV contain inverted dimer repeats that provide intramolecular double-stranded DNA. scAAV can be constructed by mutating the ITR terminal degradation site so that the rep cannot introduce a nick into the terminal degradation site. shAAV can utilize a short-chain hairpin to construct double-stranded AAV nucleic acid. Being double-stranded DNA, scAAV and shAAV offer the advantage of avoiding the DNA synthesis step required for single-stranded rAAV nucleic acid during cell entry. A possible drawback of scAAV and shAAV is the size of the DNA insert that can be incorporated, which is reduced to about half compared to single-stranded rAAV nucleic acids (each of them is incorporated herein by reference in whole, U.S. Patent No. 10,457,940; Xie et al., Mol Ther. (2017) 25(6): pp. 1363-1374; and McCarty Mol. Ther. (2008) 16(10): pp. 1648-1656).

[0118]

[0150] Naturally occurring AAV capsids contain viral proteins VP1, VP2, and VP3 in a ratio of approximately 1:1:10. AAV vectors can be constructed with all three viral proteins based on a specific serotype, or with one, two, or all three viral proteins based on different serotypes.

[0119]

[0151] Recombinant AAV capsids and nucleic acids may be based on the same serotype (or subgroup or mutant) or may be different from each other. In certain embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITR) as the capsidating capsid protein.

[0120]

[0152] In different embodiments, the rAAV capsid is at least 80% and at least 85% of VP1, VP2, or VP3 of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 12 or SEQ ID NO: 15 The product contains proteins that have sequence identity of at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, or at least 99.9%, or are 100% identical.

[0121]

[0153] A recombinant AAV capsid containing VP1 of Sequence ID No. 12 is described, for example, in U.S. Patent No. 9,840,719; an rAAV capsid containing VP1 of Sequence ID No. 15 is described, for example, in U.S. Patent No. 9,169,299; both of these patents are incorporated herein by reference.

[0122]

[0154] In certain embodiments, the AAV capsid includes VP1, VP2, and VP3, each independently having at least 80%, at least 90%, at least 95%, or 100% sequence identity with any of the following variants (e.g., amino acid insertions, additions, substitutions, and deletions, etc., capsid variants): AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 12 or SEQ ID NO: 15; and any of the following variants (e.g., amino acid insertions, additions, substitutions, and deletions, etc., capsid variants). (See, for example, U.S. Patent Nos. 9,909,142 and 9,840,719 disclosing RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6, whose disclosures are incorporated in their entirety herein; U.S. Patent Application Publication Nos. 2013 / 0059732 and 9,169,299 disclosing LK01, LK02 and LK03; and U.S. Patent No. 11,110,153.)

[0123]

[0155] In a particular embodiment, the capsid includes VP1 having the sequence of SEQ ID NO: 12; VP2 having the sequence of SEQ ID NO: 13; and VP3 having the sequence of SEQ ID NO: 14.

[0124]

[0156] In certain embodiments, AAV capsids can cross the blood-brain barrier and result in CNS expression. Examples of such AAV capsids and designs of AAV capsids capable of giving CNS expression are provided in Chen et al., (2021) J. Control. Release 333, pp. 129-138 (e.g., AAV9, AAV-PHP·B, AAV-PHP.eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent No. 9,585,971, each of which is incorporated herein in its entirety by reference; and Goertsen et al., (2022) Nat. Neurosci. 25, pp. 106-115 (2022).

[0125]

[0157] The AAV genome contains two main types of genes: rep and cap. Transcription from the rep genes is initiated by two different promoters, resulting in the production of non-structural proteins named Rep78, Rep68, Rep52, and Rep40. The rep proteins function in genome replication and / or capsidation. The cap genes encode structural proteins that constitute the capsid (VP1, VP2, and Vp3); non-structural assembly activating proteins (APP) that exert functions related to capsid assembly; and membrane-bound accessory proteins that can be associated with the growth phase of the replication cycle (the entire text is incorporated herein by reference, Maurer and Weitzman (2020) Hum. GeneTher. 31(9-10): pp. 499-511).

[0126]

[0158] AAV requires helper virus function to complete its replication cycle. Helper virus function can be supplied by various viruses in tolerant cell lines. Tolerant cell lines are cell lines that can support viral replication. Examples of helper viruses for AAV include adenoviruses, HSV-1, HPV-16, and HBoV1, which can be used with tolerant primate cells, for example; and baculoviruses, which can be used with tolerant insect cells such as sf9 (both of which are incorporated herein in their entirety by reference, Maurer and Weitzman (2020) Hum. GeneTher. (2020) 31(9-10): pp. 499-511 and Meier et al., (2020) Viruses 19; 12(6): p. 662).

[0127]

[0159] Recombinant AAV can be generated, for example, by supplying the viral proteins necessary for vector production in trans using a suitable helper virus or plasmid and cell line. In certain embodiments, rAAV is generated using an rAAV vector genome plasmid. The plasmid contains a portion of the rAAV nucleic acid that is ultimately packaged or capsidated to form a viral (e.g., rAAV) vector. The "plasmid backbone" contains elements important for reproduction and recombinant virus production. Except for possible 3'ITR and / or 5'ITR cloning residues, the plasmid backbone is not packaged or capsidated into viral particles itself.

[0128]

[0160] A vector genome plasmid may contain regions such as the origin of replication and selectable markers. Additional possible sites include cloning sites.

[0129]

[0161] Recombinant AAVs can be generated from various types of cell lines, including HeLa, A549, BHK, Vero, and HEK293, or their derivatives. In certain embodiments, HEK293 cells are used (American Type Culture Collection accession number ATCC CRL1573). Other host cell lines suitable for rAAV vector generation are described, for example, in Robert et al., (2017) Biotechnol. J. (2017) 12(3):1600 pp. 193; and international application PCT / US2017 / 024951, the disclosure of which is incorporated in its entirety herein.

[0130]

[0162] Recombinant AAVs can be cultured under a variety of different conditions suitable for cell growth and gene expression. References describing rAAV production include Clement and Grieger (2016) Mol.Ther.Methods Clin.Dev. 16;3:1600 p. 2; Robert et al., (2017) Biotechnol.J.12(3), 1600 p. 193; and Adeno-Associated Virus Vectors (2019), Castle, ed., 1st ed., Springer New York, New York, NY.

[0131]

[0163] In certain embodiments, AAV helper function is introduced into host cells by transfecting them with an AAV helper construct, either prior to or concurrently with the transfection of an AAV expression vector. Host cells possessing AAV helper function may be referred to as “helper cells” or “packaging helper cells.” Thus, AAV helper constructs are sometimes used to provide at least transient expression of AAV rep and / or cap genes in order to compensate for missing AAV function necessary for proliferative AAV transduction. AAV helper constructs often lack AAV ITR and are unable to replicate and package themselves. These constructs may take the form of plasmids, phages, transposons, cosmids, viruses, or virions, for example. Numerous AAV helper constructs are described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both rep and cap expression products. Numerous other vectors encoding rep and / or cap expression products are known. Recombined AAVs can be fabricated, for example, as described in U.S. Patent No. 9,408,904, and International Application Numbers PCT / US2017 / 025396 and PCT / US2016 / 064414, the entirety of which is incorporated herein by reference.

[0132]

[0164] In certain embodiments, the rAAV vector is produced by rAAV-producing cells containing rAAV helper virus activity. The genome of the rAAV-producing cells includes rAAV nucleic acid, a rep gene, and a cap gene.

[0133]

[0165] In certain embodiments, the rAAV vector is produced by culturing rAAV-tolerant cells containing an AAV genome plasmid, the rAAV-tolerant cells further comprising rep and cap genes provided either as part of the cell genome and / or by one or more separate plasmids; and helper virus activity provided either as part of the cell genome and / or by one or more separate plasmids. In further embodiments, (a) the rAAV-tolerant cell line is a packaging cell, and the genome of the packaging cell contains cap and rep genes; (b) the rep gene, cap gene, and helper activity are provided from the same plasmid; or (c) the rep gene and cap gene are provided by a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

[0134]

[0166] In certain embodiments, including the use of HSV helper functions, the helper functions are provided by genes encoding at least UL5, UL8, UL52, and ICP8.

[0135]

[0167] In certain embodiments, including the use of adenovirus helper functions, the helper functions are provided by genes encoding at least E1A, E1B19K, E1B55K, E2A, E4orf6, and VA RNA. In certain embodiments, E1, E2A, and VR RNA functions are provided by helper plasmids, and additional helper functions are provided by the host strain.

[0136]

[0168] In certain embodiments, the rAAV vector is obtained by the steps of generating rAAV using a method described herein and purifying the rAAV. Purification of rAAV can be performed using techniques such as gradient-based purification, column-based methods, and composite methods (see, for example, Ayuso et al., (2010), Curr Gene Ther. (2010) 10(6): pp. 423-436, which is incorporated herein by reference in its entirety.

[0137] VC Retrovirus Vector

[0169] Retroviruses are enveloped single-stranded RNA viruses containing 5' and 3' LTRs, as well as signal packaging sequences located immediately outside the LTRs. Different types of retroviral vectors may contain different amounts of viral genome. In certain embodiments, a retroviral vector is an HIV-based lentiviral vector that retains all cis-acting sequences necessary for viral RNA packaging, reverse transcription, and proviral DNA integration, but removes all HIV protein-coding genes. Lentiviral vectors have a packaging capacity of up to approximately 9 kb. If necessary, stuffer sequences can be used to increase rAAV nucleic acid size and packaging efficiency. Lentiviral vectors can be produced by supplying the viral proteins necessary for vector production in trans using appropriate plasmids and cell lines (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6: p. 53).

[0138] VI. Nonviral vectors

[0170] In certain embodiments, the gene delivery vehicle is a non-viral vector. Examples of non-viral vectors include nanoparticles and naked nucleic acids. A preferred non-viral vector is nanoparticles. A wide variety of nanoparticles can be used, including lipid nanoparticles (LNPs), polymeric nanoparticles, lipid polymer nanoparticles (LPNPs), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes (for example, each of these is incorporated herein by reference in whole: Riley and Vermerris, Nanomaterials (2017) pp. 201, 7, 94; Thomas et al., Molecules (2019) pp. 24, 374; Bochicchio et al., (2021) pp. 13, 198; Munagala et al., Cancer Letters (2021) pp. 505, 58; Fu et al., (2020) NanoImpact 20 pp. 100261; Neshat et al., (2020) Current See Opin. Biotechnol. 66: pp. 1-10; Ouranidis et al., (2022) Biomedicines, pp. 10, 50; and Qin et al., Signal Transduct Target Ther. (2022) May 21; 7(1): p. 166).

[0139]

[0171] If desired, nanoparticles can target cell types, for example, by targeting ligands that recognize target cell receptors. Examples of targeted ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies and proteins / peptides (e.g., RGD, epidermal growth factor, and low-density lipoproteins), and peptides (e.g., Teo et al., Advanced Drug Delivery Reviews (2016), pp. 98, 41).

[0140]

[0172] Nanoparticles can be used to deliver PPT1-coding polynucleotide constructs to cells. In different embodiments, nanoparticles can deliver additional therapeutic compounds; and one or more additional compounds are provided in different nanoparticles. References to compounds include small and large molecules (e.g., therapeutic proteins and antibodies).

[0141]

[0173] The preparation of different nanoparticles and the incorporation of nucleic acids and other compounds are known in the art. Examples of publications illustrating the incorporation of nucleic acids in specific nanoparticles such as LPNPs and LNPs include Teo et al., Advanced Drug Delivery Reviews (2016) 98, p. 41; Bochicchio et al., Pharmaceutics (2021) 13, p. 198; Mahzabin and Das, IJPSR (2021) 12(1), p. 65; and Teixeira et al., (2017) Prog. Lipid Res. October; 68: pp. 1-11 (each of these is incorporated herein by reference in whole). Factors that may affect the incorporation of small molecules into nanoparticles include hydrophobicity and the presence of ionizable moieties (see, for example, Nii and Ishii, Int. J. Pharm. (2005) 298: pp. 198-205; and Chen et al., J. Control. Release (2018) 286: pp. 46-54).

[0142] VI.A. Lipid-based delivery systems

[0174] Lipid-based delivery systems involve the use of lipids as components. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.

[0143]

[0175] "Lipid nanoparticles" or "LNPs" refer to lipid-based vesicles that are useful for the delivery of nucleic acid molecules and have nanoscale dimensions. In different embodiments, the nanoparticles are approximately 10 nm to 1000 nm, approximately 50 nm to 500 nm, or approximately 50 nm to 200 nm.

[0144]

[0176] DNA is negatively charged. Therefore, the inclusion of cationic lipids, such as aminolipids, may be beneficial for LNPs. Exemplary aminolipids are U.S. Patents 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, and 8,466,12, all of which are incorporated in their entirety herein. This is described in U.S. Patent No. 2, and No. 7,745,651, and U.S. Patent Application Publications 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125. In certain embodiments, the LNP includes an aminolipid described in U.S. Patent No. 9,512,073, which is incorporated in whole herein.

[0145]

[0177] The terms “cationic lipid” and “aminolipid” are used interchangeably herein to include lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and pH titrable amino groups (e.g., alkylamino or dialkylamino groups). Cationic lipids are typically protonated (i.e., positively charged) at pH below their pKa and substantially neutral at pH above their pKa. Cationic lipids may also be titrable cationic lipids. In certain embodiments, cationic lipids include a protonable tertiary amine (e.g., pH titrable) group; a C18 alkyl chain in which each alkyl chain may independently have one or more double bonds, one or more triple bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0146]

[0178] Examples of cationic lipids include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-C2K-DMA, XTC2, and also known as DLin-K-C2-DM). A) Examples include 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-M-C3-DMA, also known as MC3), salts thereof, and mixtures thereof. Other cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0147]

[0179] Further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino (methylpepiazino)-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy (dilinoleyoxy)-3-(dimethylamino)acetoxypropane (DLin-DAC), and 1,2-dilinoleyo Dilinoleoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3- (Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristiloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium Nium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-β-oxy)-3'-oxapene Examples include toxic)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS), and disubstituted spermine (D2S), or mixtures thereof.

[0148]

[0180] Numerous commercially available preparations of cationic lipids can be used, such as LIPOFECTIN® (including DOTMA and DOPE available from GIBCO / BRL) and LIPOFECTAMINE® (including DOSPA and DOPE available from GIBCO / BRL).

[0149]

[0181] Additional ionizable lipids that can be used include C12-200, 306Oi10, MC3, cKK-E12, bCKK-E12, Lipid 5, Lipid 9, ATX-002, ATX-003, and Merck-32. U.S. Patent Application Publication No. 2017 / 0367988 describes Merck-32.

[0150]

[0182] In further embodiments, cationic lipids may be present in amounts ranging from about 10% to about 85% based on the molar ratio of LNPs, or from about 50% to about 75% based on the molar ratio of LNPs.

