Compositions and methods for treating Niemann-Pick disease type A

Abstract

Provided herein are polynucleotide sequences encoding human acid sphingomyelinase (SMPD1), and expression cassettes comprising these coding sequences. Also provided are vectors, e.g., recombinant adeno-associated virus (rAAV) vectors, having a vector genome comprising an engineered SMPD1 coding sequence operably linked to one or more regulatory sequences. Additionally provided are compositions comprising these expression cassettes and rAAV, as well as methods of using these compositions for the treatment of Niemann-Pick type A disease.

Description

[Background technology]

[0001] Niemann-Pick disease is an inherited condition associated with lipid metabolism, the breakdown, transport, and use of lipids and cholesterol in the body. In people with this condition, abnormal lipid metabolism causes harmful amounts of lipids to accumulate in the spleen, liver, lungs, bone marrow, and brain. Niemann-Pick disease types A and B are caused by a different set of mutations in the SMPD1 gene. This gene codes for an enzyme called acid sphingomyelinase. This enzyme is found in lysosomes, compartments in cells that break down and recycle different types of molecules. Acid sphingomyelinase is responsible for converting a fat (lipid) called sphingomyelin into another type of lipid called ceramide. Mutations in SMPD1 result in a deficiency of acid sphingomyelinase, which results in reduced breakdown of sphingomyelin and the accumulation of this fat in cells. This accumulation of fat causes cells to malfunction and ultimately leads to death. Over time, cell loss impairs the function of tissues and organs, including the brain, lungs, spleen, and liver, in people with Niemann-Pick disease types A and B.

[0002] Niemann-Pick type A develops during infancy and is characterized by enlargement of the liver and spleen (hepatosplenomegaly), failure to gain weight and grow at the expected rate (failure to thrive), and progressive deterioration of the nervous system. Due to the involvement of the nervous system, Niemann-Pick type A is also known as the neurological type. Infants with Niemann-Pick type A usually develop enlargement of the liver and spleen (hepatosplenomegaly) by the age of 3 months, and an inability to gain weight and grow at the expected rate (failure to thrive). Affected children develop normally until about 1 year of age, after which they experience a progressive loss of mental ability and movement (psychomotor regression). Children with Niemann-Pick type A also develop widespread lung damage (interstitial lung disease) that can cause recurrent lung infections and ultimately lead to respiratory failure. All affected children have an eye abnormality called cherry red spots, which can be identified on an ophthalmologic exam. Children with Niemann-Pick disease type A (NPA) generally do not survive beyond childhood. Currently, there is no effective treatment for the condition.

[0003] There is a need in the art for compositions and methods for the safe and effective treatment of patients with NPA disease. Summary of the Invention

[0004] In one aspect, a recombinant AAV (rAAV) is provided, comprising an AAVhu68 capsid having a vector genome packaged therein, the vector genome comprising a human sphingomyelin phosphodiesterase 1 (hSMPD1) coding sequence and a regulatory sequence that directs expression of human acid sphingomyelinase (hSMPD1) in a cell.

[0005] Recombinant AAV (rAAV) is provided, comprising an AAV capsid and a vector genome packaged therein. In certain embodiments, the vector genome comprises an engineered nucleic acid sequence encoding human acid sphingomyelinase (hSMPD1) operably linked to a regulatory sequence directing expression of hSMPD1, and an AAV 3'ITR, wherein the hSMPD1 coding sequence is SEQ ID NO:22, or a coding sequence at least 90% identical to SEQ ID NO:22 (also reproduced in SEQ ID NO:23) encoding hSMPD1 of SEQ ID NO:3, or a coding sequence at least 90% identical to SEQ ID NO:3 (also reproduced in SEQ ID NO:23) encoding hSMPD1 of SEQ ID NO:2. In certain embodiments, the hSMPD1 coding sequence encodes hSMD1 with an Ala at position 36 and / or a G at position 506 of the human sequence, with reference to the numbering of SEQ ID NO:2 (or 23). ... The hSMPD1 protein has the sequence of SEQ ID NO:2 (or 23) or SEQ ID NO:3. In other embodiments, the hSMPD1 protein comprises an exogenous leader sequence fused to an hSMPD1 protein comprising amino acids 47-631 of SEQ ID NO:2 (SEQ ID NO:23) or SEQ ID NO:3. The regulatory sequence may comprise a UbC or CB7 promoter and may comprise an SV40 late or rabbit beta globin polyadenylation site. In certain embodiments, the AAV vector genome comprises a CB7 promoter, an intron, an hSMPD1 coding sequence, and a rabbit beta globin sequence. In certain embodiments, the vector genome comprises a sequence of SEQ ID NO:21, 19, 10, or 8, or comprises a sequence of SEQ ID NO:20, 18, 11, or 9. In certain embodiments, the AAV vector genome comprises a UbC promoter, an hSMPD1 coding sequence, and an SV40 late polyadenylation sequence. In certain embodiments, the AAV vector genome further comprises a full-length AAV2 inverted terminal repeat (ITR) sequence. In a specific embodiment, the AAV capsid is an AAVhu68 capsid.

