WO2025166048A1 - Synthetic gba1 genes - Google Patents

Abstract

Synthetic polynucleotides (coGBA1) encoding human glucocerebrosidase (GCase) and exhibiting augmented expression in cell culture and/or in a mammal are described herein. Related recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the coGBA1 polynucleotides are also described. Methods of treating a disease or condition mediated by GCase, such as Gaucher disease type 1, 2, or 3, Parkinson's disease, or a Lewy body disorder associated with GBA1 mutations, are also described.

Description

SYNTHETIC GBA1 GENES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63/627,542, filed January 31, 2024, which is incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project numbers 1ZIAHG200318-19 and ZIA HG200336-19 by the National Institutes of Health, National Human Genome Research Institute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 97,662 Byte XML file named “772399.XML,” dated January' 30, 2025.

BACKGROUND OF THE INVENTION

[0004] Gaucher disease (GD) is an inherited deficiency of the lysosomal enzy me glucocerebrosidase (GCase) resulting from mutations in GBA1. This gene, located on human chromosome 1 q21, has 11 exons, with a highly homologous pseudogene located 16 kilobases (kb) downstream. Over 500 different disease-associated rare and common pathogenic variants have been identified. Recombination events within GBA1 and both upstream and downstream are the source of some mutations. GD is a lysosomal storage disorder and is classically divided into three types based on the presence and rate of neurological manifestations: Gaucher disease type 1 (GDI) is non-neuronopathic. Gaucher disease type 2 (GD2), a neurodegenerative disorder of infancy, is the acute neuronopathic form. Gaucher disease type 3 (GD3) is the chronic neuronopathic form with diverse manifestations that include abnormal eye movements, myoclonic epilepsy or learning disabilities. Enzyme replacement therapy (ERT) for GD was approved in 1992, and several thousands of patients have now received this therapy. It is a life-long treatment, administered intravenously (IV), that costs about $100,000- $400,000 per patient per year. ERT can successfully reverse the visceral and hematological manifestations of Gaucher disease. However. GCase does not cross the blood-brain-barrier when delivered IV. Therefore, IV-delivered GCase does not impact brain involvement in the neuronopathic forms of GD.

[0005] Moreover, there is a need for central nervous system (CNS)-directed therapies to treat patients with GBA1 mutations. Mutations in GBA1 , even in the heterozy gous state, are the most commonly known genetic risk factor for the development of Parkinson’s disease and related Lewy body disorders. Between 5-10% of patients with Parkinson’s disease carry a variant in GBA1. Therefore, there is a large, unmet need in the field of Gaucher therapeutics to develop efficacious therapies for neuronopathic GD as well as the larger group of patients who have Parkinson’s disease and related Lewy body disorders caused by GBA1 mutations.

BRIEF SUMMARY OF THE INVENTION

[0006] An aspect of the invention provides a synthetic GBA1 (glucosylceramidase beta 1) polynucleotide (coGBA l) selected from the group consisting of: (a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 2-4; (b) a polynucleotide having a nucleic acid sequence with at least 85% identity⁷ to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 15 and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1; (c) a polynucleotide having a nucleic acid sequence with at least 85% identity to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 16, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 118-1611 of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 118-1611 of SEQ ID NO: 1; (d) a polynucleotide having a ribonucleic acid (RNA) sequence encoded by a polynucleotide that is complementary⁷ to the polynucleotide of any' one of (a)-(c); and (e) a peptide-modified polynucleotide comprising the polynucleotide of any one of (a)-(d).

[0007] Further aspects of the invention provide recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the coGBAl polynucleotides of the invention.

[0008] Still further aspects of the invention provide methods of treating a disease or condition mediated by GCase. [0009] Another aspect of the invention provides a method of detecting the presence of a synthetic GBA1 polynucleotide (coGBAl) in a biological sample from a mammal, the method comprising: (a) obtaining at least one test sample comprising isolated nucleic acid from a biological sample from a mammal; (b) contacting any of the inventive synthetic polynucleotide described herein with the at least one test sample under conditions allowing for a complex to form between the synthetic polynucleotide and the isolated nucleic acid of the test sample; (c) detecting the complex; and (d) comparing a presence of the complex in the at least one test sample with an absence of complex from a negative sample that lacks the synthetic polynucleotide, wherein detection of the complex is indicative of the presence of the synthetic polynucleotide in the biological sample from the mammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0010] Figures 1A-1F show an alignment of the sequences of human GBA1 NCBI Reference Sequence: NM_001005742.3 (hGBA l) (SEQ ID NO: 1) against each codon optimized GBA1 sequence including codon optimized GBA1.1 (coGBAl.l) (SEQ ID NO: 2), codon optimized GBA1.2 (coGBA1.2) (SEQ ID NO: 3). and codon optimized GBA1.3 (coGBAl.3) (SEQ ID NO: 4). Figure 1 A shows the alignment at positions 1 -300, Figure IB at positions 301-600, Figure 1C at positions 601-900, Figure ID at positions 901-1200, Figure IE at positions 1201-1500, and Figure IF at positions 1501-1611.

[0011] Figure 2 shows a phylogenetic tree of GBA1 polynucleotides.

[0012] Figures 3A-3C show an alignment of the sequences of human GBA1 NCBI

Reference Sequence: NM_001005742.3 (hGBAl) (SEQ ID NO: 1) against codon optimized GBA1 sequence GBA1. 1 (coGBAl. 1) (SEQ ID NO: 2). Figure 3A shows the alignment at positions 21-495, Figure 3B at positions 496-1095, and Figure 3C at positions 1096-1611. [0013] Figures 4A-4C show an alignment of the sequences of human GBA1 NCBI Reference Sequence: NM_001005742.3 (hGBAl) (SEQ ID NO: 1) against codon optimized GBA1 sequence GBA1.2 (coGBA1.2) (SEQ ID NO: 3). Figure 4A shows the alignment at positions 21-500, Figure 4B at positions 501-1100, and Figure 4C at positions 1101-1599. [0014] Figures 5A-5C show an alignment of the sequences of human GBA1 NCBI Reference Sequence: NM_001005742.3 (hGBAl) (SEQ ID NO: 1) against codon optimized GBA1 sequence GBA1.3 (coGBAl.3) (SEQ ID NO: 4). Figure 5A shows the alignment at positions 25-504, Figure 5B at positions 505-1104, and Figure 5C at positions 1105-1611. [0015] Figure 6 is an image of a western blot showing the level of GCase expression in HEK 293T cells transfected with GFP control plasmid (GFP), hGBA pcDNA3. 1(+) plasmid (hGBAl), CoGBAl.l pcDNA3.1(+) plasmid (coGBAl.l), CoGBA1.2 pcDNA3.1(+) plasmid (coGBA1.2), or CoGBA1.3 pcDNA3.1(+) plasmid (coGBAl.3).

[0016] Figure 7 is an image of a western blot showing the level of GCase expression in HEK 293T cells transfected with GFP control plasmid (GFP), pAAV.Camk2a.hGBAl plasmid (SEQ ID NO: 14). pscAAV-EFIScoGBAl. l plasmid (SEQ ID NO: 6) (“pscAAV- Efs-coGABAl. l” in the figure), or pAAV-CAG hGBAl plasmid (SEQ ID NO: 12).

[0017] Figure 8 is a drawing of a map of the EFl S coGBAl. 1 HPRE plasmid (SEQ ID NO: 5).

[0018] Figures 9A-9B are an image of a western blot for expression of GCase (Figure 9A) and a bar graph showing the results of GCase enzyme activity (Figure 9B) in H4 GBAl^+,+ cells and H4 GBAT ' cells given no treatment (no transfection=NT), or transfected with pscAAV-EFIS-coGBAl.l plasmid (“EFs-coGBAl. l” in the figure) (SEQ ID NO: 6) or pAAV-CAG-hBGAl(CAG-hBGAl) (SEQ ID NO: 12) plasmid (Figure 9B).

[0019] Figures 10A-10B are images of western blots for expression of GCase in H4 GBA^+I+ cells and H4 GBA" cells given no treatment, and H4 GBA" cells infected with either AAV9 (Figure 10A) or AAVrhlO (Figure 10B) serotyped EFlS.coGBAl. l.HPRE (SEQ ID NO: 5) (“EFs.coGABAl. l.HPRE” in the figure) virus at a concentration of 5e⁴ or 5e⁵genome copies per cell (GCs/cell).

[0020] Figures 1 1A-1 IB are line graphs showing the probability of survival for gba K14- Inl/lnl mice (a lethal neuronopathic GBA mouse model) administered with EF 1 ScoGBA 1.1 HPRE (SEQ ID NO: 5) (“Efs. coGABA 1. I . HPRE" in the figure) in an AAV9 (Figure 11A) or AAVrhlO (Figure 1 IB) serotyped virus. Figure 11A shows the probability of survival of untreated gba K14-lnl/lnl gba KO) mice, and gba KO mice treated with AAV9- EF1 S.coGBAl . 1 .HPRE.Kana at a dose of 1 x 10¹¹ genome copies per pup (GCs/pup), 2 x 10¹¹ GCs/pup, and 4 x 10¹¹ GCs/pup. Figure 1 IB shows the probability of survival of untreated gba K14-lnl/lnl (gba KO) mice, and gba KO mice treated with AAVrhlO- EF1S. coGBAl. l. HPRE.Kana at a dose of4 x 10¹¹ GCs/pup.