[0151]

[0183] LNPs may contain neutral lipids. These neutral lipids may include lipid molecular species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Examples of such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by considerations including particle size and stability. In certain embodiments, the neutral lipid component may be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0152]

[0184] Lipids with diverse acyl chain groups of various chain lengths and degrees of saturation are available, or can be isolated or synthesized. In certain embodiments, lipids containing saturated fatty acids having carbon chain lengths in the range of C14 to C22 can be used. In certain embodiments, lipids containing mono- or diunsaturated fatty acids having carbon chain lengths in the range of C14 to C22 can be used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or phosphatidylcholine. Neutral lipids may also consist of phospholipids having other head groups such as sphingomyelin, dihydrosphingomyelin, or serine and inositol.

[0153]

[0185] In further embodiments providing neutral lipids, the neutral lipids may be present in amounts ranging from about 0.1% to about 99% by weight of LNPs or from about 5% to about 15% by weight of LNPs, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0154]

[0186] LNP may contain additional components such as sterols and polyethylene glycol. Sterols can impart fluidity to the LNP. As used herein, “sterol” means naturally occurring sterols of plant (plant sterols) or animal (zoosterol) origin, as well as synthetic sterols that do not exist naturally, all characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. Preferred sterols include those conventionally used in the field of liposomes, lipid vesicles or lipid particle preparations, most commonly cholesterol. Examples of plant sterols include campesterol, sitosterol, and stigmasterol. Other examples of sterols include sterol-modified lipids, such as those described in U.S. Patent Application Publication 2011 / 0177156. In different embodiments providing sterols, the sterols are present in amounts of about 1% to about 80% by weight of the LNP, or about 10% to about 25% by weight of the LNP.

[0155]

[0187] Polyethylene glycol (PEG) is a water-soluble polymer of ethylene PEG repeating units having terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG2000 has an average molecular weight of approximately 2,000 daltons, and PEG5000 has an average molecular weight of approximately 5,000 daltons. Commercially available PEGs from Sigma Chemical Co. and other companies include monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol torecylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM).

[0156]

[0188] In certain embodiments relating to PEG, PEG has an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In further embodiments, PEG is substituted with methyl groups at the terminal hydroxyl positions. In further embodiments, PEG has an average molecular weight of about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or from about 2,000 daltons, or from about 750 daltons.

[0157]

[0189] Examples of PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAAs) described in U.S. Patents 8,936,942 and 7,803,397. PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have various "anchor" lipid moieties for fixing the PEG portion to the surface of lipid vesicles. Suitable examples of PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates described in U.S. Patent 5,820,873 (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. In certain embodiments, the PEG-modified lipids may be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, the amount of PEG may be about 0.1% to about 50% by weight of LNP, or about 5% to about 15% by weight of LNP.

[0158]

[0190] In further embodiments relating to LNP size, prior to the step of encapsulating the nucleic acid, the LNPs have a size range of about 10 nm to 500 nm, or about 50 nm to about 200 nm, or 75 nm to about 125 nm.

[0159]

[0191] In certain embodiments relating to LNPs, LNPs are described in Billingsley et al., Nano Lett. 2020, 20, page 1578 or Billingsley et al., International Publication No. 2021 / 077066 (both of which are incorporated herein in whole by reference). Billingsley et al., and International Publication No. 2021 / 077066 describe LNPs containing lipid-anchored PEG, cholesterol, phospholipids and ionizable lipids. In certain embodiments, LNPs contain a C14-4 polyamine core and / or have a particle size of about 70 nm. C14-4 has the following structure: [ka]

[0160]

[0192] In certain embodiments, the LNP comprises a cationic lipid or lipopeptide as described in U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374, or International Publication No. 2021 / 077066 (each of which is incorporated herein in whole by reference). In certain embodiments, the LNP contains a cationic lipid, a cholesterol-based lipid, and / or one or more PEG-modified lipids. In certain embodiments, the LNP contains cKK-E12 (Dong et al., PNAS (2014) 111(11), p. 3955): [ka]

[0161]

[0193] In certain embodiments, the LNP includes a modified form of cKK-E12, referred to herein as "bCKK-E12", having the following structure: [ka]

[0162]

[0194] In certain embodiments, the LNP includes lipids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described by Sabnis et al., Molecular Therapy 2018, 26:6, pp. 1509–1519 (which are incorporated herein by reference in their entirety). In certain embodiments, the LNP includes lipids 5, 8, 9, 10, or 11 as described by Sabnis et al.

[0163]

[0195] Sabnis Other Lipid 5, Structure: [ka] It holds.

[0164]

[0196] Sabnis Other Lipid 9, structure: [ka] It holds.

[0165]

[0197] Additional lipids that may be used are, each of which is incorporated herein in whole by reference: Roces et al., Pharmaceutics, 2020, 12, p. 1095; Jayaraman et al., Angew. Chem. Int. Ed., 2012, 51, pp. 8529-8533; Maier et al., www.moleculartherapy.org, 2013, Vol. 21, No. 8, pp. 1570-1578; Liu et al., Adv. M. Examples include those described in ater.2019, 31, 1902575, e.g., BAMEA-O16B; Cheng et al., Adv.Mater., 2018, 30, 1805308, e.g., 5A2-SC8; Hajj et al., Small, 2019, 15, 1805097, e.g., 306Oi10; Du et al., U.S. Patent Application Publication No. 20160376224; and Tanaka et al., Adv.Funct.Mater., 2020, 30, 1910575.

[0166]

[0198] In further embodiments, the nanoparticles are LNPs. In further embodiments, the LNPs in molar percentage consist of, or substantially consist of, the following components: (1) about 20% to 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipids, about 0.1% to 10% of one or more PEG-conjugated lipids, and about 0% to about 70% of cholesterol; or (2) about 20% to 50% of one or more cationic lipids, about 5% to about 20% of one or more phospholipids, about 0.1% to 5% of one or more PEG-conjugated lipids, and about 20% to about 60% of cholesterol. In further embodiments, the phospholipids are neutral lipids; and the phospholipids are DOPE or DSPC.

[0167]

[0199] In certain embodiments, LNP in molar percent contains, substantially consists of, or consists of the following components: (1) cKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (2) bCKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (3) lipid 9, about 50%; C (4) Lipid 5, approximately 50%; C14-PEG2000, approximately 1.5%; cholesterol, approximately 38.5%; and DSPC, approximately 10%; (5) Ionizable lipids, approximately 50%; DSPC, approximately 10%; cholesterol, approximately 37.5%; and stabilizer (PEG-lipid), approximately 2.5%; or (6) is GenVoy-ILM® LNP (Precision NanoSystems).

[0168] VI.B. Polymer-based nanoparticles

[0200] Polymer-based delivery systems can be manufactured from a variety of natural and synthetic materials. DNA and other compounds can be captured within the polymer matrix of polymeric nanoparticles, or adsorbed or conjugated onto the surface of the nanoparticles. Examples of commonly used polymers for nucleic acid delivery include poly(lactic acid-coglycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI) and PEI derivatives, chitosan, dendrimers, polyanhydrides, polycaprolactone, polymethacrylate, poly-L-lysine, pullulan, dextran, and hyaluronic acid and poly-β-aminoesters (Thomas et al., (2019) Molecules 24, p. 3744).

[0169]

[0201] Polymer-based nanoparticles can have a variety of sizes ranging from approximately 1 nm to approximately 1000 nm, approximately 10 nm to approximately 500 nm, approximately 50 nm to approximately 200 nm, approximately 100 nm to approximately 150 nm, and below approximately 150 nm.

[0170] VI.C. Lipid Polymer Nanoparticles

[0202] Lipid polymer nanoparticles are hybrid nanoparticles that provide both lipid and polymer components and can be considered as LNPs or LPNPs in themselves. LPNP configurations can provide an outer polymer and inner lipid or an outer lipid and inner polymer. The presence of two different types of materials facilitates the design of nanoparticles to provide delayed release of components. Different lipid and polymer components can be selected considering the material to be delivered (see, for example, Teo et al., Advanced Drug Delivery Reviews (2016) 98, p. 41; Bochicchio et al., Pharmaceutics (2021) 13, p. 198; Mahzabin and Das, IJPSR (2021) 12(1), p. 65; and Teixeira et al., (2017) Prog. Lipid Res. October; 68: pp. 1-11).

[0171] IV.D. Protein and Peptide-Based Nanoparticles

[0203] Protein- and peptide-based systems can utilize a variety of different proteins and peptides. Examples of proteins that can be utilized include gelatin and elastin. Peptide-based systems, such as cell-penetrating peptides (CPPs), can be used.

[0172]

[0204] CPPs are short peptides (6 - 30 amino acid residues) with the potential for intracellular penetration to deliver therapeutic molecules. Most CPPs consist mainly of arginine and lysine residues, which makes them cationic and hydrophilic, but CPPs can also be amphiphilic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., HIV-1 Tat protein) or obtained by synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996 - 2006). Examples of CPPs include cationic CPPs such as Tat peptide, penetratin, protamine, poly-L-lysine, and polyarginine (highly positively charged); amphiphilic CPPs such as transportan, VT5, bacteriocin-7 (Bac7), proline-rich peptide (PPR), SAP(VRLPPP)3, TP10, pep-1, and MPG (chimeric or fusion peptides constructed from different sources containing both positively and negatively charged amino acid sequences); membrane-directed CPPs such as H625, SPIONs-PEG-CPP, and NP (showing both hydrophobic and amphiphilic characteristics and containing both large aromatic residues and small residues); and hydrophobic CPPs such as SG3, PFVYLI, pep-7, and fibroblast growth factor (containing only nonpolar motifs or residues).

[0173]

[0205] Protein and peptide nanoparticles can be provided in a variety of sizes, for example, in the range from about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or about 150 nm and below.

[0174]

[0206] VI.E. Peptide Cage Nanoparticles

[0207] Delivery systems based on peptide cages can be manufactured from proteinaceous materials that can assemble into cage-like structures to form a confined internal environment. Peptide cages can include a proteinaceous shell that self-assembles to form a protein cage (e.g., a structure having an internal cavity that is either naturally solvent-accessible or can be made solvent-accessible by changing the solvent concentration, pH, or equilibrium ratio). The monomers of the protein cage can be in their naturally occurring form or variant forms (e.g., fragments) including amino acid substitutions, insertions, and deletions.

[0175]

[0208] Various types of protein “shells” can assemble and can be loaded with various types of materials. Protein cages can be generated using viral coat protein(s) (e.g., from the protein coat of the bean chlorotic mottle virus), as well as non-viral proteins (e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Application Publication Nos. 20040028694 and 20090035389, each of which is incorporated herein by reference in its entirety).

[0176]

[0209] Examples of protein cages derived from non-viral proteins include: eukaryotic or prokaryotic ferritins and apolipoproteins such as 12 and 24 subunit ferritins; and heat shock proteins (HSPs) such as the class of 24 subunit heat shock proteins that form an internal core space, the small HSP of Methanococcus jannaschii, the 12-mer Dsp HSP of Escherichia coli (E. coli); and MrgA proteins.

[0177]

[0210] Protein cages can have various core sizes, such as in the range of approximately 1 nm to 1000 nm, approximately 10 nm to 500 nm, approximately 50 nm to 200 nm, approximately 100 nm to 150 nm, or below approximately 150 nm.

[0178] VI.F. Exosomes

[0211] Exosomes are small biological membrane vesicles that have been used to deliver a variety of cargoes, including small molecules, peptides, proteins, and nucleic acids. Exosomes are generally in the size range of approximately 30 nm to 100 nm and can be taken up by cells to deliver their cargo. The cargo can either associate with the exosome surface structure or be encapsulated within the exosome bilayer.

[0179]

[0212] Various modifications can be made to exosomes to facilitate cargo delivery and cell targeting. Modifications to facilitate cargo delivery include structures for associating with cargo, such as protein scaffolds and polymers. Modifications for cell targeting include ligand targeting and alteration of surface charge. Publications describing the preparation, modification, and use of exosomes for the delivery of various cargoes include Munagala et al., Cancer Letters (2021), 505, pp. 58; Fu et al., (2020) NanoImpact 20, pp. 100-261; and Dooley et al., (2021) Molecular Therapy 29(5), pp. 1729 (each of which is incorporated herein by reference).

[0180] VII. Pharmaceutical Compositions

[0213] The pharmaceutical composition comprises a pharmaceutically acceptable carrier that facilitates the administration and / or storage of a PPT1 polypeptide, a coding polynucleotide, a viral vector, or a non-viral vector. The term "pharmaceutically acceptable" indicates that the components do not cause substantially undesirable biological effects in the amounts used. A pharmaceutically acceptable carrier may contain various components, such as one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include salts, sugars, buffers, solvents, preservatives, proteins, and surfactants. A particular excipient may have two or more functions. pharmaceutically acceptable excipients and carriers that can be used with viral vectors are provided, for example, in International Publication No. 2021 / 071835.

[0181]

[0214] Pharmaceutical compositions can be formulated to be compatible with specific administration or delivery routes. Suitable compositions for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions, the preparations of which are typically sterile and may be isotonic with the blood of the intended recipient. Exemplary examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal oils, vegetable oils, and synthetic oils. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0182]

[0215] In embodiments, the pharmaceutical composition contains a formulation that can be injected into a target. Examples of injectable formulation components include isotonic sterile saline solution, salts (e.g., monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, and mixtures thereof), buffered saline solution, sugars (e.g., dextrose), and water for injection. The pharmaceutical composition may include a dry, for example, freeze-dried composition, which allows for a suitable solution composition for administration when sterile water or saline solution is added.

[0183]

[0216] Additionally, the suspension can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound to facilitate the preparation of a concentrated solution.

[0184]

[0217] "Effective dose" or "sufficient dose" means the amount that produces the indicated or desired effect. An effective dose may be administered as a single or multiple doses, alone or in combination with one or more other compositions (e.g., additional therapeutic agents or immunosuppressants), treatments, protocols, or therapeutic regimens, and may produce a long-term or short-term response.

[0185]

[0218] A pharmaceutical composition containing a transgene encoding the PPT1 polypeptide can be delivered to a subject to enable the production of the encoded protein. Delivery may be in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains sufficient genetic material to enable the recipient to produce a therapeutically effective amount of the protein in the subject.

[0186]

[0219] "Therapeutic dose" refers to the amount of active ingredient or component that produces the desired or indicated biological or medical response in the subject. The therapeutic dose can be determined based on the observed symptoms and / or through the use of biomarkers associated with a particular disease or disorder. The selection of a specific effective dose can be optimized by considering various factors, including the disease or disorder being treated or prevented, the symptoms involved, stability and efficacy in animal models, the patient's body weight, and the patient's immune status. The optimal dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and can be estimated according to the patient's condition. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0187]

[0220] In certain embodiments, the pharmaceutical composition containing the rAAV vector also contains an empty AAV capsid. In certain embodiments, the ratio of the empty AAV capsid to the rAAV vector in the pharmaceutical composition containing the rAAV vector and the empty AAV capsid is within the range of about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100 or between these ranges. In certain embodiments, the ratio of the empty AAV capsid to the rAAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0188]

[0221] Additional guidance and examples of pharmaceutical compositions and delivery systems are provided, for example, in Remington: The Science and Practice of Pharmacy (2020), 23rd edition, University of the Sciences in Philadelphia, Elsevier; The Merck Index (2013), 15th edition, edited by Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; and Ansel and Stoklosa, Pharmaceutical Calculations (2001), 11th edition, Lippincott Williams & Wilkins, Baltimore, MD.

[0189] VIII. Administration and Treatment

[0222] PPT1 polypeptides and coding polynucleotide constructs, viral vectors and nonviral vectors can be administered to, preferably, human subjects to provide prophylactic measures to reduce the likelihood or severity of a disease or disorder (order), and / or treatment of a diagnosed disease or disorder. In certain embodiments, specific therapeutic agents, routes of administration, and / or pharmaceutical compositions are selected in consideration of the specific disease or disorder being treated.

[0190]

[0223] Subjects having a particular disease or disorder or having an increased risk of a particular disease or disorder can be identified, for example, based on symptoms, PPT1 activity, biomarkers, and genetic markers. Treatment can be carried out, for example, in subjects having an increased risk of developing NCL1 symptoms and in subjects diagnosed with NCL1.

[0191]

[0224] NCL1 is an autosomal recessive progressive neurodegenerative disease whose symptoms vary depending on onset. The main symptoms are as follows: onset at birth (congenital), microcephaly, dysmorphic features, seizures and hyperactive activity; onset at 6 - 18 months of age (infantile), decreased head growth, regression of neurogenesis and seizures; onset at 2 - 4 years of age (variant), seizures, regression of neurogenesis and behavioral disorders; and onset at 5 - 7 years of age (juvenile), blindness and cognitive decline (Simonati and Williams (2022) Front. Neurol. 11; 13: 811686).

[0192]

[0225] Various mutations are associated with NCL1. References providing examples of mutations associated with NCL1 are incorporated herein in their entirety by reference, including Sheth et al. (2018) BMC Neurol. 12; 18(1): 203; Kumar et al., Advances in Protein Chemistry and Structural Biology (2022) 132: 89 - 109; Kousi et al. (2012) Hum. Mutat. 33(1): 42 - 63; and hyperlink: / / www.uniprot.org / uniprotkb / P50897 / entry#disease_variants (January 17, 2023).

[0193]

[0226] Due to the progressive nature of NCL1, early treatment is very important. In certain embodiments, treatment is carried out prior to the identification of the main symptoms. Such patients can be identified, for example, based on PPT1 enzyme activity levels and / or the presence of PPT1 mutations.

[0194]

[0227] In certain embodiments, treatment is performed in patients diagnosed with NCL1. Diagnosis may be based on symptoms, genetic testing, and / or enzyme activity measurements.

[0195]

[0228] Potential routes of administration include subcutaneous, epithelial, intradermal, subarachnoid, intraorbital, intramucosal, intranasal, intraperitoneal, intravenous, intrapleural, intraarterial, intracavitary, oral, intrahepatic, via portal vein, intramuscular, intraparenchymal, intracisional, intracranial, intracisional, intraventricular, lateral ventricle, or intraventricular administration. In certain embodiments, the virus or non-viral vector is administered to the patient via infusion in a pharmaceutical carrier.

[0196]

[0229] The preferred route of administration should result in therapeutic delivery to the CNS. Delivery to the CNS can be achieved using a variety of routes of initial administration, including intravenous administration outside the CNS, e.g., outside the brain and spinal cord, and administration to the eye (e.g., intravitreous and subretinal); and direct administration to the brain (e.g., intraparenchymal, lateral ventricle and cisterna magna) and / or the spine (e.g., subarachnoid space) (the entire text is incorporated herein by reference, Zhu et al. (2021) Trends Mol. Med. 27(6): pp. 524-537).

[0197]

[0230] If the initial administration site is outside the brain, administration to the brain can be facilitated by using techniques that facilitate transport across the blood-brain barrier. Examples of such techniques include disruption of the blood-brain barrier and the use of blood-brain barrier carriers (each of these is incorporated herein in whole by reference, Chen et al. (2021) J. Control. Release 333: pp. 129-138; Bellettato and Scrapa, Italian Journal of Pediatrics (2018) 44(Suppl 2): ​​p. 131; Haumann et al. (2020) CNS Drugs 34, pp. 1121-1131; and Camalleri et al. (2020) J. Clin. Neurophysiol. 37(2): pp. 104-117). Techniques that facilitate blood-brain barrier crossing can be used for the delivery vehicle and / or PPT1 protein.

[0198]

[0231] In certain embodiments, the treatment is carried out using an expression system that gives polypeptide expression outside the CNS (e.g., high hepatic expression) in combination with a technique that promotes PPT1 transport across the blood-brain barrier.

[0199]

[0232] In certain embodiments, treatment is performed using techniques to facilitate the passage of the delivery vehicle across the blood-brain barrier. In further embodiments, passage across the blood-brain barrier is facilitated using focused ultrasound combined with microbubbles (see, for example, Camalleri et al., (2020) J Clin Neurophysiol. 37(2): pp. 104-117, which is incorporated herein by reference in whole).

[0200]

[0233] In certain embodiments, AAV capsids that are delivered to the CNS or cross the blood-brain barrier are used. Examples of such capsids are provided in Chen et al., (2021) J. Control. Release 333: pp. 129-138 (e.g., AAV9, AAV-PHP·B, AAV-PHP.eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent No. 9,585,971, and U.S. Patent Application Publication No. 202 / 1214749, each of which is incorporated herein in its entirety by reference.

[0201]

[0234] CNS administration can also be performed, for example, by direct administration to the brain using a needle or catheter (for example, International Publication No. 2021 / 108809; Cohen-Pferrer et al., Pediatric Neurology 67(2017) pp. 23-35; and U.S. Patent No. 10,369,329, each of which is incorporated herein in whole by reference).

[0202]

[0235] Another example of a technique for CNS administration is convection-enhanced delivery. Convection-enhanced delivery involves surgical exposure of the brain followed by direct catheter placement into a target area, and subsequently the injection of the therapeutic agent (for example, both of which are incorporated herein in whole by reference, U.S. Patent Application Publication 2022 / 010001; and Debinski et al., (2009) Expert Rev Neurother. 9(10): pp. 1519-27).

[0203]

[0236] Other examples of CNS delivery devices, systems, and technologies include those described in U.S. Patent No. 8,128,600, U.S. Patent Application Publication No. 2020 / 0324089, U.S. Patent No. 1,1129643, U.S. Patent No. 1,1154377, U.S. Patent Application Publication No. 2021 / 0343397, U.S. Patent Application Publication No. 2021 / 0282866, U.S. Patent No. 9,572,928, U.S. Patent No. 8337458, U.S. Patent No. 10722265, and U.S. Patent Application Publication No. 2021 / 214749, each of which is incorporated in whole herein by reference.

[0204]

[0237] Delivery of PPT1 polypeptides and coding nucleic acids may also provide benefits in the treatment of PPT1 deficiency or lysosomal storage outside the CNS. In certain embodiments, administration results in systemic delivery. In further embodiments, treatment involves the use of expression cassettes or viral vectors containing ubiquitous or promiscuous promoters. In further embodiments, CNS entry is facilitated by the use of techniques and / or vectors that facilitate transport across the blood-brain barrier.

[0205]

[0238] In certain embodiments, the expression cassette includes a PGK promoter, a CBh promoter, or an E1F alpha promoter.

[0206]

[0239] The optimal dose may vary depending on various factors such as the specific therapeutic agent, the desired endpoint, and the route of administration. The dose, amount, frequency, or duration may be proportionally increased or decreased in consideration of adverse side effects, complications, or other risk factors for the treatment or therapy, as well as the patient's condition.

[0207]

[0240] A "unit dosage form" refers to a physically distinct unit containing a predetermined effective amount of active ingredient in combination with a pharmaceutically acceptable carrier. Unit dosage forms can be provided, for example, in ampoules and vials that can contain a pharmaceutically acceptable carrier, or in a freeze-dried or lyophilized composition. In the case of a lyophilized composition, a sterile liquid carrier may be added prior to administration. Individual unit dosage forms can be included in multi-dose kits or containers.

[0208]

[0241] An "effective dose" achieves the desired or indicated effect. For example, an effective dose for treatment reduces one or more adverse symptoms, reduces the likelihood of one or more symptoms associated with a disease or disorder, or reduces the progression of a disease or disorder. A preferred effective dose for treatment is effective in reducing several or all adverse symptoms.

[0209]

[0242] In certain embodiments, the pharmaceutical composition is administered to a subject in a dose suitable for increasing PPT1 activity. In certain embodiments, the dose is sufficient to increase PPT1 activity to levels of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% normal activity; about 10% to 200% average activity, 20% to 150% average activity, 30% to 100% average activity; or about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, or about 150% average activity. In certain embodiments, PPT1 activity is increased to 10×, 100×, or 1000× average activity. Average activity refers to the average activity that occurs in a population.

[0210]

[0243] In different embodiments, suitable dosages are vectors per kg of the subject's body weight of about 0.01 mg / kg to about 10 mg / kg, vectors per kg of the subject's body weight of about 0.01 mg / kg to about 0.1 mg / kg, vectors per kg of the subject's body weight of about 0.1 mg / kg to about 1.0 mg / kg, or vectors per kg of the subject's body weight of about 1.0 mg / kg to about 10 mg / kg.

[0211]

[0244] Generally, the rAAV dosage is at least 1×10 8 vector genomes (vg / kg) per kg of the subject's body weight to achieve a therapeutic effect, for example, 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 or 1×10 14 vector genomes (vg / kg) or more. In different embodiments, the rAAV dosage is greater than about 5×10 11 rAAV vg / kg or greater than about 5×10 11 rAAV vg / kg; greater than about 1×10 12 rAAV vg / kg or greater than about 1×10 12 rAAV vg / kg; greater than about 2×10 12 rAAV vg / kg or greater than about 2×10 12 rAAV vg / kg; greater than about 3×10 12 rAAV vg / kg or greater than about 3×10 12 rAAV vg / kg; greater than about 4×10 12 rAAV vg / kg or greater than about 4×10 12 rAAV vg / kg; greater than about 5×10 12 rAAV vg / kg or greater than about 5×10 12 rAAV vg / kg; greater than about 1×10 13 rAAV vg / kg or greater than about 1×10 13 rAAV vg / kg; greater than about 2×10 13 rAAV vg / kg or greater than about 2×10 13 rAAV vg / kg; greater than about 3×10 13 rAAV vg / kg or greater than about 3×10 13rAAV vg / kg; approx. 4×10 13 rAAV vg / kg or approximately 4 × 10 13 rAAV over vg / kg; approx. 5×10 13 rAAV vg / kg or approximately 5 × 10 13 rAAV vg / kg; approx. 6×10 13 rAAV vg / kg or approximately 6 × 10 13 rAAV is greater than vg / kg.

[0212]

[0245] An example of a dose range for rAAV vg / kg is approximately 5 × 10 11 ~about 6×10 13 rAAV dose range vg / kg; approximately 5 × 10 11 ~Approx. 5.5×10 11 rAAV dose range vg / kg: approximately 5.5 × 10 11 ~about 6×10 11 rAAV dose range vg / kg; approximately 6 × 10 11 ~Approx. 6.5×10 11 rAAV dose range vg / kg: approximately 6.5 × 10⁻⁶ 11 ~Approx. 7×10 11 rAAV dose range vg / kg; approximately 7 × 10 11 ~Approx. 7.5×10 11 rAAV dose range vg / kg: approximately 7.5 × 10 11 ~Approx. 8×10 11 rAAV dose range vg / kg; approximately 8 × 10 11 ~Approx. 8.5×10 11 rAAV dose range vg / kg: approximately 8.5 × 10 11 ~Approx. 9×10 11 rAAV dose range vg / kg; approximately 9 × 10 11 ~Approx. 9.5×10 11 rAAV dose range vg / kg: approximately 9.5 × 10 11 ~Approx. 1×10 12 rAAV dose range vg / kg; approximately 1 × 10⁻⁶ 12 ~Approx. 1.5×10 12 rAAV dose range vg / kg: approximately 1.5 × 10⁻⁶ 12 ~about 2×10 12 rAAV dose range vg / kg; approximately 2 × 10 12 ~Approx. 2.5×1012 The dosage range of rAAV vg / kg; about 2.5×10 12 ~ about 3×10 12 The dosage range of rAAV vg / kg; about 3×10 12 ~ about 3.5×10 12 The dosage range of rAAV vg / kg; about 3.5×10 12 ~ about 4×10 12 The dosage range of rAAV vg / kg; about 4×10 12 ~ about 4.5×10 12 The dosage range of rAAV vg / kg; about 4.5×10 12 ~ about 5×10 12 The dosage range of rAAV vg / kg; about 5×10 12 ~ about 5.5×10 12 The dosage range of rAAV vg / kg; about 5.5×10 12 ~ about 6×10 12 The dosage range of rAAV vg / kg; about 6×10 12 ~ about 6.5×10 12 The dosage range of rAAV vg / kg; about 6.5×10 12 ~ about 7×10 12 The dosage range of rAAV vg / kg; about 7×10 12 ~ about 7.5×10 12 The dosage range of rAAV vg / kg; about 7.5×10 12 ~ about 8×10 12 The dosage range of rAAV vg / kg; about 8×10 12 ~ about 8.5×10 12 The dosage range of rAAV vg / kg; about 8.5×10 12 ~ about 9×10 12 The dosage range of rAAV vg / kg; about 9×10 12 ~ about 9.5×10 12 The dosage range of rAAV vg / kg; about 9.5×10 12 ~ about 1×10 13 The dosage range of rAAV vg / kg; about 1×10 13 ~ about 1.5×10 13 The dosage range of rAAV vg / kg; about 1.5×10 13 ~ about 2×10 13 The dosage range of rAAV vg / kg; about 2×10 13 ~ about 2.5×10 13The dosage range of rAAV vg / kg; about 2.5×10 13 ~ about 3×10 13 The dosage range of rAAV vg / kg; about 3×10 13 ~ about 3.5×10 13 The dosage range of rAAV vg / kg; about 3.5×10 13 ~ about 4×10 13 The dosage range of rAAV vg / kg; about 4×10 13 ~ about 4.5×10 13 The dosage range of rAAV vg / kg; about 4.5×10 13 ~ about 5×10 13 The dosage range of rAAV vg / kg; about 5×10 13 ~ about 5.5×10 13 The dosage range of rAAV vg / kg; about 5.5×10 13 ~ about 6×10 13 The dosage range of rAAV vg / kg; about 6×10 13 ~ about 1×10 14 The dosage range of rAAV vg / kg may be included.

[0213]

[0246] In certain embodiments, rAAV vg / kg is about 5×10 11 vg / kg, about 6×10 11 vg / kg, about 7×10 11 vg / kg, about 8×10 11 vg / kg, about 9×10 11 vg / kg, about 1×10 12 vg / kg, about 2×10 12 vg / kg, about 3×10 12 vg / kg, about 4×10 12 vg / kg, about 5×10 12 vg / kg, about 6×10 12 vg / kg, about 7×10 12 vg / kg, about 8×10 12 vg / kg, about 9×10 12 vg / kg, about 1×10 13 vg / kg, about 2×10 13 vg / kg, about 3×10 13 vg / kg, about 4×10 13 vg / kg, about 5×10 13 vg / kg, or about 6×10 13It is administered at a dose of vg / kg.