[0021] Figures 12A-12B are line graphs showing the probability of survival for gba K14- Inl/lnl mice administered with EFIS coGBAl. l HPRE (SEQ ID NO: 5) (“EFs.coGABAl. l.HPRE” in the figure) in an AAV9 (Figure 12A) or AAVrhlO (Figure 12B) serotyped virus. Untreated Gba KO mice (nGD mice) were used as controls (closed circle). The graph depicts the percent survival of different cohorts of animals. All four nGD mice injected with high dose AAV9-hGBAl (3.1 x 10¹⁴ vg/kg, closed square) survived longer than 1 year. High dose AAVrhlO-hGBAl injection also extended lifespan of the nGD mice longer than 7 months while two out of four injected mice died at 6-7 months age. Lower doses of the AAV9-hGbal (1.5 x io¹⁴ and 7.7 x io¹³ vg/kg) did not extend the lifespan of nGD mice past 1~2 weeks.

[0022] Figures 13A and 13B are line graphs showing the probability of survival for Gbal^fox/b^lox;^rT g^CAGCreER mice (TAM-GNα KO mice) administered with EFIS coGBAl.l HPRE (SEQ ID NO: 5) (“EFs.coGABAl. l.HPRE” in the figure) in an AAV9 (Figure 13A) or AAVrhlO (Figure 13B) serotyped virus. TAM-GAu KO mice not treated with AAVs were used as controls (closed circle). Both AAV-treated and AAV-untreated mice were fed TAM chow from 6 to 10 weeks of age to induce Gbal KO. The graphs depict the percent survival of different cohorts of animals. Both AAV9-hGbal and AAVrhlO-hGBAl injection successfully extended the lifespan of the TAM-Gba KO mice for at least 8 weeks longer than the untreated control mice.

[0023] Figure 14 is a drawing of a map of the pAAV EF1L coGBAl. 1 HPRE plasmid (SEQ ID NO: 18) generated from full plasmid nanopore sequencing.

[0024] Figure 15 presents a Western blot of H4 cells (neuroblastoma) transfected with GBA plasmid constructs as indicated in lanes 1-8; non-transfected H4 cells were employed as a control. Shown are results when blots were probed with anti-GCase and p-actin.

[0025] Figure 16 presents a Western blot of H4 cells (neuroblastoma) infected with AAV9 EF1L coGBAl . l at either 0, 5e⁴, 5e⁵, or le⁶ genome copies per cell (GCs/cell) as indicated. Shown are results when blots w ere probed with anti-GCase and P-actin.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Aspects of the invention provide synthetic GBA1 polynucleotides (coGBAl polynucleotides) that may provide any one or more of a variety of advantages. For example, the inventive coGBAl polynucleotides may provide enhanced expression in mammalian cells. The inventive coGBAl polynucleotides may be expressed in any of a wide variety of mammalian tissues (e.g.. ubiquitous expression) or may be targeted for expression in a particular mammalian tissue (e.g., liver, bone marrow', or neuronal tissue). Expression in multiple types of tissues may be useful for the treatment of GD, w hich may affect multiple tissues in the body. Compared to wild-type (WT) human polynucleotides encoding GCase, the inventive coGBAl polynucleotides are codon-optimized and then individually adjusted to enhance expression upon administration. The inventive coGBAl polynucleotides may provide one or both of improved survival and increased GCase activity, e.g., in a mouse model of model of neuronopathic GCase deficiency.

[0027] The inventive coGBAl polynucleotides may, advantageously, be useful as a therapeutic, via viral or non-viral mediated gene delivery, to restore GCase function in patients with a disease or condition associated with a GBA1 mutation, e.g., GD. The inventive coGBAl polynucleotides may increase GCase activity through enhanced expression and may ameliorate disease progression. The inventive coGBAl polynucleotides may also be useful for the in vitro production of GCase for use in enzy me replacement therapy for diseases or conditions associated with a GBA1 mutation, e.g., GD. The inventive coGBAl polynucleotides may also be useful for mRNA therapeutics. DNA-based non-viral gene therapies, or non-viral gene therapy with AAV, lentiviral and editing vectors, and other vectors, such as neurotrophic vectors.

[0028] The inventive coGBAl polynucleotides may provide increased expression of the GBA1 gene relative to naturally occurring human GBA1 sequences. In aspects, the inventive coGBAl polynucleotides were designed to not alter the naturally occurring human GCase amino acid sequence. They were also designed to have any one or more of increased transcription, translation, and stability. This design was accomplished by, e.g., any one or more of the evaluation of human codon biases, evaluation of GC, CpG. and negative GpC content, evaluation of the interaction between the codon and anti-codon, reducing or eliminating cryptic splicing sites and RNA instability motifs, and compatibility with the vector backbones.

[0029] Human GCase (also referred to as "glucosy Icerami dase beta 1” or “glucocerebrosidase’^?) is a lysosomal membrane protein that cleaves the beta-glucosidic linkage of glycosylceramide, an intermediate in glycolipid metabolism. The amino acid sequence of human WT GCase is set forth in SEQ ID NO: 15 (GenBank® Accession No. NP_000148.2). The gene encoding naturally occurring human GCase is referred to as GBA1. The WT human GBA1 nucleic acid sequence is set forth in SEQ ID NO: 1 (GenBank® Accession No. NM_001005742.3). The polynucleotides encoding synthetic GCase are referred to as coGBAl. Naturally occurring human GBA1 is referred to as GBA1 , while synthetic GBA1 is designated as coGBAl, even though the two are identical at the amino acid level.

[0030] The WT, human GCase (SEQ ID NO: 15) includes a 39-amino acid residue, N- terminal signal peptide. The mature GCase (GCase) is composed of 497 amino acids (amino acid residues 40-536). The amino acid sequence of the WT GCase signal peptide is SEQ ID NO: 17. The amino acid sequence of the mature GCase is SEQ ID NO: 16.

[0031] An aspect of the invention provides a synthetic GBA1 polynucleotide (coG A l) selected from the group consisting of: (a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 2-4; (b) a polynucleotide having a nucleic acid sequence with at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 15, and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1; (c) a polynucleotide having a nucleic acid sequence with at least 85% identity to the nucleic acid sequence of nucleotides 1 18-1611 of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 16, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 118-1611 of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 118-1611 of SEQ ID NO: 1; (d) a polynucleotide having a ribonucleic acid (RNA) sequence encoded by a polynucleotide that is complementary to the polynucleotide of any one of (a)-(c); and (e) a peptide-modified polynucleotide comprising the polynucleotide of any one of (a)-(d).

[0032] In one aspect of the invention, codon optimization was employed to create a highly active and synthetic GBA1 polynucleotide. Accordingly, in an aspect, the synthetic polynucleotide is codon-optimized. “Codon optimization” refers to the process of altering a naturally occurring polynucleotide sequence to enhance expression in the target organism, e.g., humans. Codon optimization involves determining the relative frequency of a codon in the protein-encoding genes in the human genome. For example, isoleucine can be encoded by AUU, AUC, or AU A, but in the human genome, AUC (47%), AUU (36%), and AUA (17%) are variably used to encode isoleucine in proteins. Therefore, in the proper sequence context, AUA would be changed to AUC to allow this codon to be more efficiently translated in human cells.

[0033] In the subject application, the human GBA1 gene has been altered to replace codons that occur less frequently in human genes with those that occur more frequently and/or with codons that are frequently found in highly expressed human genes. A series of synthetic codon optimized and adjusted GBA1 genes were developed (Table 1). Alignments (Figures 1A-1F; 3A-3C; 4A-4C; and 5A-5C) and phylogenetic analysis prepared with CLUSTAL W (Figure 2) reveals that the coGBAl polynucleotides are highly divergent at the nucleotide level while maintaining WT GCase amino acid sequence identity.

[0034] Thus, an aspect of the invention comprises GCase-encoding, synthetic polynucleotides comprising the nucleic acid sequence of any one of SEQ ID NOs: 2-4.

[0035] Another aspect of the invention provides a polynucleotide having a nucleic acid sequence with at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 15 and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1 . Additional aspects of the invention provide a polynucleotide having a nucleic acid sequence with at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%. at least 98%, at least 99%, or 100% identity to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence 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%, or at least 99% , or 100% identical to SEQ ID NO: 15, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1.

[0036] Another aspect of the invention provides a polynucleotide having a nucleic acid sequence with at least 85% identity to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 16, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 118-1611 of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 118-1611 of SEQ ID NO: 1. Additional aspects of the invention provide a polynucleotide having a nucleic acid sequence with at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence 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%, or at least 99% , or 100% identical to SEQ ID NO: 16. wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 118-1611 of SEQ ID NO: 1. [0037] Another aspect of the invention provides a polynucleotide having a ribonucleic acid (RNA) sequence encoded by a polynucleotide that is complementary to the polynucleotide of any one of (a)-(c): (a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 2-4; (b) a polynucleotide having a nucleic acid sequence with at least 85% identity (such as at least 86%. at least 87%, at least 88%, at least 89%, 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%, or at least 99%, or 100% identity) to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical (such as at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. or 100% identical) to SEQ ID NO: 15, and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1; and (c) a polynucleotide having a nucleic acid sequence with at least 85% identity (such as 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%, or 100% identity ) to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity) to SEQ ID NO: 16, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 118-1611 of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 118-1611 of SEQ ID NO: 1. In this regard, the inventive coGBAl polynucleotides may be useful for preparing a variety of RNA molecules, including, but not limited to, mRNA, modified mRNA, lipid nanoparticle-mRNA (LNP-mRNA), and circRNAs. Such RNA molecules could be programmed per the canonical rules of transcription with natural (U) and/or modified ribonucleotides. Modified ribonucleotides may include, but are not limited to, 2-thiouridine (s2U), pseudouridine ( ), Nl- methylpseudouridine (ml'P) , N6-methyladenosine (m6A), 5-methylcytosine (m5C) and other modified ribonucleotides known to practitioners of the art.