[0214]

[0247] In certain embodiments, doses and dose ranges for other viral vectors are as provided herein with respect to rAAV. For example, in certain embodiments, doses and dose ranges for recombinant adenovirus vectors, recombinant retrovirus vectors (e.g., lentiviruses), and recombinant herpes simplex virus vectors are the same as those exemplified above with respect to rAAV.

[0215]

[0248] In different embodiments, preferred doses for PPT1 administration are approximately 0.01 mg / kg to approximately 25 mg / kg of protein per kg of body weight of the subject, or approximately 0.1 mg / kg to approximately 1.0 mg / kg of protein per kg of body weight of the subject.

[0216]

[0249] In certain embodiments, the polypeptide constructs, polynucleotide constructs, viral vectors, and nonviral vectors described herein are administered in combination with additional compounds or therapies for specific diseases or disorders; and / or in combination with compounds that reduce the immune response produced against the provided or constructed polypeptides, polynucleotides, and / or delivery vehicles. The additional compounds or therapies may be provided in various modalities, such as being administered separately; and being administered or performed substantially simultaneously with, prior to, or after the administration of the polypeptide constructs, coding polynucleotide constructs, viral vectors, and nonviral vectors described herein.

[0217]

[0250] In certain embodiments, the administration of polypeptide constructs, coding polynucleotide constructs, viral vectors, and non-viral vectors described herein is in combination with immunosuppressants or regimens. Such agents and regimens may be used, if necessary, to achieve immune tolerance to the produced PPT1 protein, the provided polynucleotide, or the provided delivery vehicle, or to mitigate the immune response. Examples of immunosuppressants and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particle (SVP)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles)), ImmTOR-IL® (ImmTOR with a Treg-selective IL-2 agonist), B-cell depletion, immunoadsorption, and plasmapheresis.

[0218]

[0251] In certain embodiments, a viral vector or nonviral vector is administered in combination with one or more immunosuppressants, where one or more immunosuppressants are administered prior to, substantially simultaneously with, or after the administration of the vector or nonviral vector. In certain embodiments, one or more immunosuppressants are administered at the same time as the vector or nonviral vector. In certain embodiments, one or more immunosuppressants are administered 1 to 12, 12 to 24, or 24 to 48 hours prior to the administration of the viral or nonviral vector; or 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 14, 14 to 20, 20 to 25, 25 to 30, 30 to 50 days, or more than 50 days prior. In certain embodiments, one or more immunosuppressants are administered 1–12, 12–24, or 24–48 hours after administration of a viral or nonviral vector; or 2–4, 4–6, 6–8, 8–10, 10–14, 14–20, 20–25, 25–30, 30–50 days, or more than 50 days. Administration of immunosuppressants after the period following administration of a vector or nonviral vector may be performed, for example, over the period following administration of a vector or nonviral vector, e.g., 20–25, 25–30, 30–50, 50–75, 75–100, 100–150, 150–200, or more than 200 days, if a decrease in the encoded protein after the initial expression level is observed.

[0219]

[0252] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid, for example, a corticosteroid. In certain embodiments, the immunosuppressant is prednisone, prednisolone, calcineurin inhibitors (e.g., cyclosporine, tacrolimus), MMF (mycophenolic acid, for example, CellCept®, Myfortic®), CD52 inhibitors (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, beratacept), anti-CD3 mAb, anti-LFA-1 mAb (e.g., ephalizumab), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epratuzumab), anti-CD20 mAbs (e.g., rituximab, orelizumab, ofatumumab, beltuzumab), proteasome inhibitors (e.g., bortezomib), TACI-Ig (e.g., atacicept), anti-C5 mAbs (e.g., eculizumab), mycophenolate esters, azathioprine, sirolimus, everolimus, TNFR-Ig, anti-TNF mAbs, tofacitinib, anti-IL-2R (e.g., basiliximab), anti-IL-17 mAbs (e.g., secukinumab), anti-IL-6 mAbs (e.g., anti-IL-6 antibody silkumab, anti-IL-6 receptor antibody tocilizumab (Actemra®)), IL-10 inhibitors, TGF-β inhibitors, B-cell targeted antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particles (SVP®)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles), intravenous gamma globulin (IVIG), omalizumab, methotrexate, tyrosine kinase inhibitors (e.g., ibrutinib), cyclophosphamide, fingolimod, B-cell activator (BAFF) inhibitors (e.g., anti-BAFF mAbs, e.g., belimumab), proliferation-inducing ligand inhibitors (APRIL), anti-IL-1β These include mAbs (e.g., canakinumab (Haris®)), C3a inhibitors, tregitopes (see, for example, U.S. Patent No. 10,213,496), or combinations and / or derivatives thereof.

[0220]

[0253] Immunosuppressive protocols, including the use of rapamycin alone or in combination with IL-10, can be used to reduce, mitigate, inhibit, prevent, or block humoral and cellular immune responses to the PPT1 protein. Liver gene transfer using viral vectors (e.g., rAAV) and non-viral vectors can be used to induce immune tolerance to the PPT1 protein through the induction of regulatory T cells (Tregs).

[0221]

[0254] Strategies to reduce (overcome) or evade humoral immunity to viral vectors such as rAAV in systemic gene transfer include: administration of high vector doses; use of empty AAV capsids as decoys to adsorb anti-AAV antibodies; administration of immunosuppressants to reduce, mitigate, inhibit, prevent, or eradicate the humoral immune response to rAAV; alteration of rAAV capsid serotype or genetic modification of rAAV capsids to reduce susceptibility to neutralizing antibodies; use of plasma exchange cycles to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers; and use of delivery techniques such as balloon catheters and subsequent saline flushing. Such strategies are described in Mingozzi et al., (2013) Blood, 122: pp. 23-36. An additional strategy is the use of an AAV-specific plasmapheresis column to selectively deplete anti-AAV antibodies from plasma without depleting the total immunoglobulin pool, as described by Bertin et al., 2020, Sci.Rep.10:864. Similar techniques and strategies can be used for other types of viral vectors.

[0222]

[0255] Empty capsids used as decoy probes can be supplied in different ratios to the viral vector. The amount of empty capsid administered can be adjusted based on the amount (titer) of antibodies produced in a particular target. In certain embodiments, the ratio of empty AAV capsid to rAAV vector is within the range of approximately 100:1 to 50:1, approximately 50:1 to 25:1, approximately 25:1 to 10:1, approximately 10:1 to 1:1, approximately 1:1 to 1:10, approximately 1:10 to 1:25, approximately 1:25 to 1:50, or approximately 1:50 to 1:100 or between these ranges. In certain embodiments, the ratio of empty AAV capsid to rAAV vector administered is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Preferably, the serotype of the empty capsid is the same as the rAAV serotype.

[0223]

[0256] Strategies for reducing humoral immunity to rAAV (which can be applied to other viral vectors) include methods for removing, depleting, capturing, and / or inactivating AAV antibodies, commonly referred to as apheresis, and more specifically plasmapheresis when blood products are involved. Apheresis or plasmapheresis is a process of ex vivo (outside the body) circulation of a human subject's plasma through a device that alters the plasma by adding, removing, and / or replacing components before returning it to the patient. Plasmapheresis can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). This procedure can be used to deplete, capture, inactivate, reduce, or remove AAV-binding immunoglobulins (antibodies) that may contribute to rAAV neutralization, thereby reducing the titer of AAV antibodies in the target being treated. Examples include using a device consisting of an AAV capsid affinity matrix column and passing blood products (e.g., plasma) through an AAV capsid affinity matrix that provides binding affinity for various isotypes of AAV antibodies (see, for example, Bertin et al., (2020) Sci. Rep. 10, p. 864, which is incorporated herein in its entirety by reference).

[0224]

[0257] In certain embodiments, polypeptide constructs, coding polynucleotide constructs, viral vectors, and non-viral vectors may be used in combination with agents that block, inhibit, or reduce the interaction between neonatal Fc receptor (FcRn) and IgG, such as anti-FcRn antibodies, to reduce IgG recycling and enhance IgG clearance in vivo, and / or agents that reduce circulating antibodies bound to PPT1 polypeptides, coding nucleic acids, or delivery vehicles. In certain embodiments, antibody binding is reduced or inhibited by agents that reduce the interaction between IgG and FcRn, proteases, or glycosidases.

[0225]

[0258] In certain embodiments, the polypeptide constructs, polynucleotide constructs, viral vectors, and non-viral vectors described herein are used in combination with endopeptidases (e.g., IdeS derived from Streptococcus pyogenes) or modified variants thereof, or with endoglycosidases (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof. Such therapies can be carried out, for example, to reduce or eliminate neutralizing antibodies and to enable the treatment of patients who were previously considered unsuitable for treatment. Such strategies are described, for example, by Leborgne et al., (2020) Nat. Med., 26: pp. 1096-1101.

[0226]

[0259] In certain embodiments, the therapeutic method in a subject is carried out in combination with a compound that reduces the expression of native mutant PPT1, resulting in mutant PPT1 with reduced activity. Mutant PPT1 expression can be inhibited, for example, using inhibitory nucleic acids that selectively target the mutant PPT1 coding sequence. The reference to “selectively targeting” mutant PPT1 activity indicates that the expression of the polynucleotide encoding the PPT1 protein, resulting in increased activity, is not significantly affected. The inhibitory nucleic acid can be provided on the same polynucleotide and / or vector encoding the PPT1 protein, or using a separate viral or non-viral vector. Examples of inhibitory nucleic acids include short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), ribozymes, and antisense RNA.

[0227] IX. Kit

[0260] The present invention includes a kit comprising a packaging material and one or more components thereof. The kit typically includes a label or accompanying document, including a description or instructions for the in vitro, in vivo, or ex vivo use of the components thereof. The kit may include such components, for example, a PPT1 polypeptide, a virus or nonviral vector, and optionally a second active ingredient such as another compound, drug, or composition.

[0228]

[0261] A kit refers to a physical structure that contains one or more components. The packaging material can keep the components sterile and may be made of materials commonly used for such purposes, such as paper, cardboard, glass, plastic, foil, ampoules, vials, and test tubes.

[0229]

[0262] The label or package insert may include identification information, dosage, mechanism of action, pharmacokinetics, and pharmacodynamics of one or more of its components, as well as clinical pharmacology of the active ingredient(s). The label or package insert may include manufacturer information, lot number, place and date of manufacture, and information identifying the expiration date. The label or package insert may include information about diseases for which the kit components can be used. The label or package insert may include instructions for clinicians or subjects on how to use one or more of the kit components in a method, use, or treatment protocol or treatment regimen. The instructions may include dosage, frequency, or duration, and instructions on how to perform any of the methods, uses, treatment protocols, or prophylactic or treatment regimens described herein.

[0230]

[0263] Labels or package inserts may include information about one or more benefits that the components may offer, such as preventive or therapeutic benefits. Labels or package inserts may include information about possible adverse side effects, complications, or reactions, such as notes to the subject or clinician about situations in which the use of a particular composition is inappropriate. Adverse side effects or complications may also occur if the subject has taken, will take, or is currently taking one or more other drugs that may be incompatible with the composition, or if the subject has taken, will take, or is currently taking another treatment protocol or treatment regimen that may be incompatible with the composition, and therefore the instructions may include information about such incompatibility.

[0231]

[0264] Labels or accompanying documents include "printed materials," such as paper or cardboard, that are separate from or attached to the components, kit, or packaging material (e.g., a box), or attached to the ampoules, test tubes, or vials containing the kit components. Labels or accompanying documents may additionally include computer-readable media such as barcode printed labels, discs, CDs or DVD-ROM / RAM, optical discs such as DVDs and MP3s, magnetic tapes, or electrical storage media such as RAM and ROM, or magnetic / optical storage media, flash media, or hybrids thereof such as memory cards.

[0232] X mRNA therapeutic agents

[0265] In certain embodiments, RNA versions of the nucleic acids encoding the PPT1 polypeptide described herein are provided as mRNA constructs capable of expressing the encoded polypeptide within a cell. The mRNA construct comprises a 5' cap, a 5' UTR, coding RNA, a 3' UTR, and a poly(a) tail. The UTR and poly(a) tail can provide various functions, such as being involved in the intracellular localization of mRNA and regulating translation efficiency and mRNA stability. The design and generation of mRNA constructs, including various modifications, are exemplified in various publications, each of which is incorporated herein in its entirety by reference; Ouranidis et al., (2022) Biomedicines, 10, p. 50; Qin et al., Signal Transduct Target Ther. (2022) 21;7(1):166; and U.S. Patent Application Publication No. 2013 / 0259924.

[0233]

[0266] In certain embodiments, mRNA constructs are delivered to cells or targets using nanoparticles. Examples of nanoparticles include those provided in Section VI (including VI.A. to V.E.) above, Ouranidis et al., (2022) Biomedicines, 10, pp. 50, and U.S. Patent Application Publication No. 2013 / 0259924.