[0038] Another aspect of the invention provides a peptide-modified polynucleotide comprising the polynucleotide of any one of (a)-(d): (a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 2-4; (b) a polynucleotide having a nucleic acid sequence with at least 85% identity (such as at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%, or 100% identity) to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical) to SEQ ID NO: 15, and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1; (c) a polynucleotide having a nucleic acid sequence with at least 85% identity (such as at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%, or 100% identity) to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical) to SEQ ID NO: 16, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 118-1611 of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 118-1611 of SEQ ID NO: 1; and (d) a polynucleotide having a ribonucleic acid (RNA) sequence encoded by a polynucleotide that is complementary to the polynucleotide of any one of (a)-(c). Peptide-modified polynucleotides (Peptide Nucleic Acids (PNAs)) are DNA/RNA analogues in which sugar-phosphate backbone is replaced by N-2-aminoethylglycine repeating units.

[0039] In one aspect, the inventive synthetic polynucleotide encodes a polypeptide with 100% sequence identity to the naturally occurring, human GCase (SEQ ID NO: 15) or the mature WT GCase of SEQ ID NO: 16.

[0040] In another aspect, the inventive synthetic polynucleotide encodes a polypeptide with at least 90% identity to SEQ ID NO: 15 or the mature WT GCase of SEQ ID NO: 16 and which retains the naturally occurring human GCase function z.e., the capacity to cleave the beta-glucosidic linkage of glycosylceramide. Additional aspects provide a synthetic polynucleotide that encodes a polypeptide with at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity- to SEQ ID NO: 15 or the mature WT GCase of SEQ ID NO: 16 and which retains the naturally occurring human GCase function.

[0041] In one aspect, the GCase encoded by the inventive polynucleotides retains at least 90% of the naturally occurring human GCase function, z.e., the capacity- to cleave the betaglucosidic linkage of glycosylceramide. In another aspect, the encoded GCase retains at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the naturally occurring human GCase function. This enzyme function can be measured, for example, by measuring the capacity to cleave the betaglucosidic linkage of glycosylceramide and/or the ability to provide one or both of improved survival and increase in GCase activity, e.g., in a mouse model of neuronopathic GCase deficiency, for example, as described in the Examples below.

[0042] In some aspects, the synthetic polynucleotide exhibits improved expression in a mammalian host cell relative to the expression of naturally occurring human GBA1 polynucleotide sequence. The improved expression is due to the polynucleotide comprising codons that have been optimized relative to the naturally occurring human GBA1 polynucleotide sequence of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1). In one aspect, the synthetic polynucleotide has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of less commonly used codons replaced with more commonly used codons. In additional aspects, the polynucleotide has at least 85%, at least 90%, or at least 95% of less commonly used codons replaced with more commonly used codons, and demonstrates equivalent or enhanced expression of GCase in a mammalian cell as compared to the expression of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1) in the same mammalian host cell.

[0043] In some aspects, the synthetic polynucleotide sequences of the invention provide equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1) (as demonstrated by expression of the polynucleotide of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: Q in the same mammalian host cell). In some aspects, the inventive polynucleotide exhibits an increase in expression in a mammalian host cell relative to the expression of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1) in the same mammalian host cell. In some aspects, the inventive polynucleotide preferably encodes a polypeptide that retains at least 80% of the enhanced GCase expression (as demonstrated by expression of the polynucleotide of any one of SEQ ID NOs: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4) in a mammalian host cell). In additional aspects, the polypeptide retains at least 85%, at least 90%, at least 95% or 100% of the enhanced expression observed with the polynucleotide of any one of SEQ ID NOs: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4) in a mammalian host cell. In aspects, the mammalian host cell is a human host cell or a mouse host cell. In some aspects, the mammalian host cell is a neuronal cell, a hepatic cell, or a bone marrow cell from human or mouse.

[0044] In designing the coGBAl of the present invention, the following considerations were balanced. For example, the fewer changes that are made to the nucleotide sequence of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1), the less potential there is to alter the secondary structure of the sequence, which can have a significant impact on gene expression. The introduction of undesirable restriction sites was also reduced, facilitating the subcloning of coGBAl into the recombinant expression vector. However, a greater number of changes to the nucleotide sequence of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1) allows for more convenient identification of the translated and expressed message, e.g. mRNA, in vivo. Additionally, a greater number of changes to the nucleotide sequence of SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1) provides for increased likelihood of greater expression. Homopolymeric strings of cytosines (C) and guanines (G) were also removed. These considerations were balanced when arriving at the nucleic acid sequences of SEQ ID NO: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4). The polynucleotide sequences encoding coGBAl allow for increased expression of the coGBAl polynucleotide relative to naturally occurring human GBA1 polynucleotides. Because the sequences are novel, they may facilitate detection using nucleic acid-based assays.

[0045] GCase has a total of 536 amino acid residues, and coGBA 1 contains approximately 536 codons corresponding to said amino acid residues. In SEQ ID NOs: 2-4, codons were changed from that of the natural human GBA1. However, despite changes from SEQ ID NO: 1, SEQ ID NOs: 2-4 encode the amino acid sequence SEQ ID NO: 15 of WT, human GCase. Codons for SEQ ID NOs: 2-4 were changed in accordance with the equivalent amino acid positions of SEQ ID NO: 15. In this aspect, the amino acid sequence for natural human GCase has been retained.

[0046] Similarly, in nucleotides 118-1611 of SEQ ID NOs: 2-4, codons were changed from that of the natural human GBA1. However, despite changes from nucleotides 118-1611 of SEQ ID NO: 1, nucleotides 118-1611 of SEQ ID NOs: 2-4 encode the amino acid sequence SEQ ID NO: 16 of WT, mature human GCase. Codons for nucleotides 118-1611 of SEQ ID NOs: 2-4 were changed in accordance with the equivalent amino acid positions of SEQ ID NO: 16. In this aspect, the amino acid sequence for natural human mature GCase has been retained.

[0047] It can be appreciated that partial reversion of the designed coGBAl to codons that are found in GBA1 can be expected to result in nucleic acid sequences that, when incorporated into appropriate vectors, can also exhibit the desirable properties of any one or more of SEQ ID NOs: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4). For example, such partial reversion or hybrid variants can have GCase expression from a vector inserted into an appropriate host cell that is equivalent to that of any one or more of SEQ ID NOs: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4). For example, aspects of the invention include nucleic acids in which at least 1 altered codon, at least 2 altered codons, at least 3, altered codons, at least 4 altered codons, at least 5 altered codons, at least 6 altered codons, at least 7 altered codons, at least 8 altered codons, at least 9 altered codons, at least 10 altered codons, at least 11 altered codons, at least 12 altered codons, at least 13 altered codons, at least 14 altered codons, at least 15 altered codons, at least 16 altered codons, at least 17 altered codons, at least 18 altered codons, at least 20 altered codons, at least 25 altered codons, at least 30 altered codons, at least 35 altered codons, at least 40 altered codons, at least 50 altered codons, at least 55 altered codons, at least 60 altered codons, at least 65 altered codons, at least 70 altered codons, at least 75 altered codons, at least 80 altered codons, at least 85 altered codons, at least 90 altered codons, at least 95 altered codons, at least 100 altered codons, at least 110 altered codons, at least 120 altered codons, at least 130 altered codons, at least 130 altered codons, at least 140 altered codons, at least 150 altered codons, at least 1 0 altered codons, at least 170 altered codons, or at least 180 altered codons, in any one of SEQ ID NOs: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4) are reverted to native codons according to SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1). and having equivalent or increased expression in a mammalian host cell as compared to expression of any one of SEQ ID NOs: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4) in the same mammalian host cell. Alternately, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the altered codon positions in any one of SEQ ID NOs: 2-4 (or nucleotides 118-1611 of any one of SEQ ID NOs: 2-4) are reverted to the native sequence according to SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1), and having equivalent or increased expression in a mammalian host cell as compared to expression of any one of SEQ ID NOs: 2-4 (or nucleotides 1 18-1611 of any one of SEQ ID NOs: 2-4) in the same mammalian host cell.

[0048] The exact differences between SEQ ID NOs: 2-4 and SEQ ID NO: 1 demonstrate that the coGBAl polynucleotides are distinct from WT human GBA1. Table 2 depicts the pairwise nucleotide identity between SEQ ID NOs: 2-4 and SEQ ID NO: 1. The values range between 76% and 81%. The exact positions of variation are depicted in a base-by-base comparison in Figures 1 A-1F; 3A-3C; 4A-4C; and 5A-5C vs Table 2, and a phylogenetic analysis presented in Figure 2 further demonstrates that SEQ ID NOs: 2-4 are distinct.

[0049] In some aspects, polynucleotides of the present invention do not share 100% identity with SEQ ID NO: 1 (or nucleotides 118-1611 of SEQ ID NO: 1). In other words, in some aspects, polynucleotides having 100% identity with SEQ ID NO: 1 (or nucleotides 118- 1611 of SEQ ID NO: 1) are excluded from the aspects of the present invention.

[0050] In one aspect of a synthetic polynucleotide according to the invention, the nucleic acid sequence is a DNA sequence (e.g., a cDNA sequence). In another aspect, the nucleic acid sequence is an RNA sequence or peptide-modified nucleic acid sequence.

[0051] In another aspect, the invention is directed to a recombinant expression vector comprising any of the coGBAl polynucleotides described herein. In aspects of the invention, the recombinant expression vector may be single-stranded or self-complementary.

[0052] In an aspect of the invention, the recombinant expression vector may be a viral vector, such as a lentiviral vector, a retroviral vector, an alphaviral vector, a vaccinial viral vector, an adenoviral vector, a herpes viral vector, a fowl pox viral vector, or an adeno- associated viral vector (e.g., AAV9, AAVrhlO vector, or a vector derived from other AAV serotypes known in the art). In other aspects of the invention, the recombinant expression vector may be a non-viral vector, such as a transposon (e.g., PiggyBac (PB) transposon, Sleeping Beauty transposon), retrotransposon, plasmid DNA, liposome-DNA complex (lipoplexes), or polymer-DNA complex (polyplexes).

[0053] In aspects of the invention, the vector can be or comprise a neurotropic vector, such as have been described in the art (see, e.g., Huang et al., Science 384(6701): 1220-1227 (2024) (incorporated herein in its entirety), describing an AAV capsid, BI-hTFRl , which binds human transferrin receptor (TfRl)). Additionally, herpesviral vectors (e.g., Herpes Simplex Virus (HSV)-l. HSV-2, etc.), which exhibit neurotropism, can be used. In aspects of the invention, the vector can be or comprise an RNA only integrating LNP delivery systems, such as are known in the art (see. e.g., Zhang et al., Nat. Biotechnol., 43(1): 42-51 (2025) (incorporated herein in its entirety), describing precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus and Wang et al., EMBO Rep., doi.org/10. 1038/s44319-024-00364-7 (2025) (incorporated herein in its entirety), describing a CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery, which can be used to deliver the synthetic polynucleotide according to the invention as the “payload.”).

[0054] In certain aspects, the recombinant expression vector comprises an expression cassette. The expression cassette may, for example, include any one or more of a 5’ inverted terminal repeat (ITR), a promoter, one or more introns, any of the inventive coGBAl polynucleotides described herein, an mRNA stability element, such as the WPRE (woodchuck post-transcriptional regulatory element) or HPRE (hepatitis B derived post- translational response element), a polyadenylation signal (e.g., bovine growth hormone polyadenylation signal (BGH A) or the rabbit beta-globin polyadenylation signal (rBGA)), and a 3’ ITR.

[0055] In another aspect of the recombinant expression vector according to the invention, the synthetic polynucleotide is operably linked to an expression control sequence (e.g.. a promoter). The promoter may vary with the type of viral recombinant expression vector used. For example, the promoter may be a viral promoter. Viral promoters include, for example, the ubiquitous cytomegalovirus immediate early (CMV-IE) promoter, the chicken beta-actin (CBA) promoter, the simian virus 40 (SV40) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, the Moloney murine leukemia virus (MoMLV) LTR promoter, and other retroviral LTR promoters. In a preferred aspect, the promoter is the EFIS (elongation factor 1 promoter, short) constitutive promoter, EF1L promoter (elongation factor 1 promoter, long) constitutive promoter, Camk2a neurological promoter, or the CMV enhanced, chicken beta actin heterologous (CAG) constitutive promoter.

[0056] In one specific aspect, the inventive coGBAl polynucleotide could be placed under the transcriptional control of a ubiquitous or tissue-specific promoter. In an aspect of the invention, the recombinant expression vector is configured for ubiquitous expression of the coGBAl polynucleotide. The use of a tissue-specific promoter can restrict unwanted GBA1 expression, as well as facilitate persistent GBA1 expression. The inventive coGBAl polynucleotide could then be delivered into the portal vein, systemic circulation, ventricles, or directly injected into a tissue or organ, such as the brain. In an aspect of the invention, the recombinant expression vector is configured for expression of the coGBAl polynucleotide in neuronal tissue, hepatic tissue, or bone marrow tissue. In addition to the neuronal tissue, the kidney, pancreas, eye, heart, lungs, and muscle may constitute targets for therapy. Other tissues or organs may be additionally contemplated as targets for therapy.

[0057] Tissue-specific promoters include, without limitation, Camk2a, Apo A-I, ApoE, hAAT, transthyretin, liver-enriched activator, albumin, PEPCK, and RNAPu promoters (liver), PAI-1, ICAM-2 (endothelium), MCK, SMC a-actin, myosin heavy-chain, and myosin light-chain promoters (muscle), cytokeratin 18, CFTR (epithelium), GFAP, NSE, Synapsin I, Preproenkephahn, d0H, prolactin, and myelin basic protein promoters (neuronal), and ankyrin, a-spectrin, globin, HLA-DRa, CD4, glucose 6-phosphatase, and dectin-2 promoters (erythroid).

[0058] Regulatable promoters (for example, ligand-inducible or stimulus-inducible promoters) are also contemplated for expression constructs according to the invention. The inventive recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

[0059] In yet another aspect, the coGBAl polynucleotide could be used in ex vivo applications via packaging into a recombinant expression vector to create an integrating recombinant expression vector that could be used to permanently correct any cell type from a patient with GCase deficiency. The coG/M /-transduced and corrected cells could then be used as a cellular therapy. Examples might include CD34+ stem cells, primary hepatocytes, or fibroblasts derived from patients with GCase deficiency. Fibroblasts could be reprogrammed to other cell types using induced pluripotent stem cell (iPS) methods well known to practitioners of the art. In yet another aspect, the coGBAl polynucleotide could be recombined using genomic engineering and editing techniques that are well known to practitioners of the art. such as ZFNs, TALENS and Cas/CRISPR, into the GBA1 locus, a genomic safe harbor site, such as AAVS 1, or into another advantageous location, such as into rDNA, the albumin locus, SERPINA 5 or 7, PCSK9, GAPDH, or a suitable expressed pseudogene.

[0060] In an aspect of the invention, the recombinant expression vector comprises the nucleic acid sequence of any one of SEQ ID NOs: 5-8 and 18. [0061] Another aspect of the invention further provides a host cell comprising any of the polynucleotides or any of the recombinant expression vectors described herein. As used herein, the term “host cell” refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Examples of host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a cell. For purposes of producing a GCase, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be, for example, a neuronal cell, a hepatic cell, or a bone marrow cell from human or mouse.

[0062] Also provided by an aspect of the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described herein, in addition to at least one other cell, e.g., a host cell (e.g.. a kidney or liver cell), which does not comprise any of the recombinant expression vectors. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one aspect of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0063] The inventive synthetic polynucleotides, recombinant expression vectors, host cells (including populations thereof), all of which are collectively referred to as “inventive coGBAl material(s)” hereinafter, can be formulated into a composition, such as a pharmaceutical composition. In this regard, an aspect of the invention provides a pharmaceutical composition comprising any of the inventive synthetic polynucleotides, recombinant expression vectors, or host cells (including populations thereof), described herein, and a pharmaceutically acceptable carrier. [0064] A pharmaceutical composition for treating a mammal by gene therapy may comprise a therapeutically effective amount of a recombinant expression vector comprising the coGBAl polynucleotide or a viral particle produced by or obtained from same. The pharmaceutical composition may be for human or non-human mammal usage. Typically, a physician will determine the actual dosage which will be most suitable for an individual mammal, and it will vary with the age, weight, and response of the particular individual mammal.

[0065] The composition may, in specific aspects, comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. Such materials should be non-toxic and should not interfere with the efficacy of the coGBAl polynucleotide. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy, 23^rd Ed., Academic Press (2020).

[0066] As used herein, a '‘pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In certain aspects, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0067] The choice of pharmaceutical carrier, excipient, or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient, or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system). For oral administration, excipients such as starch or lactose may be used. For parenteral administration, a sterile aqueous solution may be used, optionally containing other substances, such as salts or monosaccharides to make the solution isotonic with blood.

[0068] A pharmaceutical composition according to the invention may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The pharmaceutical compositions may be administered to a patient alone, or in combination with other agents, modulators, or drugs (e.g., antibiotics).

[0069] The pharmaceutical composition may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Additional dosage forms contemplated include: in the form of a suppository or pessary; in the form of a lotion, solution, cream, ointment or dusting powder; by use of a skin patch; in capsules or ovules; in the form of elixirs, solutions, or suspensions; in the form of tablets or lozenges.

[0070] Routes of delivery of a coGBAl polynucleotide according to the invention may include, without limitation, injection (systemic or at target site), for example, intradermal, subcutaneous, intravenous, intraperitoneal, intraocular, subretinal, renal artery, hepatic vein, intramuscular injection, intraventricular, intracranial; physical, including ultrasound (- mediated transfection), electric field-induced molecular vibration, electroporation, transfection using laser irradiation, photochemical transfection, gene gun (particle bombardment); parenteral and oral (including inhalation aerosols and the like).

[0071] Vehicles for delivery of a synthetic GBA1 polynucleotide (coGBAB) according to the invention may include, without limitation, viral vehicles (for example, AAV, adenovirus, baculovirus, retrovirus, lentivirus, foamy virus, herpes vims, Moloney murine leukemia vims, Vaccinia virus, and hepatitis virus) and non- viral vehicles (for example, naked DNA, mini- circules, liposomes, ligand-polylysine-DNA complexes, nanoparticles, cationic polymers, including poly cationic polymers such as dendrimers, synthetic peptide complexes, artificial chromosomes, and poly dispersed polymers). Thus, dosage forms contemplated include injectables, aerosolized particles, capsules, and other oral dosage forms.