[0234] XI. Additional aspects and embodiments

[0267] Examples of additional aspects, embodiments, and combinations thereof include the following: 1) A polynucleotide comprising a nucleic acid sequence encoding a palmitoyl protein thioesterase-1 (PPT1) polypeptide, wherein the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity, at least 97% identity, or at least 99% identity with respect to the sequence of SEQ ID NO: 1, (a) The PPT1 polypeptide further comprises a variant thereof having any of the signal sequences from SEQ ID NOs. 16 to 27 or one amino acid substitution, deletion, or insertion; and / or (b) The PPT1 amino acid sequence includes a glycine (G), valine (V), or leucine (L) substitution of aspartic acid (D) at its amino terminus; and / or (c) The PPT1 sequence contains the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus; and / or (d) The nucleic acid sequence includes a PPT1 coding sequence that has at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity with any of sequence numbers 61 to 94. Polynucleotide. 2) The polynucleotide according to claim 1, wherein the PPT1 polypeptide further comprises the signal sequence which includes any of the sequences of sequence numbers 16 to 27. 3) The polynucleotide according to 2, wherein the nucleic acid contains one of the signal coding sequences among sequence numbers 43 to 58. 4) The polynucleotide according to 2, wherein the polypeptide contains one of the signal sequences from SEQ ID NOs. 16-21 and 24-27. 5) The polynucleotide according to 4, wherein the signal sequence includes the sequence of SEQ ID NO: 16 or 19. 6) The polynucleotide according to 5, wherein the signal sequence includes the sequence of SEQ ID NO: 16 and the nucleic acid sequence includes the signal coding sequence of SEQ ID NO: 43, or the signal peptide includes the sequence of SEQ ID NO: 19 and the nucleic acid sequence includes the signal coding sequence of SEQ ID NO: 50. 7) The polynucleotide according to 2, wherein the polypeptide comprises the signal coding sequence of SEQ ID NO: 23. 8) The polynucleotide according to 7, wherein the nucleic acid sequence includes the signal coding sequence of SEQ ID NO: 54. 9) The polynucleotide according to any one of 1 to 6, wherein the PPT1 amino acid sequence includes a G, V, or L substitution of aspartate D at its amino terminus, and the PPT1 amino acid sequence has at least 95% identity, at least 97% identity, or at least 99% identity with SEQ ID NO: 1; or includes SEQ ID NO: 1. 10) The polynucleotide according to 9, wherein the PPT1 amino acid sequence includes the sequence of SEQ ID NO: 2, and X is G, X is V, or X is L. 11) The polynucleotide according to any one of 1 to 3, 7, or 8, wherein the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, and the PPT1 amino acid sequence has at least 95% identity, at least 97% identity, or at least 99% identity with respect to the sequence of SEQ ID NO: 1. 12) The polynucleotide according to 11, wherein the PPT1 sequence includes sequence number 4. 13) The nucleic acid containing the PPT1 coding sequence contains a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to any of sequence numbers 61 to 94, or contains any of sequence numbers 61 to 94; in a further embodiment, the PPT1 coding sequence contains a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to any of sequence numbers 62 to 64, 71, 74, 78, 79, or 83; in a further embodiment, the PPT1 coding sequence has at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to any of sequence numbers 62 to 64, 71, 74, 78, 79, or 83 A polynucleotide according to any one of 1 to 12, comprising a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity, or comprising any of the sequences of SEQ ID NOs. 62-64, 71, 74, 78, 79, or 83; in a further embodiment, the PPT1 coding sequence comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the sequence of SEQ ID NO. 64, or comprising the sequence of SEQ ID NO. 64; in a further embodiment, the PPT1 coding sequence comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the sequence of SEQ ID NO. 79, or comprising the sequence of SEQ ID NO. 79. 14) The polynucleotide according to claim 1, wherein the PPT1 polypeptide contains a sequence having at least 99% identity with any of SEQ ID NOs. 31 to 42, or contains any of SEQ ID NOs. 31 to 42. 15) The polynucleotide according to 14, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31 or SEQ ID NO: 34. 16) The polynucleotide according to 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO 31, and the nucleic acid comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to any of SEQ ID NOs 107-125 and 168; or the PPT1 polypeptide comprises the sequence of SEQ ID NO 31, X is G, and the nucleic acid comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to any of SEQ ID NOs 107-125, or comprises any of SEQ ID NOs 107-125 and 168. In a further embodiment, the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, where X is G, and the nucleic acid comprises a sequence having at least 85% identity, at least 90% identity, at least 95%, and at least 99% identity with respect to SEQ ID NO: 64, or comprises the sequence of SEQ ID NO: 64; in a further embodiment, the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, where X is G, and the nucleic acid comprises a sequence having at least 85% identity, at least 90% identity, at least 95%, and at least 99% identity with respect to SEQ ID NO: 79, or comprises the sequence of SEQ ID NO: 79. 17) The polynucleotide according to 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO 34, and the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with respect to any of SEQ ID NOs 126-140 and 161-167; or the PPT1 polypeptide comprises the sequence of SEQ ID NO 34, where X is G, and the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with respect to any of SEQ ID NOs 126-140 and 161-167, or comprises any of SEQ ID NOs 126-140 and 161-167. 18) The polynucleotide according to claim 1, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 38. 19) A palmitoyl protein thioesterase-1 (PPT1) polypeptide comprising a PPT1 amino acid sequence having at least 95% identity, at least 97% identity, and at least 99% identity with respect to the sequence of Sequence ID No. 1, (a) The PPT1 polypeptide further comprises a variant thereof having any of the signal sequences from SEQ ID NOs. 16 to 27 or one amino acid substitution, deletion, or insertion; and / or (b) The PPT1 amino acid sequence includes a glycine (G), valine (V), or leucine (L) substitution of aspartic acid (D) at its amino terminus; and / or (c) The PPT1 sequence contains the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, Polypeptide. 20) The polypeptide according to 19, wherein the PPT1 polypeptide further comprises a signal sequence containing any of the sequences 16 to 27. 21) The polypeptide according to 20, wherein the polypeptide contains any of the signal sequences among sequence numbers 16-21 and 24-27. 22) The polypeptide according to 21, wherein the signal sequence includes either sequence number 16 or 19. 23) The polypeptide according to 20, wherein the polypeptide comprises the signal sequence of SEQ ID NO: 23. 24) The polypeptide according to any one of 19 to 23, wherein the PPT1 amino acid sequence includes a G, V, or L substitution of aspartic acid D at its amino terminus, and the PPT1 amino acid sequence has at least 97% identity or at least 99% identity with the sequence of SEQ ID NO: 1. 25) The polypeptide according to 24, wherein the PPT1 amino acid sequence includes the sequence of SEQ ID NO: 2, and X is G. 26) The polypeptide according to 19, wherein the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, and the PPT1 amino acid sequence has at least 97% or at least 99% identity with the sequence of SEQ ID NO: 1. 27) The polypeptide according to 26, wherein the PPT1 sequence includes sequence number 4. 28) The polypeptide according to 19, wherein the PPT1 polypeptide comprises a sequence having at least 99% or 100% identity with any of sequence numbers 31 to 42. 29) The polypeptide according to 28, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, SEQ ID NO: 34, or SEQ ID NO: 38, where X is G. 30) A polynucleotide comprising a PPT1 polypeptide coding nucleic acid sequence, wherein the PPT1 coding nucleic acid sequence encodes the PPT1 polypeptide described in any one of 19 to 29. 31) A polynucleotide comprising two or more exons and one or more introns that collectively encode the PPT1 polypeptide described in any one of 19 to 29. 32) A polynucleotide according to any one of 1 to 18, 30, or 31, which is an expression cassette comprising one or more expression regulatory elements operably linked to the nucleic acid encoding the PPT1 polypeptide. 33) The polynucleotide according to 32, wherein the nucleic acid encoding the PPT1 polypeptide is operably linked to an upstream promoter and a downstream polyadenylation signal. 34) The polynucleotide according to 32, wherein the expression cassette comprises a promoter, a Kozak sequence, the PPT1 polypeptide coding nucleic acid sequence, and a polyadenylation signal, wherein the expression cassette is operably ligated from 5' to 3' to the nucleic acid encoding the PPT1 polypeptide. 35) The polynucleotide according to 33 or 34, wherein the promoter contains a sequence having at least 95% identity, at least 97% identity, or at least 99% identity with respect to the sequence of SEQ ID NO: 5, or contains SEQ ID NO: 5, or the promoter contains a sequence having at least 95% identity, at least 97% identity, or at least 99% identity with respect to the sequence of SEQ ID NO: 173, or contains SEQ ID NO: 173. 36) The polynucleotide according to any one of 33 to 35, wherein the polyadenylation signal operably linked to the PPT1 coding nucleotide sequence includes a sequence having at least 95% identity, at least 97% identity, or at least 99% identity with respect to the sequence of SEQ ID NO: 6, or includes SEQ ID NO: 6. 37) The polynucleotide according to any one of 32 to 36, wherein the expression cassette contains a nucleotide sequence having at least 95% identity with any of sequence numbers 141 to 143, or at least 97% identity with any of sequence numbers 141 to 143; or the expression cassette contains a nucleotide sequence having at least 95% identity with any of sequence numbers 169, or at least 97% identity with any of sequence numbers 169; or the expression cassette contains a nucleotide sequence having at least 95% identity with any of sequence numbers 170, or at least 97% identity with any of sequence numbers 170. 38) A polynucleotide, which is DNA, as described in any one of 1-18 and 30-37. 39) Recombinant viral vector nucleic acid comprising a polynucleotide as described in any one of 1-18 and 30-38, and a 5' and / or 3' viral element that results in viral packaging and replication. 40) The recombinant viral vector nucleic acid according to 39, wherein the DNA comprises an adeno-associated virus (AAV) inverted repeat (ITR) adjacent to the 5' end of the polynucleotide and an AAV ITR adjacent to the 3' end of the polynucleotide. 41) The recombinant viral vector nucleic acid according to 40, wherein the recombinant viral vector nucleic acid comprises the 5'ITR and 3'ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, or AAV3B. 42) The recombinant viral vector nucleic acid according to 40, wherein the 5'ITR contains a sequence having at least 95% identity, at least 97% identity, or at least 99% identity with respect to the sequence of sequence number 8, or contains sequence number 8; and the 3'ITR contains a sequence having at least 95% identity, at least 97% identity, or at least 99% identity with respect to the sequence of sequence number 9, or contains sequence number 9. 43) A recombinant viral vector nucleic acid according to any one of 39 to 42, further comprising a polyadenylated sequence operably linked to the 3'ITR. 44) Recombinant viral vector nucleic acid according to any one of 39 to 43, further comprising one or more stuffer sequences. 45) The recombinant viral vector nucleic acid according to any one of 39 to 44, wherein the recombinant viral vector nucleic acid contains a sequence that has at least 95% identity, at least 97% identity, at least 99% identity or 100% identity with any of the sequences of sequence numbers 144 to 154; or the recombinant viral vector nucleic acid contains a sequence that has at least 95% identity, at least 97% identity, at least 99% identity or 100% identity with the sequence of sequence number 171; or the recombinant viral vector nucleic acid contains a sequence that has at least 95% identity, at least 97% identity, at least 99% identity or 100% identity with the sequence of sequence number 172. 46) A gene delivery vehicle comprising a virus or non-viral vector, and a polynucleotide described in any one of 1-18 or 30-38, or a recombinant viral vector nucleic acid described in any one of 39-45. 47) A viral vector, the gene delivery vehicle described in 46. 48) The gene delivery vehicle according to 47, wherein the viral vector is a recombinant AAV vector, a recombinant lentiviral vector, or a recombinant adenovirus vector. 49) The gene delivery vehicle according to 48, wherein the viral vector is a recombinant AAV vector, and the recombinant AAV vector comprises a capsid containing a VP1, VP2, or VP3 having at least 90% identity, at least 95% identity, or 100% identity to any VP1, VP2, or VP3 sequence from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 12, or SEQ ID NO: 15. 50) The gene delivery vehicle according to 49, wherein the capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 capsid; or the capsid comprises VP1 of SEQ ID NO: 12 or SEQ ID NO: 15; in a further embodiment, the gene delivery vehicle according to 49, wherein the capsid comprises VP1 comprising the sequence of SEQ ID NO: 12, VP2 comprising the sequence of SEQ ID NO: 13, and VP3 comprising the sequence of SEQ ID NO: 14. 51) A nonviral vector, as described in 46, for gene delivery. 52) The gene delivery vehicle according to 51, wherein the nonviral vector is a nanoparticle selected from the group consisting of lipid nanoparticles (LNPs), polymeric nanoparticles, lipid polymer nanoparticles (LPNPs), protein or peptide-based nanoparticles, DNA dendrimers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. 53) The gene delivery vehicle according to 52, wherein the nonviral vector is an LNP or an LPNP. 54) A pharmaceutical composition comprising a polynucleotide according to any one of 1 to 18 or 30 to 38, a polypeptide according to any one of 19 to 29, a recombinant viral vector nucleic acid according to any one of 39 to 45, or a gene delivery vehicle according to any one of 46 to 53, and a pharmaceutically acceptable carrier. 55) A method for increasing PPT1 in a subject, comprising the step of administering to the subject a polynucleotide according to any one of 1 to 18 or 30 to 38, a polypeptide according to any one of 19 to 29, a recombinant viral vector nucleic acid according to any one of 39 to 45, or a gene delivery vehicle according to any one of 46 to 53, or a pharmaceutical composition according to 54. 56) A method for treating neuronal ceroid lipofuscinosis 1 in a subject, comprising the step of administering to the subject a polynucleotide according to any one of 1 to 18 or 30 to 38, a polypeptide according to any one of 19 to 29, a recombinant viral vector nucleic acid according to any one of 39 to 45, or a gene delivery vehicle according to any one of 46 to 53, or a pharmaceutical composition according to 54. 57) The method according to 55 or 56, wherein the administration step includes intracerebral, intracisional, or intraventricular administration. In further embodiments, the administration is intraventricular; the administration is intraventricular and results in significant rAAV delivery to at least the prefrontal cortex, parietal cortex, temporal cortex, occipital cortex, thalamus, cerebellar cortex, hippocampus, corpus callosum, spinal cord, caudate nucleus, choroid plexus, optic chiasm, fornix, periaqueductal gray matter, olfactory bulb, and optic nerve. 58) The method according to 55 or 56, wherein the administration step includes an initial administration outside the central nervous system (CNS). 59) The method according to any one of 55 to 58, wherein the administration is systemic. 60) The method according to any one of 55 to 59, wherein the subject is a human. 61) An AAV vector genome plasmid containing a recombinant viral vector nucleic acid as described in any one of 39-45. 62) The AAV genome plasmid described in 61, lacking the rep and cap genes. 63) A method for generating an rAAV vector, comprising the step of culturing an rAAV-producing cell line containing rAAV helper virus activity, wherein the genome of the producing cell contains one recombinant viral vector nucleic acid, rep gene and cap gene from among 39 to 45, and the rAAV vector is generated. 64) A method for generating an rAAV vector, comprising the step of culturing rAAV-tolerant cells comprising the AAV genome plasmid of 61 or 62, wherein the rAAV-tolerant cells further comprise (a) rep and cap genes provided either as part of a cell genome and / or by one or more separate plasmids; and (b) helper virus activity provided by the cell genome and / or by one or more separate plasmids. 65) The method according to 64, wherein the rAAV-tolerant cells are packaging cells, and the genome of the packaging cells includes a cap gene and a rep gene. 66) The method according to 64, wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid; or (b) the rep gene and the cap gene are provided by a rep / cap plasmid and the helper activity is provided by a helper plasmid. 67) A method for obtaining an rAAV vector, comprising the steps of (a) generating rAAV using one of the methods in 63 to 67, and (b) purifying rAAV.

[0235] XII. Array

[0268] Table 2 provides various nucleic acid and amino acid sequences. In some cases, variable sequences are described herein. Underlined sequences describe signal sequences when present in the full-length PPT1 sequence. Some nucleic acid sequences shown in bold present codons. A reference to “origin” for a signal amino acid sequence indicates that it has been modified from the native sequence.

[0236]

[0269] In a different embodiment, the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the nucleic acid sequences provided in Table 2; the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the nucleic acid sequences provided in Table 2, with the stop codons shown in bold being absent and / or replaced with different stop codons; or the polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the amino acid sequences provided in Table 2.