[0072] In another aspect, the invention comprises a method of treating a disease or condition mediated by GCase. The disease or condition can, in one aspect, be GD. a GCase deficiency, Parkinson’s disease, or a Lewy body disorder caused by a GBA1 mutation. In an aspect of the invention, the disease or condition is neuronopathic GD. In an aspect of the invention, the disease or condition is Gaucher disease type 1 (GDI), Gaucher disease type 2 (GD2), or Gaucher disease type 3 (GD3). This method may comprise administering to a mammal in need thereof a therapeutic amount of any of the inventive synthetic polynucleotides, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions described herein.

[0073] The GCase (SEQ ID NO: 15) contains an N-terminal signal peptide, SEQ ID NO: 17, that is removed to produce the mature GCase SEQ ID NO: 16. In another aspect of this invention, the respective nucleic acids within nucleotides 1-117 of SEQ IDs: 2-4 may be used to generate heterologous signal peptides. Nucleotides 1 18-1611 of SEQ ID NOs: 2-4 may be used to generate the mature GCase that could be used for enzyme replacement therapy or as a source of processed GCase that might be suitable for use in enzymology and as the active component of enzyme replacement therapy.

[0074] Enzyme replacement therapy involves the administration of the functional enzyme (GCase) to a mammal in a manner so that the enzyme administered will catalyze the reactions in the body that the mammal’s own defective or deleted enzyme cannot. In enzyme replacement therapy, the defective enzyme can be replaced in vivo or repaired in vitro using the synthetic polynucleotide according to the invention. The functional enzyme molecule can be isolated or produced in vitro, for example. Enzyme replacement therapy may be accomplished by administration of the synthetic GCase orally, sub-cutaneously, intramuscularly, intravenously, or by other therapeutic delivery routes.

[0075] Accordingly, an aspect of the invention provides a method of treating a disease or condition mediated by GCase. comprising: producing the GCase. with or without the signal peptide, by expressing any of the inventive synthetic polynucleotides or recombinant expression vectors described herein by a host cell, and purifying the enzy me from the host cell; and administering to a mammal in need thereof the purified GCase. The host cells may be as described herein with respect to other aspects of the invention. Methods of purifying enzymes are known in the art.

[0076] In another aspect, the invention includes the mature GCase, e.g., of SEQ ID NO: 16, attached to a carrier, sy nthetic or heterologous signal peptide, charged or lipophilic small molecule to direct toward the lysosome; conjugated or covalently modified to a peptide that targets the lysosome; or encapsulated to deliver the mature GCase to a subcellular organelle, cell type or tissue. [0077] Methods for producing recombinant enzymes in vitro are known in the art. In vitro enzyme expression systems include, without limitation, cell-based systems (bacterial (for example, Escherichia coli, Corynebacterium, Pseudomonas fluorescens), yeast (for example, Saccharomyces cerevisiae, Pichia Pastor is), insect cell (for example, Baculovirus- infected insect cells, non-lytic insect cell expression), and eukaryotic systems (for example, Leishmania)) and cell-free systems (using purified RNA polymerase, ribosomes, tRNA, ribonucleotides). Recombinant GCase can be produced in plant cells (e.g., carrot). Viral in vitro expression systems are likewise known in the art. The enzyme isolated or produced according to the above-iterated methods exhibits, in specific aspects, 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%, or at least 99%, or 100% homology to the naturally occurring human GCase (with or without the signal peptide).

[0078] Gene therapy can involve in vivo gene therapy (direct introduction of the genetic material into the cell or body) or ex vivo gene transfer, which usually involves genetically altering cells prior to administration of the genetically altered cells. In one aspect, genome editing, or genome editing with engineered nucleases (GEEN) may be performed with the coGBAl polynucleotides of the present invention allowing coGBAl DNA to be inserted, replaced, or removed from a genome using artificially engineered nucleases. Any known engineered nuclease may be used such as Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR-Cas system, and engineered meganuclease re-engineered homing endonucleases. Alternately, the coGBAl polynucleotides of the present invention, in combination with a CRISPR-CASP, ZFN, or TALEN, can be used to engineer correction at the locus in a mammaFs cell either in vivo or ex vivo, then, in one aspect, use that corrected cell, such as a fibroblast or lymphoblast, to create an iPS or other stem cell for use in cellular therapy.

[0079] Accordingly, an aspect of the invention provides a method of treating a disease or condition mediated by GCase, comprising administering to a cell of a mammal in need thereof any of the inventive polynucleotides described herein, wherein the polynucleotide is inserted into the cell of the mammal via genome editing on the cell of the mammal. The genome editing may, for example, use prime editing or a nuclease selected from the group of ZFNs, TALENs, the clustered regularly interspaced short palindromic repeats (CRISPR-Cas system) and meganuclease re-engineered homing endonucleases. In an aspect of the invention, the method comprises administering the polynucleotide to an isolated cell of the mammal, and the method further comprises administering the cell to the mammal. In another aspect of the invention, the method comprises administering the polynucleotide to the cell of the mammal in vivo.

[0080] The inventive methods of treating a disease or condition mediated by GCase may comprise administering a therapeutically effective amount of the coGBAl material (or the genetically altered cell) to the mammal. A ‘’therapeutically effective amount’’ refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the coGBAl material may vary according to factors such as the disease state, age, sex, and weight of the mammal, and the ability of the coGBAl material to elicit a desired response in the mammal. A therapeutically effective amount is also one in which any toxic or detrimental effects of the coGBAl material are outweighed by the therapeutically beneficial effects.

[0081] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of the coGBAl material calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0082] The term “treat,’’ as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary' skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment of the disease or condition in a mammal. Furthermore, the treatment provided by the inventive method can include treatment of one or more conditions or symptoms of the disease or condition being treated. In another aspect, the invention is directed to the preclinical amelioration or rescue from the condition or disease state, for example, GD, that the afflicted mammal exhibits.

[0083] The mammal referred to in the inventive methods can be any mammal. As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, mammals of the order Lagomorpha, such as rabbits, and sheep (e.g., a sheep model of GD). It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0084] Another aspect of the invention provides a method of detecting the presence of a synthetic GBA1 polynucleotide (coGBAl) in a biological sample from a mammal. Such methods may be useful for any of a variety’ of applications, for example, detecting the presence of the coGBAl polynucleotide in a biological sample from a mammal that has been administered any of the coGBAl materials as described herein.

[0085] The method may comprise obtaining at least one test sample comprising nucleic acid from a biological sample from a mammal. The biological sample may be a sample of any tissue from the mammal, for example, blood, kidney, liver, or any of the other tissues and organs described herein with respect to other aspects of the invention. In an aspect, the nucleic acid is isolated or purified from the biological sample to form the test sample. In another aspect, the test sample comprises the biological sample, and the nucleic acid in the biological sample is tested in situ.

[0086] The method may further comprise contacting any of the inventive coGBAl polynucleotides described herein with the at least one test sample under conditions allowing for a complex to form betw een the coGBAl polynucleotide and the nucleic acid of the test sample. In this regard, the method comprises contacting the nucleic acid of the test sample with the inventive coGBAl polynucleotide under conditions which allow the inventive coGBA 1 polynucleotide to specifically hybridize with the nucleic acid of the test sample as is known in the art. The method may comprise amplifying the inventive coGBAl polynucleotide and the nucleic acid of the test sample nucleic acid using any suitable ty pe of polymerase chain reaction (PCR) as is known in the art.

[0087] In an aspect, the inventive coGBAl polynucleotide further comprises a detectable label. The label may be any' label suitable for detecting hybridization, e.g., a complex, of the inventive coGBAl polynucleotide with the nucleic acid of the test sample. Exemplary detectable labels may include any one or more of radioactive labels, non-radioactive labels, fluorescent labels, and chemiluminescent labels. [0088] The method may further comprise detecting the complex. The complex may be detected using, for example, a radioactive label or a dye as is known in the art. In a preferred embodiment, the method comprises measuring light emitted from a fluorescent dye using, e.g., a laser. Detecting the complex may, optionally, further comprise measuring the amount of complex formed.

[0089] The method may further comprise comparing a presence of the complex in the at least one test sample with an absence of complex from a negative sample that lacks the inventive coGBAl polynucleotide. The presence of complex from the at least one test sample is indicative of the presence of the inventive coGBAl polynucleotide in the test sample and the absence of complex from the at least one test sample is indicative of the absence of the inventive coGBAl polynucleotide in the test sample. In an aspect of the invention, the method comprises determining a background level of signal generated by the label in the negative sample that lacks the inventive coGBAl polynucleotide and comparing the background level of signal with the level of signal detected in the test sample. A level of signal that is higher or lower in the test sample as compared to that measured in the negative sample may be indicative of the presence of the inventive coGBAl polynucleotide in the test sample.

[0090] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0091] This example demonstrates the development of synthetic, codon optimized GBA1 polynucleotides (coGBAl).

[0092] A series of synthetic codon optimized and adjusted GBA1 polynucleotides, designated GBA1.1 (SEQ ID NO: 2), GBA1.2 (SEQ ID NO: 3). and GBA1.3 (SEQ ID NO: 4), were developed using different algorithms and then manually adjusted. The sequences of the coGBAl polynucleotides and the sequence of human WT GBA1 are show n in Table 1.