[0237]

[0270] [Table 2] TIFF2026511472000009.tif136149 TIFF2026511472000010.tif142149 TIFF2026511472000011.tif160149 TIFF2026511472000012.tif148149 TIFF2026511472000013.tif121149 TIFF2026511472000014.tif126149 TIFF2026511472000015.tif137149 TIFF2026511472000016.tif139149 TIFF2026511472000017.tif134149 TIFF2026511472000018.tif134149 TIFF2026511472000019.tif134149 TIFF2026511472000020.tif134149 TIFF2026511472000021.tif134149 TIFF2026511472000022.tif134149 TIFF2026511472000023.tif134149 TIFF2026511472000024.tif134149 TIFF2026511472000025.tif134149 TIFF2026511472000026.tif134149 TIFF2026511472000027.tif134149 TIFF2026511472000028.tif134149 TIFF2026511472000029.tif134149 TIFF2026511472000030.tif134149 TIFF2026511472000031.tif134149 TIFF2026511472000032.tif134149 TIFF2026511472000033.tif139149 TIFF2026511472000034.tif144149 TIFF2026511472000035.tif144149 TIFF2026511472000036.tif144149 TIFF2026511472000037.tif148149 TIFF2026511472000038.tif121149 TIFF2026511472000039.tif142149 TIFF2026511472000040.tif135149 TIFF2026511472000041.tif126149 TIFF2026511472000042.tif126149 TIFF2026511472000043.tif126149 TIFF2026511472000044.tif126149 TIFF2026511472000045.tif126149 TIFF2026511472000046.tif126149 TIFF2026511472000047.tif126149 TIFF2026511472000048.tif126149 TIFF2026511472000049.tif126149 TIFF2026511472000050.tif126149 TIFF2026511472000051.tif126149 TIFF2026511472000052.tif126149 TIFF2026511472000053.tif126149 TIFF2026511472000054.tif126149 TIFF2026511472000055.tif126149 TIFF2026511472000056.tif126149 TIFF2026511472000057.tif138149 TIFF2026511472000058.tif141149 TIFF2026511472000059.tif138149 TIFF2026511472000060.tif138149 TIFF2026511472000061.tif137149 TIFF2026511472000062.tif141149 TIFF2026511472000063.tif137149 TIFF2026511472000064.tif138149 TIFF2026511472000065.tif137149 TIFF2026511472000066.tif138149 TIFF2026511472000067.tif138149 TIFF2026511472000068.tif137149 TIFF2026511472000069.tif138149 TIFF2026511472000070.tif137149 TIFF2026511472000071.tif141149 TIFF2026511472000072.tif137149 TIFF2026511472000073.tif138149 TIFF2026511472000074.tif137149 TIFF2026511472000075.tif138149 TIFF2026511472000076.tif130149 TIFF2026511472000077.tif138149 TIFF2026511472000078.tif138149 TIFF2026511472000079.tif137149 TIFF2026511472000080.tif138149 TIFF2026511472000081.tif137149 TIFF2026511472000082.tif137149 TIFF2026511472000083.tif131149 TIFF2026511472000084.tif126149 TIFF2026511472000085.tif126149 TIFF2026511472000086.tif126149 TIFF2026511472000087.tif138149 TIFF2026511472000088.tif137149 TIFF2026511472000089.tif137149 TIFF2026511472000090.tif138149 TIFF2026511472000091.tif137149 TIFF2026511472000092.tif137149 TIFF2026511472000093.tif137149 TIFF2026511472000094.tif142149 TIFF2026511472000095.tif127149

[0238] [Related applications]

[0001] This application claims priority under U.S. Provisional Patent Application No. 63 / 491,205 filed on March 20, 2023, U.S. Provisional Patent Application No. 63 / 584,000 filed on September 20, 2023, and U.S. Provisional Patent Application No. 63 / 600,153 filed on November 17, 2023, each of which is incorporated in its entirety herein by reference.

[0239] [References to electronically submitted sequence listings]

[0002] The contents of the electronic sequence listing (065830.23WO1.xml; size: 315,901 bytes; and creation date: March 8, 2024) are incorporated in their entirety within this specification by reference. [Examples]

[0240]

[0271] Examples illustrating various features of the present invention and methodologies for carrying it out are provided below. The examples provided are not limited to the claimed invention.

[0241]

[0272] Example 1: PPT1 expression construct having an exogenous signal peptide

[0273] Intracellular and secreted PPT1 activity was measured in cells transfected with plasmids containing various PPT1 rAAV expression constructs. To evaluate the depalmitoylation activity of palmitoyl protein thioesterase (PPT1), 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (gluctopyranoside) (MUTG) was used as a substrate. The release of MU from MU-6SPalm-βGal cannot be achieved by the action of PPT1 alone, because this enzyme only hydrolyzes the palmitoyl thioester linkage, producing a non-fluorescent reaction intermediate. Therefore, exogenous β-galactosidase or β-glucosidase was used to hydrolyze the intermediate, resulting in the release of fluorescent 4MU detectable by a fluorescence plate reader. Runs with known amounts of free 4MU were performed in parallel and used for plotting standard curves. The levels of PPT1 in the samples were quantified using a standard curve or simply expressed as relative fluorescence use (RFU).

[0242]

[0274] Figure 1 is a schematic diagram of the basic structure of a PPT1 rAAV expression construct having the following nucleic acid regions described: 5'-ITR, EF-1α promoter (EF1a), Kozak sequence (Kozak), signal sequence (SS), mature PPT1 sequence (human PPT1), bovine polyadenylated sequence (bGH-pA), stuffer (stuffer sequence), synthetic polyadenylated sequence (synthetic pA), and 3'-ITR.

[0243]

[0275] Table 3 provides construct naming for AAV nucleic acids, referring to the sequence numbers of the encoded signal peptide sequence, the encoded mature PPT1 amino acid sequence, the encoded polynucleotide signal sequence, the encoded mature PPT1 polynucleotide sequence, and the complete rAAV sequence.

[0244]

[0276] [Table 3]

[0245]

[0277] Table 3 characterizes various constructs as belonging to one of the following groups: Group 1, Group 2, or Group 3. Group 1 provides polypeptides in which (a) the N-terminus of mature PPT1 is substituted and (b) a heterologous signal peptide is included. Group 2 provides polypeptides in which (a) the N-terminus of mature PPT1 is substituted and (b) a heterologous signal peptide is modified (derived from) it. Group 3 provides polypeptides in which (a) mature PPT1 has an N-terminal addition (tPA) or deletion (OSM) and (b) a heterologous signal peptide is modified.

[0246]

[0278] Figure 2A provides a bar graph showing the PPT1 enzyme activity levels detected in total cell lysates (intracellular) from PPT1 knockout HeLa cells transfected with various AAV plasmid constructs. The x-axis describes the signal sequence present in full-length PPT1. The data illustrate the ability of various constructs to express functional PPT1 protein. Values ​​are expressed relative to native type (unmodified PPT1) set to 100%. Data are normalized based on transfection efficiency. The number of experiments was n=3-4, except for tPA (n=2). Values ​​are mean ± SEM.

[0247]

[0279] Figure 2B provides a bar chart showing the PPT1 enzyme activity levels detected in secretion medium (secretion) collected from PPT1 knockout HeLa cells transfected with AAV plasmids containing various manipulated human PPT1 candidates. The x-axis describes the secretion signal present in full-length PPT1. The data illustrate the ability of various constructs to express functional PPT1 protein. Values ​​are expressed relative to the native type (unmodified PPT1) set to 100%. Data are normalized based on transfection efficiency. Experimental size n=3-4. Values ​​are mean ± SEM. *p<0.05, native type vs. others, Mann-Whitney test.

[0248]

[0280] Example 2: PPT1 uptake by non-transfected cells

[0281] The uptake of secreted PPT1 when expressed from various constructs was measured using plasmids containing various rAAV nucleic acids and a TdTomato (red fluorescent protein) reporter. Figure 3A shows a schematic diagram of a plasmid describing the following rAAV nucleic acid regions: 5'-ITR, EF-1α promoter (EF1a), Kozak sequence (Kozak), signal sequence (SS), mature PPT1 sequence (human PPT1), bovine growth hormone polyadenylation sequence (bGH-pA), stuffer (stuffer sequence), synthetic polyadenylation sequence (synthetic pA), and 3'-ITR; and the following tdTomato region: UbC (ubiquitin C promoter), 5'UTR, tdTomato, 3'UTR, and RbGpA (rabbit beta-globin polyadenylation sequence).

[0249]

[0282] Table 4 provides construct naming for rAAV nucleic acids, referring to the sequence numbers of the encoded signal peptide sequence, the encoded mature PPT1 amino acid sequence, the encoded polynucleotide signal sequence, the encoded mature PPT1 polynucleotide sequence, and the complete rAAV sequence.

[0250]

[0283] [Table 4]

[0284] PPT1 knockout HeLa cells were transfected with various plasmids, fixed with 4% paraformaldehyde 48 hours after transfection, and immunostained with mouse anti-PPT1 (Novus Biologicals (OTI1F10)) and Alexa 488 conjugate anti-mouse secondary antibody (green fluorescence). The nuclei were labeled with Hoechst and therefore observed as blue.

[0251]

[0285] Images were acquired using the Opera Phenix Plus HCS system (Perkin Elmer) (image data not shown). Cells emitting green light indicated the presence of PPT1, while cells emitting yellow light indicated the presence of both PPT1 and TdTomato (plasmid-transfected cells). Cells emitting only green light suggested the uptake of PPT1 present in the culture medium, secreted by transfected (red) cells. Figure 3B is a bar graph reflecting the image analysis of immunohistochemistry, showing the ratio of cells with PPT1 (green) to transfected cells (red). The increased ratios for Sp7-F, SPARC, and tPA compared to PPT1 with the native signal peptide suggest an increased number of PPT1-positive cells due to increased uptake of engineered PPT1 possessing exogenous signal sequences. Each circle (average of 30 fields of view) represents an independent transfection (obtained from 2-3 experiments). Values ​​are mean ± SD. P-values ​​obtained by one-way ANOVA: ****p<0.0001, ***p<0.001, **p<0.01.

[0252]

[0286] Figure 3C provides a bar chart showing the results of Figure 3B, normalized for the natural-type construct. White circles indicate independent transfections. Each point (average of 30 fields of view) is an independent transfection (obtained from 2-3 experiments). Values ​​are mean ± SD. P-values ​​by one-way ANOVA: ****p<0.0001, ***p<0.001, **p<0.01.

[0253]

[0287] Example 3: In vitro expression of PPT1 by cortical neurons transduced by recombinant AAV

[0288] Dose-dependent PPT1 expression and secretion from primary cortical neurons of AAV-transduced rats were evaluated. The overall design of the recombinant AAV vector is shown in Figure 1. The recombinant rAAV vector was named "Sp7-F.PPT1" or "death PPT1". Sp7-F.PPT1 corresponds to Sp7-F as described in Table 3. Death PPT1 encodes a mutated full-length PPT1, which catalytically inactivates the protein.

[0254]

[0289] Recombinant rAAV vectors were prepared by triple transfection using capsids containing VP1 from SEQ ID NO: 12, VP2 from SEQ ID NO: 13, and VP3 from SEQ ID NO: 14 in human fetal kidney cells, and then purified by CsCl purification.

[0255]

[0290] Cultured neurons were transduced using rAAVs of three MOIs (low, 1E+5; medium, 5E+5; and high, 1E+6), and PPT1 activity in the culture medium was analyzed on days 3, 4, or 6 post-transduction. The results are shown in Figure 4. Cells transduced with rAAVs that catalytically encode death-type PPT1 showed no activity. Untreated or diluent-treated cells were used as negative controls. Purified recombinant PPT1 was used as a positive control for the assay. Each circle represents a value obtained from independently transduced cells in a single well. Data are mean ± SD.

[0256]

[0291] Secreted PPT1 bands from rat primary cortical neurons transduced with moderate and high doses were examined for glycosylation on day 6. PPT1 protein was not observed in vehicle or non-transduced cells. Glycosylated PPT1 was detected in cells transduced with the rAAV vector (data not shown).

[0257]

[0292] Example 4: In vivo expression of PPT1 by recombinant AAV

[0293] Sp7-1. PPT1 expression and secretion were measured in C57BL6 / J mice administered rAAV containing viral vector nucleic acid encoding either PPT1 viral vector nucleic acid (SEQ ID NO: 143, see Table 4) or dead PPT1 (enzymatically inactive PPT1). Recombinant rAAV vectors were prepared using capsids containing VP1 (SEQ ID NO: 12), VP2 (SEQ ID NO: 13), and VP3 (SEQ ID NO: 14), and purified by CsCl purification. Administration was performed directly into the hippocampus by stereotactic injection or via the IV route. Table 5 summarizes the experimental protocol.

[0258]

[0294] [Table 5]

[0259]

[0295] Brain analysis was performed 6 weeks after injection. Animals that received only the diluent were used as negative controls.

[0260]

[0296] Figures 5A, 5B, and 5C illustrate serum PPT1 expression and activity at various time points in mice administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid. Figure 5A illustrates PPT1 production in serum of mice IV-administered with AAV containing Sp7-F.PPT1 viral vector nucleic acid or dead PPT1 viral vector nucleic acid. Serum samples were collected at 2, 4, or 6 weeks post-administration and analyzed by automated capillary-based immunoassay (WES; Protein Simple) to detect PPT1. Mice administered with diluents were used as negative controls. The presence of PPT1 (indicated by arrowheads in Figure 5A) was detected at 2 weeks in mice administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid, and at further increased levels at 4 weeks. The signal saturated at 6 weeks. Purified recombinant PPT1 was used as a positive control. Figure 5B shows serum activity in IV-administered mice. Figure 5C shows serum activity in mice administered IPA (intra-hippocampal via stereotactic injection). ND indicates undetectable. Data are mean ± SD. ANOVA P values ​​are *P<0.05, ***P<0.001, ****P<0.0001.

[0261]

[0297] Figure 6 illustrates liver PPT1 activity from mice (n=4, indicated by circles) IV-administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid. Mice injected with a diluent were used as negative controls.

[0262]

[0298] Figures 7, 8, 9, and 10 illustrate the localization, expression, glycosylation, and activity of PPT1 expressed in the brain after hippocampal administration of rAAV containing Sp7-F.PPT1 viral vector nucleic acid in mice. Figure 7 illustrates immunohistochemical analysis showing increased PPT1 staining in the hippocampus (indicated by asterisks). Figure 8 shows the detection of PPT1 protein in hippocampal protein lysates analyzed by JESS. The double lines observed at low exposure (indicated as low) indicate two glycosylation forms of PPT1. Figure 9 confirms that PPT1 expressed in the hippocampus was glycosylated. Hippocampal protein extracts were treated with deglycosylase and analyzed by JESS assay. The reduced molecular weight after deglycosylase treatment (lanes marked with +) suggested that PPT1 was glycosylated. Figure 10 provides a hippocampal lysate PPT1 activity assay demonstrating that PPT1 expressed in the brain was biologically active, as described in Example 1.

[0263]

[0299] Example 5: CNS distribution of rAAV encoding PPT1

[0300] Wild-type C57BL / 6J male mouse offspring on day 1 were administered rAAV containing Sp7-F.PPT1 viral vector nucleic acid by bilateral intraventricular (ICV) injection. The recombinant AAV containing Sp7-F.PPT1 viral vector nucleic acid is described in Example 3. The injection involved administering 1E+10 vector genome (vg) in the low-dose group and 1E+11vg in the high-dose group, with a total of 6 μL, 3 μL per side. Six weeks after injection, the brain was excised and the left hemisphere was dissected into specific regions: cortex, hippocampus, thalamus, brainstem, and cerebellum. Each of these regions was further divided into two equal sections. One section was processed for vector genome analysis, while the other was used for testing of transgene (PPT1 protein) expression. The right hemisphere of the brain was fixed and subjected to histological evaluation. The spinal cord was dissected coronally into cervical, thoracic, and lumbar segments, and half of each segment was used for vector genome analysis. Cerebrospinal fluid (CSF) was collected by cisterna magna puncture and subsequently analyzed for PPT1 levels.