TABLE 1

[0093] An alignment created with MUSCLE (Figures 1A-1F) and phylogenetic analysis prepared with CLUSTAL W (Figure 2) revealed that the coGBAl alleles are highly divergent at the nucleotide level while maintaining wild-type GCase amino acid sequence identity (Table 2). The phylogenetic analysis also shows that coGBAl. 1 is distinct from the other polynucleotides. The pairwise BLASTN analysis between wild-type GBA1 (hGBAl denoted hGBA, and coGBAl.1 in Figures 3A-3C showed only 80% nucleotide identity, with changes distributed throughout the entire length of the gene. Similarly, the pairwise BLASTN analysis between wild-type GBA1 (hGBAl), denoted hGBA, and coGBAl.2 in Figures 4A-4C showed 76% nucleotide identity and between wild-type GBA1 (hGBAl) and coGBAl.3 (Figures 5A-5C) showed 81% nucleotide identity. These analyses revealed that the GBA1 synthetic codon optimized genes are highly divergent at the nucleotide level while maintaining wild-type GCase amino acid sequence identity. TABLE 2

EXAMPLE 2

[0094] This example demonstrates the development of AAV vectors to express the synthetic GBA1 polynucleotides.

[0095] To examine the relative difference in translation efficiency between the coGBAl alleles, each gene was cloned into a conventional expression vector, pcDNA3. 1, to examine GCase protein after transfection into HEK 293T cells (Figure 6).

[0096] Each cDNA of the 4 GBA1 polynucleotides (hGBAl (SEQ ID NO: 1), coGBAl. 1

(SEQ ID NO: 2), coGBAl.2 (SEQ ID NO: 3), and coGBAl.3 (SEQ ID NO: 4)) was cloned into a pcDNA3. 1 (+) backbone in order to test for optimized expression. HEK 293T cells, which are embryonic kidney fibroblasts, were seeded into 6-well plates at a density of 4xl0⁵ cells per well. Each well was transiently transfected with 4 pg of plasmid DNA (hGBA pcDNA3.1(+), coGBAl.1 pcDNA3.1(+), coGBAl.2 pcDNA3.1(+), coGBAl.3 pcDNA3.1(+), or GFP control plasmid) with LIPOFECTAMINE™ 3000 reagent (INVITROGEN) and OPTI-MEMTM (THERMOFISHER SCIENTIFIC) according to the manufacturer's instructions. Each transfection was performed in duplicate or triplicate. Cells were harvested 72 hours post transfection and lysed in M-PER (ThermoFisher Scientific) supplemented with fresh protease inhibitors (COMPLETE™ PROTEASE INHIBITOR COCKTAIL, EDTA-free SIGMA). Homogenates were centrifuged at 16.000 RCF for 10-15 minutes. Supernatant was collected and measured for protein content by BCA assay (PIERCE™ BCA PROTEIN ASSAY KITS, THERMOFISHER SCIENTIFIC). Samples were then denatured in 5x SDS loading buffer and run on SDS-PAGE gels (5 pg of protein per lane). Resulting blots were immunoblotted (IB) with one or more of the indicated antibodies: GCase (anti-GCase R386 rabbit polyclonal antibody-made in house) and GAPDH (abeam, ab9485). The coGBAl. 1 cDNA directed the expression of the highest level of GCase protein compared to the other polynucleotides.

[0097] A parallel study was conducted to examine the ability⁷ of different promoters to direct expression of GCase in HEK293T cells. hGBAl (SEQ ID NO: 1) or coGBAl. 1 (SEQ ID NO: 2) was cloned into 3 different vector backbones in order to determine the optimal promoter for expression including one that used a neurological promoter, pAAV.Camk2a.hGBAl (SEQ ID NO: 14) (also referred to as pAAV[Exp]- Camk2a(short)>hGBA[NM_001005750.1]:oPRE in Table 3), one that used a constitutive elongation factor 1 short promoter, pscAAV.EFs.coGBAl. 1 (SEQ ID NO: 6) (also referred to as pscAAV[Exp]-EFS>{cohGBAl .1 } in Table 3), and one that used a viral enhanced constitutive promoter, pAAV.CAG.hGBAl (SEQ ID NO: 12) (Figure 7). HEK 293T cells (embryonic kidney fibroblasts) were seeded into 6-well plates at a density of 4xl0⁵ cells per well. Each well was transiently transfected with 4 pg of plasmid DNA pAAV.Camk2a.hGBAl (SEQ ID NO: 14), pscAAV-Efs-coGBAl . 1 (SEQ ID NO: 6), pAAV- CAG hGBAl (SEQ ID NO: 12), or GFP control plasmid with LIPOFECTAMINE™ 3000 reagent (INVITROGEN) and Opti-MEMTM (ThermoFisher Scientific) according to the manufacturer’s instructions. Each transfection was performed in triplicate. Cells were harvested 72 hours post transfection and lysed in RIPA buffer supplemented with fresh protease inhibitors (COMPLETE™ PROTEASE INHIBITOR COCKTAIL, SIGMA). Homogenates were centrifuged at 16,000 RCF for 10-15 minutes. Supernatant was collected and measured for protein content by BCA assay (PIERCE™ BCA PROTEIN ASSAY KITS, THERMOFISHER SCIENTIFIC). Samples were then denatured in 5x SDS loading buffer and run on SDS-PAGE gels using 5 pg of protein per lane. Resulting blots were immunoblotted (IB) with one or more of the indicated antibodies: GCase (anti-GCase R386 rabbit polyclonal antibody-made in house) and GAPDH (abeam, ab9485). The Western blotting measuring GCase levels demonstrated that both constitutive promoters directed robust expression of GCase (Figure 7).

[0098] The nucleotide sequences of the various GBA1 AAV plasmids are shown in Table 3.

TABLE 3

EXAMPLE 3

[0099] This example demonstrates the efficacy of AAV GBA1 vectors at driving expression of GCase in H4 GBA1 KO cells.

[00100] A transgene was designed to express coGBA 1. 1 under the control of the EFl S promoter in a plasmid that contains an AAV backbone designed to package a transgene in the single-strand genome configuration. The map of this plasmid is depicted in Figure 8 and the sequence is SEQ ID NO: 5.

[00101] The AAV EFIS coGBAl. 1 HPRE transgene plasmid was then used to transfect H4 cells, which are from a neuroglioma line, and H4 GBA1'¹' cells (Gehrlein et al., Nat. Commun., 14: 2057 (2023)) and compared to an AAV transgene that used the enhanced CAG promoter to drive the expression of wild t pe GBA1. The H4 cells and H4 GBA1~^!~ cells were seeded into 6-well plates at a density of 4xl0⁵ cells per well. Each well was transiently transfected with 4 pg of plasmid DNA of either pscAAV-EFIS-coGBAl . l (SEQ ID NO: 6) or pAAV-CAG hGBAl(SEQ ID NO: 12) with LIPOFECTAMINE™ 3000 reagent (INVITROGEN) and OPTI-MEMTM (THERMOFISHER SCIENTIFIC) according to the manufacturer's instructions or were left untransfected. Each transfection was performed in triplicate. Cells were harvested 48 hours post transfection and lysed in RIPA buffer supplemented with fresh protease inhibitors (COMPLETE™ PROTEASE INHIBITOR COCKTAIL, SIGMA). Homogenates were centrifuged at 16,000 RCF for 10-15 minutes. Supernatant was collected and measured for protein content by BCA assay (PIERCE™ BCA PROTEIN ASSAY KITS, THERMOFISHER SCIENTIFIC). Samples were then denatured in 5x SDS loading buffer and run on SDS-PAGE gels (5 pg of protein per lane). Resulting blots were immunoblotted (IB) with one or more of the indicated antibodies: GCase (anti- GCase R386 rabbit polyclonal antibody -made in house). The expression of GCase from both transgenes was robust (Figure 9A).

[00102] The relative GCase activity of cell lysates was also measured. Transfection was performed the same as above. Cells were harvested 48 hours post transfection and lysed in GCase activity assay buffer (0.1 IM Na2HPO4, 0.04M citrate, 0.25% Triton X-100, and 0.2% sodium taurocholate: pH 54. -5.5) supplemented with one protease inhibitor tablet (COMPLETE™ MINI PROTEASE INHIBITOR COCKTAIL, SIGMA) per 10 ml of buffer. Protein lysates were prepared in a 96 well-plate, diluting samples with GCase buffer. The concentration of protein was determined via a BCA assay and control and experimental samples were maintained at similar concentrations (0.5-1 mg/mL). 0.8 mM CBE was prepared by diluting lOOmM CBE in GCase buffer. Control buffer without CBE was also prepared by mixing DMSO with GCase buffer (9.2:0.8 GCase buffer: DMSO). 10 pL of protein lysate from the 96 well-plate was pipetted to each assay well in a 384 well-plate with quadruplicate replications using an Eppendorf multichannel pipette. 5 pL of 0.8mM CBE and 5 pL of control buffer without CBE were added and incubated for 15 min at 37°C shaking at 600 rpm. 2.5 mM 4-Methylumbelliferyl-B-D-glucoside (4-MU) was prepared by diluting IM 4-MU in GCase buffer. Following the 15 min incubation, the 384 well-plate was spun own and 15 pL of 4-MU solution was added to each assay well to reach a total volume of 30 pL per well and incubated for 1 hour at 37°C, shaking at 450 rpm. Following incubation, plates were spun down and 30 pL of IM glycine solution (pH 10.5) was added to each assay well. 4-MU fluorescence was read with a microplate reader (FLEXSTATION 3, MOLECULAR DEVICES, Excitation: 365 nm; Emission: 449 nm; Cutoff: 435nm; 3 reads/well). GCase activity was calculated for each protein lysate according to the equation: (fluorescence of CBE-free sample minus fluorescence of sample with CBE) I protein concentration. These results demonstrate that the pAAV plasmid vectors restore GCase levels and activity in the H4 neuroglioma cell line and that expression of GCase from either transgene was accompanied by supraphysiologic GCase activity (Figure 9B).