[0264]

[0301] ICV delivery of the viral vector showed a dose-dependent distribution in the brain and spinal cord of mice. Figure 11 shows the vector genome copy number (VGCN), an indicator of viral transduction, in various brain and spinal cord regions. Figure 12 shows the multiplicative change (FC) of PPT1 activity in various brain regions of rAAV-injected animals compared to mice injected with a diluent. PPT1 activity in tissue lysates was quantified using MUTG as described in Example 1. Figure 13 illustrates the FC of PPT1 activity in cerebrospinal fluid (CSF) of rAAV-injected mice compared to mice injected with a diluent. FC is the relative mean activity level of the diluent group. Figure 14 is a scatter plot illustrating the correlation between VGCN and PPT1 enzyme activity in the brain. The Spearman correlation coefficient was r=0.66, p<0.0001.

[0265]

[0302] Figure 15 shows the detection of glycosylated and deglycosylated PPT1 protein in brain lysates analyzed by JESS. Protein extracts were treated with deglycosylase and analyzed by the JESS assay. The double lines in lanes 1-2 indicate the two glycosylated forms of PPT1. The reduced molecular weight after deglycosylase treatment (lanes 3-4) suggests that PPT1 was glycosylated.

[0266]

[0303] Histological evaluation was performed using antibodies against PPT1, followed by detection with a fluorophore conjugate secondary antibody. Images were acquired using a fluorescence microscope. Animals receiving the diluent showed baseline PPT1 signaling. Animals given low doses of rAAV showed slightly increased PPT1 expression compared to the diluent, while animals receiving high doses showed significantly higher expression. PPT1 expression was prominent throughout the cortex, from the rostral to the caudal region, and was also prominent in the hippocampus, striatum, and olfactory bulb. PPT1 signaling surrounded NeuN signaling, suggesting that PPT1 was primarily expressed in neurons (data not shown).

[0267]

[0304] Example 6: In vivo distribution of rAAV and encoded PPT1 in sheep

[0305] The CNS distribution of rAAV containing Sp7-1.PPT1 (Example 3) and the encoded PPT1 was evaluated in sheep. 9-10 month old PPT1+ / - male lambs were administered rAAV containing Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep) encoded viral vector nucleic acid by unilateral intraventricular (ICV) injection. A dose of 1E+14vg in 2 mL was injected. Eight weeks after injection, the brain was excised, a coronal slab was prepared, and brain punches (3 mm wide) obtained from the target region were analyzed for vector genome and PPT1 protein. The contralateral hemisphere brain slab was fixed and used for histological evaluation. The spinal cord was dissected coronally into cervical, thoracic, and lumbar segments and subjected to vector genome and PPT1 protein analysis. Cerebrospinal fluid (CSF) was analyzed for PPT1 levels. The liver was collected and analyzed for vector genome.

[0268]

[0306] ICV administration resulted in vector delivery to various brain regions (rostral to caudal) and the spinal cord in sheep. Vector genome copy number (VGCN) as a measure of viral transduction was measured across various regions in sheep: cortical regions (Figure 16A), (Figure 16B) thalamus, (Figure 16C) cerebellar cortex, (Figure 16D) hippocampus, (Figure 16E) corpus callosum, (Figure 16F) additional shown brain regions, (Figure 16G) spinal cord, and (Figure 16H) liver. Vector genomes derived from frozen tissue DNA were quantified using quantitative PCR (qPCR) with standard curves. Vertical arrows indicate that the sample originated from a contralateral brain region. White circles correspond to sheep administered with the Sp7-F.PPT1 vector, while black circles depict results for sheep administered with a GFP-expressing vector. Data is provided for the following regions: prefrontal cortex (FC), motor cortex (CM), somatosensory cortex (SSC), piriform cortex (PC), suprasylvian gyrus (SSG), lateral gyrus (EcG), medial gyrus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaqueductal gray matter (Periaq G), olfactory bulb (Ol), optic nerve (Op), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), spinal cord - cervical (SC), spinal cord - thoracic (SC_Tho), and spinal cord - lumbar (SC_Lum).

[0269]

[0307] The PPT1 activity assay demonstrates functional PPT1 expression and secretion in the brain and CSF. PPT1 activity was measured using MUTG as described in Example 1. Figures 17A–17D illustrate PPT1 activity in Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep) rAAV-injected sheep in the cortex (Figure 17A), thalamus (Figure 17B), cerebellar cortex (Figure 17C), and caudate nucleus (Figure 17D). Each circle is the result of one tissue punch from a brain region. N indicates the number of regions from 2 GFP-injected animals and 4 PPT1-injected animals. Data are mean ± SEM. Statistical analysis by Mann-Whitney U test, *P<0.05, ***P<0.001.

[0270]

[0308] Figure 18 provides 95% confidence intervals for mean PPT1 activity across the overall treatment groups. After obtaining log-transformed activity results for all data in Figures 17A–17D and explaining the differences in brain regions in punch count and mean values, a hypothesis test for mean treatment-type differences was performed at a 0.05 alpha level. The test revealed a significant difference in mean activity levels for PPT1 over GFP. The mean estimated multiplier change for PPT1 over GFP was 3.9, with a median of 73 nmol / mg / hour for PPT1 versus 19 nmol / mg / hour for GFP. ****P<0.0001, weighted two-way ANOVA.

[0271]

[0309] Figure 19 is a bar chart showing the percentage change in PPT1 activity in cerebrospinal fluid (CSF) of sheep administered rAAV carrying Sp7-F.PPT1 (n=4 sheep) or GFP (n=2 sheep). The percentage change is relative to the mean activity of the control (GFP animals). Each circle represents one animal. N indicates the number of animals. Data are mean ± SEM.

[0272]

[0310] Figure 20 shows the results of a JESS assay detecting PPT1 expression in tissue lysates of sheep spinal cord injected with an rAAV vector containing either PPT1 (sheep 1-4) or GFP (sheep 1-2) coding nucleic acids.

[0273]

[0311] Figure 21 illustrates the results showing elevated levels of mean PPT1 activity in the thoracic and lumbar segments of the spinal cord of sheep administered with rAAV containing the nucleic acid encoding PPT1.

[0274]

[0312] Immunofluorescence microscopy images were obtained from the cerebellar lobe of sheep brains for histological evaluation to assess transduction and transgene expression. Contralateral hemisphere brain slabs were fixed, and 30-micron thick frozen sections were prepared and processed for immunohistochemical analysis. Blue fluorescent DNA staining indicating dsDNA was obtained using DAPI (4',6-diamidino-2-phenylindole). Immunostaining of PPT1 was performed using an antibody against purified PPT1 protein, followed by subsequent staining with a fluorophore conjugate secondary antibody. Images showing GFP expression and localization of PPT1 were visualized and obtained using fluorescence microscopy. Broad expression of transgenes (GFP reporter and PPT1) was observed throughout the cerebellar lobe of sheep brains.

[0275]

[0313] Example 7: Improvement of motor function

[0314] The effects of rAAVs providing (1) Sp7-PPT1 or (2) SPARC.PPT1 in PPT1 knockout (KO) mice were evaluated using rotarod evaluation. Rotarod evaluation is described, for example, in Deacon J.Vis.Exp. May 29, 2013; (75):e2609 (the entire text is incorporated herein by reference). Sp7-PPT1, SPARC.PPT1, and rAVV are listed in Table 3. KO mice were administered rAAVs containing the PPT1 gene by bilateral ICV injection on postnatal day 1 and evaluated at 7 months of age. rAAVs were prepared as described in Example 3 and administered at a dose of 1E+11vg / animal.

[0276]

[0315] The results are shown in Figure 22. Untreated (Un) or vehicle-treated (Veh) KO mice were used as negative controls. "Natural type" refers to the rAAV vector encoding natural human PPT1 (SEQ ID NO: 29). Sp7-PPT1 is described by "1", and SPARC.PPT1 is described by "2". Each circle represents one mouse. Bars are mean ± SEM. One-way ANOVA, Tukey's post-hoc test, *P<0.05, ***P<0.001, ****P<0.0001. CNS-targeted rAAV therapy successfully delivered functional human PPT1 to PPT1 KO mice, rescuing coordination and balance.

[0277]

[0316] Example 8: Improvement of motor function and balance

[0317] The ability of CNS-targeted rAAV vectors containing nucleic acids encoding Sp7-PPT1 (rAAV-Sp7-PPT1) and SPARC.PPT1 (rAAV-SPARC.PPT1), which rescue motor coordination and balance in Ppt1- / - mice, was evaluated using an accelerated rotorod instrument. rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 were prepared as described in Example 3. The rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 vector nucleic acids are listed in Table 4.

[0278]

[0318] Ppt1- / - mice were given PND1 (low dose, 1 x 10⁶). 11 (vg / animal) or PND1 and 3 (high dose, 3.82 × 10) 11rAAV-Sp7-PPT1 or rAAV-SPARC.PPT1 was administered to vg / animals via bilateral intraventricular injection and evaluated at various time points. Mice were examined over four trials at each time point. Each session included a 5-minute training trial at 4 RPM on a rotorod instrument (Rotamex, OH). One hour after the training trial, animals were examined for three consecutive 5-minute acceleration trials, during which the speed was varied over 300 seconds. The interval between trials was at least 30 minutes. Waiting time from fall from the accelerating rod was recorded and quantitative analysis was performed. Untreated or vehicle-treated Ppt1- / - mice were used as negative controls. "Natural type" refers to AAV vectors containing the unmodified human PPT1 gene. In each group, the number of mice varied from 10 to 18, except for the WT, 11mo group, where n=7. The sex distribution was generally balanced within each group.

[0279]

[0319] Figure 23 illustrates the ability of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 to improve motor coordination and balance, as evaluated by the delay time to fall from an accelerating rod (see, for example, Kovacs and Pearce, Dis Model Mech. April 2015; 8(4): pp. 351-361): PMC4381334. Both rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 reduced the delay time to fall.

[0280]

[0320] Example 9: Muscle strength

[0321] The ability of CNS-targeted rAAV vectors containing nucleic acids encoding Sp7-PPT1 (rAAV-Sp7-PPT1) or SPARC.PPT1 (rAAV-SPARC.PPT1) to improve muscle strength in Ppt1- / - mice was evaluated based on grip strength. rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 were prepared as described in Example 3. The rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 vector nucleic acids are listed in Table 4.

[0281]

[0322] Ppt1- / - mice were given PND1 (low dose, 1.00 × 10⁶). 11 (vg / animal) or PND1 and 3 (high dose, 3.82 × 10) 11 rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 were administered to vg / animals via bilateral intraventricular injection and evaluated at the indicated time points. Muscle strength in the forelimb muscles was assessed using grip strength in five consecutive trials (San Diego Instruments, San Diego, CA). The mean of all five trials was used for plotting. The animals were lowered towards the platform and gently pulled backward with consistent force by the experimenter until the grip was released.

[0282]

[0323] Figure 24 illustrates the effects of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 on grip strength at various time points. Ppt1- / - mice that were either untreated or treated with a vehicle served as the negative control group. The term "natural" refers to AAV vectors containing the unmodified human PPT1 gene. In each group, the number of mice at each time point varied from 7 to 18, and the sexes were generally balanced. Both rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 improved grip strength.

[0283]

[0324] Example 10: PPT1 activity

[0325] Ppt1- / - mice were given PND1 (low dose, 1.00 × 10⁶). 11 (vg / animal) or PND1 and 3 (high dose, 3.82 × 10) 11 rAAV-Sp7-PPT1 or rAAV-SPARC.PPT1 was administered to vg / animal (vg / animal) via bilateral lateral intraventricular injection. As described in Example 1, PPT1 activity in serum at the indicated time points was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as the substrate. PPT1 activity was determined using a standard curve (created by using 4MU of known concentration).

[0284]

[0326] The results of the PPT1 assay are shown in Figures 25A and 25B. Figure 25A illustrates the activity at various time points. Figure 25B illustrates the activity at 8 months. Increased levels of serum PPT1 were observed in AAV-treated mice, indicating sustained long-term expression of PPT1. Untreated or vehicle-treated Ppt1- / - mice were used as negative controls. "Natural type" refers to AAV vectors containing the unmodified human PPT1 gene.

[0285]

[0327] Example 11: Cortical, brainstem, and cerebellar PPT1 activity

[0328] Ppt1- / - mice were given PND1 (low dose, 1.00 × 10⁶). 11 rAAV-Sp7-PPT1 or rAAV-SPARC.PPT1 was administered to vg / animals by bilateral intraventricular injection. PPT1 activity was measured in the cortex, brainstem, and cerebellum using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate, as described in Example 1. PPT1 activity was determined using a standard curve (created by using 4MU of known concentration). Tissues were analyzed at 10 months of age, except for untreated Ppt1- / - mice tested at 8 months of age.

[0286]

[0329] Figures 26A–26C provide bar charts showing PPT1 activity in the cortex (Figure 26A), brainstem (Figure 26B), and cerebellum (Figure 26C). The elevated PPT1 activity at 10 months of age in rAAV-treated mice indicates stable expression of PPT1 in the brains of Ppt1- / - mice. "Natural" refers to AAV vectors containing the unmodified human PPT1 gene. Bars represent mean ± SEM. Each circle represents a mouse.

[0287]

[0330] Example 12: Codon-optimized PPT1 code sequence

[0331] The expression of various codon-optimized constructs was evaluated in vitro. Plasmids containing rAAV nucleic acid are illustrated in Figure 27. The constructs are summarized in Tables 6 and 7.

[0288]

[0332] [Table 6]

[0289]

[0333] [Table 7]

[0290]

[0334] Examples of full-length rAAV nucleic acid constructs are provided by SEQ ID NOs: 171 and 172. SEQ ID NOs: 171 provides the full-length rAAV nucleic acid sequence of a construct named "SpF7-co4," and SEQ ID NOs: 172 provides the full-length rAAV nucleic acid sequence of a construct named "SpF7-co19." Other rAAV sequences were constructed by swapping the signal and PPT coding sequences.

[0291]

[0335] PPT1 knockout HeLa cells were transfected with different AAV plasmids containing codon-optimized coded PPT1 sequences. PPT1 expression was evaluated by measuring enzyme activity in the culture medium 48 hours after transfection.

[0292]

[0336] Figures 28A and 28B are bar graphs depicting the expression levels of PPT1 in culture medium of in vitro cells transfected with various codon-optimized variants of human PPT1. As illustrated in Figure 27, different codon-optimized PPT1 cDNA constructs (excluding the signal sequence), labeled CO on the x-axis followed by a number, were paired with the signal sequences (B) Sp7F, SP7F (codon-optimized), and (C) SpSPARC or SpSPARC (codon-optimized) and cloned downstream of a long EF1a promoter. Each circle represents an independently transfected well in a 96-well plate. Values ​​are presented as mean ± SEM.

[0293]

[0337] Example 13: Survival Data

[0338] Ppt1- / - mice were given PND1 (low dose, 1 x 10⁶). 11(vg / animal) or PND1 and 3 (high dose, 3.82 × 10) 11 rAAVs containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 were administered to animals via bilateral intraventricular injection. The rAAV Sp7F.PPT1 and SPARC.PPT1 constructs are summarized in Table 4. The number of mice enrolled in the study was WT, n=20; KO, Un=16; KO, Veh, and native Hi, n=17. The remaining treatment groups each contained 18 mice. Untreated or vehicle-treated Ppt1- / - mice were used as negative controls. "Natural" refers to AAV vectors containing the unmodified human PPT1 gene.

[0294]

[0339] Survival data are shown in Figure 29. Mouse data were fitted using a Cox proportional hazards model to determine whether survival probabilities differed among treatment groups. The KO and Veh groups had significantly lower survival than the WT and all AAV-treated groups (all p<0.01). The KO, Veh and KO, Un groups did not differ from each other (p=0.22). AAV = adeno-associated virus; CNS = central nervous system; lo = low dose (1 × 10⁻⁶). 11 vg / animal);hi=high dose(3.82×10 11 vg / animal);KO=Ppt1- / -;PND=days old;Un=untreated;Veh=vehicle;WT=wild type.

[0295]

[0340] Example 14: Brain weight loss

[0341] Ppt1- / - mice were given PND1 (low dose, 1 x 10⁶). 11 (vg / animal) or PND1 and 3 (high dose, 3.82 × 10) 11rAAVs containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 were administered via bilateral intraventricular injection. The rAAV Sp7F.PPT1 and SPARC.PPT1 constructs are summarized in Table 4. Untreated or vehicle-treated Ppt1- / - mice were used as negative controls. "Natural type" refers to AAV vectors containing the unmodified human PPT1 gene. Mice were euthanized at 8-10 months of age, their brains were collected, and their weight was recorded. Except for KO, Veh, n=4 and lead 1, hi, n=9, the number of mice was n=6 / group.

[0296]

[0342] Figure 30 is a bar chart illustrating the effect of rAAVs containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 on brain weight in Ppt1- / - mice. Sp7F.PPT1 and SPARC.PPT1 at low and high doses inhibited brain weight loss in Ppt1- / - mice.

[0297]

[0343] Although the present invention has been described and illustrated with reference to certain embodiments thereof, those skilled in the art will understand that various adaptations, changes, modifications, substitutions, deletions, or additions to the procedures and protocols can be made without departing from the spirit and scope of the invention.

Claims

1. A polynucleotide comprising a nucleic acid sequence encoding a palmitoyl protein thioesterase-1 (PPT1) polypeptide, wherein the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, (a) The PPT1 polypeptide further comprises a variant thereof having any signal sequence from SEQ ID NOs: 16 to 27 or one amino acid substitution, deletion or insertion; and / or (b) The PPT1 amino acid sequence includes a glycine (G), valine (V), or leucine (L) substitution of aspartic acid (D) at its amino terminus; and / or (c) The PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus; and / or (d) The nucleic acid sequence includes a PPT1 coding sequence that has at least 85% identity with any of sequence numbers 61 to 94, Polynucleotide.

2. The polynucleotide according to claim 1, wherein the PPT1 polypeptide further comprises the signal sequence which includes any sequence from sequence numbers 16 to 27.

3. The polynucleotide according to claim 2, wherein the nucleic acid includes any of the signal coding sequences among sequence numbers 43 to 58.

4. The polynucleotide according to claim 2, wherein the polypeptide comprises any of the signal sequences among SEQ ID NOs: 16-21 and 24-27.

5. The polynucleotide according to claim 4, wherein the signal sequence includes either sequence number 16 or 19.

6. The polynucleotide according to claim 5, wherein the signal sequence includes the sequence of SEQ ID NO: 16, and the nucleic acid sequence includes the signal coding sequence of SEQ ID NO: 43; or the signal peptide includes the sequence of SEQ ID NO: 19, and the nucleic acid sequence includes the signal coding sequence of SEQ ID NO:

50.

7. The polynucleotide according to claim 2, wherein the polypeptide comprises the signal sequence of sequence number 23.

8. The polynucleotide according to claim 7, wherein the nucleic acid sequence includes the signal coding sequence of sequence number 54.

9. The polynucleotide according to any one of claims 1 to 6, wherein the PPT1 amino acid sequence includes a G, V, or L substitution of aspartate D at its amino terminus, and the PPT1 amino acid sequence has at least 97% identity with the sequence of Sequence ID No.

1.

10. The polynucleotide according to claim 9, wherein the PPT1 amino acid sequence includes the sequence of SEQ ID NO: 2, and X is G.

11. The polynucleotide according to any one of claims 1 to 3, 7, or 8, wherein the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, and the PPT1 amino acid sequence has at least 97% identity with the sequence of Sequence ID No.

1.

12. The polynucleotide according to claim 11, wherein the PPT1 sequence includes the sequence of sequence number 4.

13. The polynucleotide according to any one of claims 1 to 12, wherein the nucleic acid comprises a sequence having at least 85% identity with any of sequence numbers 61 to 94.

14. The polynucleotide according to claim 1, wherein the PPT1 polypeptide comprises a sequence having at least 99% identity with any of sequence numbers 31 to 42.

15. The polynucleotide according to claim 14, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31 or SEQ ID NO:

34.

16. The polynucleotide according to claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, X is G, and the nucleic acid comprises a sequence having at least 85% identity with any of the sequences of SEQ ID NOs: 107-125 and 168.

17. The polynucleotide according to claim 16, wherein the nucleic acid comprises a sequence having at least 95% identity with any of the sequences 107 to 125 and 168.

18. The polynucleotide according to claim 17, wherein the nucleic acid comprises any of the sequences 107 to 125 and 168.

19. The polynucleotide according to claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 34, X is G, and the nucleic acid comprises a sequence having 85% identity with any of the sequences of SEQ ID NOs: 126-140 and 161-167.

20. The polynucleotide according to claim 19, wherein the nucleic acid comprises a sequence having 95% identity with any of the sequences 126-140 and 161-167.

21. The polynucleotide according to claim 20, wherein the PPT1 coding sequence includes any of sequence numbers 126-140 and 161-167.

22. The polynucleotide according to claim 1, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO:

38.

23. A PPT1 polypeptide comprising a palmitoyl protein thioesterase-1 (PPT1) amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, (a) The PPT1 polypeptide further comprises a variant thereof having any signal sequence from SEQ ID NOs: 16 to 27 or one amino acid substitution, deletion or insertion; and / or (b) The PPT1 amino acid sequence includes a glycine (G), valine (V), or leucine (L) substitution of aspartic acid (D) at its amino terminus; and / or (c) The PPT1 sequence contains the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, PPT1 polypeptide.

24. The polypeptide according to claim 23, further comprising a signal sequence containing any of sequence numbers 16 to 27.

25. The polypeptide according to claim 24, comprising any signal sequence among sequence numbers 16-21 and 24-27.

26. The polypeptide according to claim 25, wherein the signal sequence includes either sequence 16 or 19.

27. The polypeptide according to claim 24, comprising the signal sequence of sequence number 23.

28. The polypeptide according to any one of claims 23 to 27, wherein the PPT1 amino acid sequence includes a G, V, or L substitution of aspartic acid D at its amino terminus, and the PPT1 amino acid sequence has at least 97% identity with the sequence of SEQ ID NO:

1.

29. The polypeptide according to claim 28, wherein the PPT1 amino acid sequence includes the sequence of SEQ ID NO: 2, and X is G.

30. The polypeptide according to claim 23, wherein the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, and the PPT1 amino acid sequence has at least 97% identity with the sequence of Sequence ID No.

1.

31. The polypeptide according to claim 30, wherein the PPT1 sequence includes sequence number 4.

32. The polypeptide according to claim 23, comprising a sequence having at least 99% identity with any of sequence numbers 31 to 42.

33. The polypeptide according to claim 32, comprising the sequence of sequence number 31, sequence number 34, or sequence number 38, where X is G.

34. A polynucleotide comprising a PPT1 polypeptide coding nucleic acid sequence, wherein the PPT1 coding nucleic acid sequence codes for the PPT1 polypeptide described in any one of claims 23 to 33.

35. A polynucleotide comprising two or more exons and one or more introns that collectively encode the PPT1 polypeptide according to any one of claims 23 to 34.

36. The polynucleotide according to any one of claims 1 to 22, 34, or 35, comprising an expression cassette containing one or more expression control elements operably linked to the nucleic acid encoding the PPT1 polypeptide.

37. The polynucleotide according to claim 36, wherein the nucleic acid encoding the PPT1 polypeptide is operably linked to an upstream promoter and a downstream polyadenylation signal.

38. The polynucleotide according to claim 36, wherein the expression cassette comprises a promoter, a kozak sequence, the PPT1 polypeptide coding nucleic acid sequence, and a polyadenylation signal, operably ligated from 5' to 3' to the nucleic acid encoding the PPT1 polypeptide.

39. The polynucleotide according to claim 37 or 38, wherein the promoter includes a sequence having at least 95% identity with the sequence of sequence number 5 or 173.

40. The polynucleotide according to any one of claims 37 to 39, wherein the polyadenylation signal operably linked to the PPT1 coding nucleotide sequence includes a sequence having at least 95% identity with respect to the sequence of SEQ ID NO:

6.

41. The polynucleotide according to any one of claims 36 to 40, wherein the expression cassette comprises a nucleotide sequence having at least 95% identity with any of the sequences 141 to 143, 169, and 170.

42. A polynucleotide according to any one of claims 1 to 22 and 34 to 41, which is DNA.

43. Recombinant viral vector nucleic acid comprising a polynucleotide according to any one of claims 1 to 22 and 34 to 42, and a 5' and / or 3' viral element resulting in viral packaging and replication.

44. The recombinant viral vector nucleic acid according to claim 43, wherein the DNA comprises an adeno-associated virus (AAV) inverted repeat (ITR) adjacent to the 5' end of the polynucleotide and an AAV ITR adjacent to the 3' end of the polynucleotide.

45. Recombinant viral vector nucleic acid according to claim 44, comprising the 5'-ITR and 3'-ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh. 10, AAVrh. 74, or AAV3B.

46. The recombinant viral vector nucleic acid according to claim 44, wherein the 5'ITR includes a sequence having at least 95% identity with the sequence of sequence number 8, and the 3'ITR includes a sequence having at least 95% identity with the sequence of sequence number 9.

47. The recombinant viral vector nucleic acid according to any one of claims 43 to 46, further comprising a polyadenylated sequence operably linked to the 3' ITR.

48. A recombinant viral vector nucleic acid according to any one of claims 43 to 47, further comprising one or more stuffer sequences.

49. Recombinant viral vector nucleic acid according to any one of claims 43 to 48, comprising a sequence having at least 95% identity with any of sequences 144-154, 171, and 172.

50. A gene delivery vehicle comprising a virus or a non-viral vector, and a polynucleotide according to any one of claims 1 to 22 and 34 to 42 or a recombinant viral vector nucleic acid according to any one of claims 43 to 49.

51. A viral vector, the gene delivery vehicle according to claim 50.

52. The gene delivery vehicle according to claim 51, wherein the viral vector is a recombinant AAV vector, a recombinant lentiviral vector, or a recombinant adenovirus vector.

53. The gene delivery vehicle according to claim 52, wherein the viral vector is a recombinant AAV vector, and the recombinant AAV vector comprises a capsid containing VP1, VP2, or VP3 having at least 90% identity with any of the VP1, VP2, or VP3 sequences among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh. 74, AAV3B, AAV-2i8, AAVrh. 10, AAVrh. 8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh. 10, SEQ ID NO: 12, or SEQ ID NO:

15.

54. The gene delivery vehicle according to claim 53, wherein the capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh. 74, AAV3B, AAV-2i8, AAVrh. 10, AAVrh. 8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh. 10 capsid; or the capsid contains VP1 of SEQ ID NO: 12 or SEQ ID NO:

15.

55. The gene delivery vehicle according to claim 54, wherein the capsid comprises VP1 containing the sequence of SEQ ID NO: 12, VP2 containing the sequence of SEQ ID NO: 13, and VP3 containing the sequence of SEQ ID NO:

14.

56. The gene delivery vehicle according to claim 50, which is a nonviral vector.

57. The gene delivery vehicle according to claim 56, wherein the non-viral vector is a nanoparticle selected from the group consisting of lipid nanoparticles (LNPs), polymeric nanoparticles, lipid polymer nanoparticles (LPNPs), protein or peptide-based nanoparticles, DNA dendrimers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes.

58. The gene delivery vehicle according to claim 56, wherein the nonviral vector is LNP or LPNP.

59. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 22 and 34 to 42, a PPT1 polypeptide according to any one of claims 23 to 33, a recombinant viral vector nucleic acid according to any one of claims 43 to 49, or a gene delivery vehicle according to any one of claims 50 to 58, and a pharmaceutically acceptable carrier.

60. A method for increasing PPT1 in a subject, comprising the step of administering to the subject a polynucleotide according to any one of claims 1 to 22 and 34 to 42, a PPT1 polypeptide according to any one of claims 23 to 33, a recombinant viral vector nucleic acid according to any one of claims 43 to 49, a gene delivery vehicle according to any one of claims 50 to 58, or a pharmaceutical composition according to claim 59.

61. A method for treating neuronal ceroid lipofuscinosis 1 in a subject, comprising the step of administering to the subject a polynucleotide according to any one of claims 1 to 22 and 34 to 42, a PPT1 polypeptide according to any one of claims 23 to 33, a recombinant viral vector nucleic acid according to any one of claims 43 to 49, a gene delivery vehicle according to any one of claims 50 to 58, or a pharmaceutical composition according to claim 59.

62. The method according to claim 60 or 61, wherein the administration step includes intracerebral, intracisional, or intraventricular administration.

63. The method according to claim 62, wherein the administration is intraventricular and results in significant rAAV delivery to at least the prefrontal cortex, parietal cortex, temporal cortex, occipital cortex, thalamus, cerebellar cortex, hippocampus, corpus callosum, spinal cord, caudate nucleus, choroid plexus, optic chiasm, fornix, periaqueductal gray matter, olfactory bulb, and optic nerve.

64. The method according to claim 60 or 61, wherein the administration step includes an initial administration outside the central nervous system (CNS).

65. The method according to claim 63, wherein the subject is a sheep.

66. The method according to any one of claims 60 to 62, wherein the administration is systemic.

67. The method according to any one of claims 60 to 63, wherein the subject is a human.

68. AAV vector genome plasmid comprising the recombinant viral vector nucleic acid according to any one of claims 43 to 49.

69. The AAV genome plasmid according to claim 68, which lacks the rep and cap genes.

70. A method for generating an rAAV vector, comprising the step of culturing an rAAV-producing cell line containing rAAV helper virus activity, wherein the genome of the producing cell contains the recombinant viral vector nucleic acid, rep gene and cap gene described in any one of claims 43 to 49, and the rAAV vector is generated.

71. A method for generating an rAAV vector, comprising the step of culturing rAAV-tolerant cells comprising the AAV genome plasmid described in claim 68 or 69, wherein the rAAV-tolerant cells further comprise (a) rep and cap genes provided either as part of a cell genome and / or by one or more separate plasmids, and (b) helper virus activity provided by the cell genome and / or by one or more separate plasmids.

72. The method according to claim 71, wherein the rAAV-tolerant cells are packaging cells, and the genome of the packaging cells includes the cap gene and the rep gene.

73. The method according to claim 71, wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid; or (b) the rep gene and the cap gene are provided by a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

74. A method for obtaining an rAAV vector, comprising the steps of (a) generating an rAAV using the method of any one of claims 70 to 73, and (b) purifying the rAAV.

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