[00103] Next, the lead AAV plasmid, pAAV EF 1 S coGBAl.1 HPRE (SEQ ID NO: 5) w as packaged as a single stranded vector and pseudoserotyped with an AAV9 (Figure 10A) or AAVrhlO (Figure 10B) capsid and tested for expression in an infection study using H4 GBA1’ ’ cells. H4 or H4 GBAT¹' cells were seeded onto 12 well plates at 50,000 cells per well. AAV9-EFIS-COGBAI.I.HPRE or AAVrhlO-EFlS.coGBAl.l.HPRE virus was added to each well at a concentration of 0, 5e⁴, or 5e⁵ genome copies per cell (GCs/cell). 72 hours later cells were harvested and lysed in M-PER (ThermoFisher Scientific) supplemented with fresh protease inhibitors (COMPLETE™ PROTEASE INHIBITOR COCKTAIL, EDTA-free SIGMA). Homogenates were centrifuged at 16,000 RCF for 10-15 minutes. Supernatant was collected and measured for protein content by Braford assay. Samples were then denatured in 5x SDS loading buffer and run on SDS-PAGE gels (50 pg of protein per lane). The resulting blots w ere immunoblotted (IB) with one or more of the indicated antibodies: GCase (anti-GCase R386 rabbit polyclonal antibody-made in house) and -actin (PROTEINTECH, 66009-1-Ig). After 72 hours, both vectors produced robust expression of GCase (Figures 10A-10B) demonstrating not only that the transgene packaged well when used with conventional triple transfection methods, yielding high titer AAV9 and AAVrhlO viral vectors, but further, that the AAV vectors directed the robust expression of a properly processed GCase.

EXAMPLE 4

[00104] This example demonstrates the efficacy of AAV9 and AAVrhlO vectors for use in in vivo gene therapy.

[00105] Having established that the AAV9 and AAVrhlO coGBAl. 1 viral vectors displayed optimal properties for further translation, a series of in vivo gene therapy experiments using a lethal mouse model of neuronopathic GCase deficiency developed by Enquist et al., PNAS, 104(44): 17483-8 (2007) was performed. The gba K14-lnl/lnl mice are a lethal model of early onset neuronopathic Gaucher’s Disease (nGD). These gba K14-lnl/lnl mice rely upon a loxp-neo-loxp cassette cloned within the intron of exon 8 in the mouse gba gene. When crossed to a mouse line that expresses Cre under the control of the K14 promoter gba Inl/lnl; TgK14Cre mice are generated and designated gba K14-lnl/lnl. These gba K14-lnl/lnl mice express GCase in the skin but are lacking GCase throughout the body, including the brain, gba K14-lnl/lnl mice develop a rapidly progressing neurological disease after an initial symptom-free period of approximately 10 days. The gba K14-lnl/lnl mice show symptoms of motor dysfunction including abnormal gait, and seizures which are also common signs of acute neuronopathic Gaucher’s disease (GD). The gba K14-lnl/lnl mice develop continuous seizures and at 2 weeks of age and need to be sacrificed due to end-stage paralysis.

[00106] In a proof of concept study that relied upon phenotypic rescue of the lethal acute neuronopathic GD phenotype, neonatal (Pl) gba K14-lnl/lnl mice were injected with the AAV9 EFIS coGBAl.l HPRE (SEQ ID NO: 5) using retro orbital injection (n of 2 at dose 4xlOⁿ GC/pup) or facial vein injection (n of 2 at a dose of 4xlOⁿ GC/pup; n of 2 at a dose of 2xlOⁿ GC/pup, and n of 2 at a dose of IxlO¹¹ GC/pup). As shown in Figure 11A, the AAV9 EFIS coGBAl. l HPRE, when delivered at a dose of 4xlOⁿ GC/pup, dramatically extended the lifespan of gba K14-lnl/lnl mice to greater than 300 days. These mice were phenotypically normal and used as breeders in the National Human Genome Research Institute (NHGRI) mouse colony, demonstrating normal fertility. However, lower doses of AAV9 EFIS coGBAl.l HPRE, while efficacious, did not extend the lifespan ofthe gfoα K14- Inl/lnl mice past 1 -2 weeks of age.

[00107] In a similarly designed study, AAVrhlO EFIS coGBAl.l HPRE, delivered at a dose of 4xlOⁿ GC/pup via facial vein injection, also extended the lifespan of gba K14-lnl/lnl mice (Figure 1 IB). The injected mice were 7 weeks old and appeared identical to untreated control littermates.

[00108] These in vivo, proof-of-principal, studies were extended to assess the longer-term survival odds for treated mice. Mice were treated the with indicated doses of AAV9-hGBAl (Figure 12A) or AAVrhlO-hGBAl (Figure 12B) at Pl either via facial vein or retroorbital injection. Untreated Gba KO mice (nGD mice) were used as controls (closed circle). All four nGD mice injected with high dose AAV9-hGBAl (3. IxlO¹⁴ vg/kg, closed square) survived longer than 1 year. High dose AAVrhlO-hGBAl injection also extended lifespan of the nGD mice longer than 7 months while two out of four injected mice died at 6-7 months age. Lower doses of the AAV9-hGBAl (1.5 x io¹⁴ and 7.7 x io¹³ vg/kg) did not extend the lifespan of nGD mice past 1~2 weeks.

[00109] In summary', the experiments described in the Examples of this application have demonstrated the development of a series of synthetic GBA1 genes that have superior expression in a human HEK 293T cell line and restored GCase activity in a H4 GBA1 '~ cell line. A lead polynucleotide (coGBAl. /) was used to develop single strand AAV9 and AAVrhlO vectors that exhibited a transgene dose response in an infection study using H4 GBAB¹' cells. Both seroty pes produced virus with high titer, and after systemic delivery' at doses that approximate those given to humans in other AAV9 indications, mediated rescue of a severe neuronopathic mouse model of GCase deficiency. Continued observation has confirmed that administration of AAV9-hGBAl extends the lifespan of the K14-lnl/lnl for more than one year and the AAVrhlO-hGBAl has extended lifespan for longer than 7 months. These results suggest that the AAV9 and AAVrhlO vectors disclosed herein can serve as a new class of gene therapeutics to treat patients with neuronopathic GCase deficiency, and potentially forms of GBA 7-related Parkinson disease, and are suitable for immediate human translation.

EXAMPLE 5

[00110] This example demonstrates the efficacy of AAV9 and AAVrhlO vectors for use in in vivo gene therapy. [00111] Having established that the AAV9 and AAVrhlO coGBAl. 1 viral vectors displayed dramatically extended the lifespan of K14-lnl/lnl mice (a lethal model of early onset neuronopathic Gaucher’s Disease (nGD)), a series of in vivo gene therapy experiments using a mouse model of later-onset nGD.

[00112] To conduct these experiments, first a mouse model of later onset nGD was developed. Specifically, a tamoxifen (TAM)-induced Gbal knockout (KO) strategy was employed. mice, carrying a LoxP sequence inserted into intron 8 and intron 11 of Gbal, were crossed to a mouse line expressing CreER under the control of the CAG promoter to generate G mice. Under normal conditions, the m_{i ce} express wildtype Gbal. However, treating these mice with TAM causes the removal of exons 9-11 of Gbal by Cre resulting in Gbal KO in the brain and other tissues.

[00113] To generate the TAM-G/xz/ KO mice, a 4 week-period of tamoxifen-chow feeding (500 mg per kg irradiated food) was used. The development of neuronopathic symptoms was observed 3-4 weeks post TAM-chow feeds. The symptoms included weight loss, slow movement, abnormal gait, and severe neuroinflammation. The manifestations became increasingly severe, and the affected mice required euthanasia due to end-stage paralysis within a week of the initiation of symptoms.

[00114] In a proof-of-concept study to evaluate the ability of AAV gene therapy to prevent the later onset lethal neuronopathic phenotype in these TAM-G/vz/ KO mice, 4-week old juvenile _{mjce were} injected with of either AAV9- hGBAl or AAVrhlO-hGBAl retro-orbitally to enter the systemic circulation. At two weeks post injection (at 6 weeks of age), the treated and the nontreated mice were fed with the TAM chow for 4 weeks to induce the Gbal KO. Both AAV9-hGBAl and AAVrhlO-hGBAl successfully extended the lifespan of all TAM-Gbal KO mice by over 14 weeks and prevented the development of symptoms (Figure 13). Both AAV9-hGBAl and AAVrhlO- hGBAl injection successfully extended the lifespan of the TAM- KO mice for at least 8 weeks longer than the untreated control mice.

EXAMPLE 6

[00115] This example demonstrates the generation and testing of another GBA1 transgene; which uses the EF1L promoter.

[00116] A plasmid (pAAV EF1L coGBAl.l HPRE), was generated, placing the GBA1 coding sequence under control of the EF1L promoter. This plasmid was generated similarly to pAAV EFIS coGBAl. l HPRE. discussed above. A plasmid map for pAAV EF1L coGBAl . l HPRE, validated by full plasmid nanopore sequencing, is presented as Figure 14. The sequence of pAAV EF1L coGBAl.l HPRE is presented in Table 4.

Table 4

[00117] The properties of pAAV EF1L coGBAl . 1 HPRE were investigated using H4 (neuroblastoma) GBA1 knock-out (KO) cells. These cells were seeded into 60 mM plates at a density of 5 million cells pre plate. Each plate was transiently transfected with one of the following GBA plasmid constructs (accession numbers are indicated): pscAAV [Exp] -[EFIS intron truncated linker] >coGB A, pscAAV[Exp]-EFlS>{cohGBA}, pAAV[Exp]- CMV>hGBA[NM_001005749. 1]:WPRE, pAAV[Exp]- Camk2a(short)>hGBA[NM_001005750. l]:oPRE. pAAV[Exp]-

C AG>hGBA[NM_001005750. 1 ] :oPRE. pscAAV [Exp] -EFl S>hGBA[NM_001005750.1 ]. pAAV EFlS.coGBAl .l.HPRE, or pAAV EF1L coGBAl. l.HPRE. For each transfection reaction, a total of 2.16 x 10¹² gene copies of plasmid (~12 pg of DNA) was added with LIPOFECTAMINE™ 2000 reagent (INVITROGEN) and Opti-MEMTM (THERMOFISHER SCIENTIFIC) using the manufacturers' instructions.

[00118] Cells were harvested 72 hours post transfection and lysed in T-PER (THERMOFISHER SCIENTIFIC) supplemented with fresh protease inhibitors (COMPLETE™ PROTEASE INHIBITOR COCKTAIL, EDTA-free SIGMA). Homogenates were centrifuged at 16,000 RCF for 15 minutes. Supernatant was collected and measured for protein content by Bradford assay (BIO-RAD PROTEIN ASSAY DYE REAGENT CONCENTRATE, 450 ml #5000006). Samples were then denatured in 5x SDS loading buffer and run on SDS-PAGE gels (50 pg of protein per lane). Resulting blots were immunoblotted (IB) with antibodies directed against GCase (anti-GCase R386 rabbit polyclonal antibody) and P-actin (PROTEINTECH. 66009-1 -Ig).

[00119] The pAAV EF1L coGBAl. 1 HP RE plasmid also was used to generate an AAV vector containing the EF1L coGBAl. l transgene (AAV9-EF1L coGBAl.l.HPRE). The properties of AAV9-EF1L coGBAl.l.HPRE were investigated, again using H4 (neuroblastoma) GBA1 knock-out (KO) cells. H4 or H4 GBA1 KO cells were seeded onto 12 well plates at 50,000 cells per well. AAV9-EF1L coGBAl. l.HPRE virus was added to each well at a concentration of 0, 5e⁴, 5e³, or le⁶ genome copies per cell (GCs/cell). 72 hours later cells were harvested and lysed in M-PER (THERMOFISHER SCIENTIFIC) supplemented with fresh protease inhibitors (COMPLETE™ PROTEASE INHIBITOR COCKTAIL. EDTA-free SIGMA). Homogenates were centrifuged at 16,000 RCF for 10-15 minutes. Supernatant was collected and measured for protein content by Braford assay.

Samples were then denatured in 5x SDS loading buffer and run on SDS-PAGE gels (50 pg of protein per lane). Resulting blots were immunoblotted (IB) with antibodies directed against GCase (anti-GCase R386 rabbit polyclonal antibody) and P-actin (PROTEINTECH, 66009-1- Ig).

[00120] The results revealed that, as an expression plasmid, the EF1L coGBAl. 1 transgene appears more potent than EFIS coGBAl.l in directing the expression of GBA1 in H4 GBA1 KO cells (Figure 15). When employed as an AAV9 viral vector, the EF1L coGBAl. 1 transgene plasmid produced an AAV9 vector that was equally potent in H4 GBA1 KO cells (Figure 16).

[00121] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00122] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i. e. , meaning “including, but not limited to.”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00123] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A synthetic GBA1 (glucosylceramidase beta 1) polynucleotide (coGBAl) selected from the group consisting of:

(a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 2-4;

(b) a polynucleotide having a nucleic acid sequence with at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 15. and having equivalent or increased expression in a mammalian host cell relative to expression of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1 ;

(c) a polynucleotide having a nucleic acid sequence with at least 85% identity to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4 and encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 16, and having equivalent or increased expression in a mammalian host cell relative to expression of nucleotides 118-1611 of SEQ ID NO: 1 in the same host cell, wherein the polynucleotide does not have the nucleic acid sequence of nucleotides 118-1611 of SEQ ID NO: 1;

(d) a polynucleotide having a ribonucleic acid (RNA) sequence encoded by a polynucleotide that is complementary to the polynucleotide of any one of (a)-(c); and

(e) a peptide-modified polynucleotide comprising the polynucleotide of any one of (a)-(d).

2. The synthetic polynucleotide of claim 1 , wherein the polynucleotide has (a) at least 90% identity to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 or (b) at least 90% identity to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4.

3. The synthetic polynucleotide of claim 1, wherein the polynucleotide has (a) at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 2-4 or (b) at least 95% identity to the nucleic acid sequence of nucleotides 118-1611 of any one of SEQ ID NOs: 2-4.

4. The synthetic polynucleotide of any one of claims 1-3, wherein the polynucleotide exhibits increased expression in a mammalian host cell relative to the expression SEQ ID NO: 1 or nucleotides 118-1611 of SEQ ID NO: 1 in the mammalian host cell.

5. The synthetic polynucleotide of any one of claims 1-4, wherein the synthetic polynucleotide comprises codons that have been optimized relative to the naturally occurring human GBA1 polynucleotide sequence of SEQ ID NO: 1 or nucleotides 118-1611 of SEQ ID NO: 1.

6. The synthetic polynucleotide of claim 5, wherein the nucleic acid sequence has at least 70% of less commonly used codons replaced with more commonly used codons.

7. A recombinant expression vector comprising the synthetic polynucleotide of any one of claims 1-6.

8. The recombinant expression vector of claim 7, wherein the recombinant expression vector is single stranded.

9. The recombinant expression vector of claim 7, wherein the recombinant expression vector is self-complementary'.

10. The recombinant expression vector of any one of claims 7-9. wherein the recombinant expression vector is a viral vector.

11. The recombinant expression vector of any one of claims 7-9, wherein the recombinant expression vector is an adeno-associated viral (AAV) vector or a herpesvirus vector.

12. The recombinant expression vector of any one of claims 7-9, wherein the recombinant expression vector is an AAV9 or AAVrhlO vector.

13. The recombinant expression vector of any one of claims 7-9. wherein the recombinant expression vector is a non-viral vector.

13. The recombinant expression vector of any one of claims 7-9, wherein the recombinant expression vector is a neurotropic vector.

15. The recombinant expression vector of any one of claims 7-14, wherein the recombinant expression vector is configured for ubiquitous expression of the coGBAl polynucleotide.

16. The recombinant expression vector of any one of claims 7-15, wherein the recombinant expression vector is configured for expression of the coGBAl polynucleotide in neuronal tissue, hepatic tissue, or bone marrow tissue.

17. The recombinant expression vector of claim 7, wherein the recombinant expression vector comprises the nucleic acid sequence of any one of SEQ ID NOs: 5-8 or 18.

18. An isolated or purified host cell comprising the recombinant expression vector of any one of claims 7-17.

19. An isolated or purified population of cells comprising the host cell of claim 18.

20. A pharmaceutical composition comprising (i) the synthetic polynucleotide of any one of claims 1-6, the recombinant expression vector of any one of claims 7-17, the host cell of claim 18. or the population of cells of claim 19 and (ii) a pharmaceutically acceptable carrier.

21. A method of treating a disease or condition mediated by glucocerebrosidase (GCase), comprising administering to a mammal in need thereof a therapeutic amount of the synthetic polynucleotide of any one of claims 1-6. the recombinant expression vector of any one of claims 7-17, the host cell of claim 18, the population of cells of claim 19, or the pharmaceutical composition of claim 20. 22. A method of treating a disease or condition mediated by glucocerebrosidase (GCase), comprising: producing the GCase by expressing the synthetic polynucleotide of any one of claims 1-6 or the recombinant expression vector of any one of claims 7-17 by a host cell, and purifying the GCase from the host cell; and administering to a mammal in need thereof the purified GCase.

23. A method of treating a disease or condition mediated by glucocerebrosidase (GCase). comprising administering to a cell of a mammal in need thereof the polynucleotide of any one of claims 1 -6, wherein the polynucleotide is inserted into the cell of the mammal via genome editing on the cell of the mammal.

24. The method of claim 23, wherein the method comprises administering the polynucleotide to an isolated cell of the mammal, and the method further comprises administering the cell to the mammal.

25. The method of claim 23, wherein the method comprises administering the polynucleotide to the cell of the mammal in vivo.

26. The method of any one of claims 21-23, wherein the disease or condition is Gaucher disease.

27. The method of claim 26, wherein the disease or condition is Gaucher disease type 1.

28. The method of claim 26, wherein the disease or condition is Gaucher disease type 2.

29. The method of claim 26, wherein the disease or condition is Gaucher disease type 3.

30. The method of any one of claims 21-23, wherein the disease or condition is Parkinson’s disease or a Lewy body disorder caused by a GBA1 mutation.

31. A method of detecting the presence of a synthetic GBA1 (glucosylceramidase beta 1) polynucleotide (coGBA l) in a biological sample from a mammal, the method comprising:

(a) obtaining at least one test sample comprising nucleic acid from a biological sample from a mammal;

(b) contacting any of the synthetic polynucleotide of any one of claims 1-6 with the at least one test sample under conditions allowing for a complex to form between the synthetic polynucleotide and the isolated nucleic acid of the test sample;

(c) detecting the complex; and

(d) comparing a presence of the complex in the at least one test sample with an absence of complex from a negative sample that lacks the synthetic polynucleotide, wherein detection of the complex is indicative of the presence of the synthetic polynucleotide in the biological sample from the mammal.