[0006] In certain embodiments, a pharmaceutical composition is provided, comprising a formulation buffer and a population of rAAV as described herein. In certain embodiments, the pharmaceutical composition is suitable for combination therapy with a functional hSMPD1 protein. In certain embodiments, the pharmaceutical composition is formulated for delivery via intracerebroventricular (ICV), intrathecal (IT), intracisternal, or intravenous (IV) injection.

[0007] In certain embodiments, rAAV and compositions are provided for use in treating and / or ameliorating symptoms of Niemann-Pick A by alleviating weight loss or cachexia, alleviating loss of motor function, alleviating loss of cognitive function, and prolonging survival.

[0008] Other aspects and advantages of the present invention will become readily apparent from the following detailed description of the invention. [Brief description of the drawings]

[0009] [Figure 1A-1B] 1 provides bar graphs showing the amount of sphingomyelin accumulated in the liver and brain across multiple strains of Smpd1 knockout (KO) mice. KO mice from strain A, strain B, and strain C showed significant amounts of sphingomyelin accumulated in the liver ( FIG. 1A ) and brain ( FIG. 1B ) compared to wild-type (WT) mice. [Figures 2A-2D] Figure 2 shows cholesterol accumulation in the brain as indicated by positive staining for filipin. (A) Smpd1 WT mice showed no presence of cholesterol in the brain, whereas Smpd1 KO mice showed abundant amounts of cholesterol accumulated in the brain (Figure 2B). This amount was reduced in Smpd1 KO mice that underwent ICV gene therapy (Figure 2C). Figure 2D shows positive staining for filipin quantified for WT, KO, and KO+ICV AAV mice. [Figure 3A]Figure 1 shows the latency to fall during rotarod performance in WT, KO, and KO+AAV treated mice. Smpd1 KO mice receiving PBS showed a steady decrease in latency to fall over time compared to WT mice. Smpd1 KO mice received AAV. CB7.hSMPD1 or AAV.Ubc.hSMPD1 gene therapy showed rescue phenotype in the behavioral deficits exhibited in Smpd1 KO mice. There was no difference between the CB7 promoter and the Ubc promoter. [Figure 3B] Average Purkinje neuron (PN) density per lobule for AAV.CB7.SMPD1 and AAV.UbChSMPD1 compared to untreated wild-type (WT) mice and knockout mice injected with phosphate buffered saline (PBS) is shown. [Figure 4A] Shown are histochemical staining of cholesterol accumulation in the brain and macrophages in the liver, spleen, and lungs. In column 2, Smpd1 KO mice showed abundant amounts of cholesterol in the brain and lipid-filled macrophages in the liver, spleen, and lungs compared to WT mice (column 1). In columns 3 and 4, Smpd1 KO mice receiving AAV.CB7.hSMPD1 or AAV.Ubc.hSMPD1 gene therapy ICV, respectively, showed reduced cholesterol accumulation in the brain and lack or reduction of lipid-filled macrophages in the liver, spleen, and lungs. [Figure 4B] 1 shows macrophages in the alveoli of SMPD knockout mice. [Figure 4C] We show an absence of lipid-laden enlarged macrophages in the alveoli (in the lung tissue) from treated mice, suggesting correction of CNS and peripheral disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, and other compositions and methods for the expression of functional hSMPD1. In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, host cells, other compositions, and methods for the production of compositions comprising either a nucleic acid sequence encoding functional hSMPD1 or an hSMPD1 polypeptide. In yet another embodiment, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vector genomes, vectors, recombinant viruses, other compositions, and methods for the delivery of a nucleic acid sequence encoding functional hSMPD1 to a subject for the treatment of NPA disease. In certain embodiments, the adeno-associated virus (AAV) vector-based methods described herein provide a new treatment option that helps restore the desired function of hSMPD1 and alleviate symptoms associated with hSMPD1 deficiency (NPA disease) by providing expression of hSmpd1 in a subject in need thereof.

[0011] As used herein, the term "therapeutic level" refers to at least about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or greater than 100% of SMPD1 (ASMase) enzyme activity relative to healthy controls.

[0012] Suitable assays for measuring hSMPD1 enzyme activity are known to those skilled in the art. Additionally or alternatively, the function of AAV-mediated delivery of SMPD1 enzyme can be evaluated by measuring the reduction of lipid accumulation in cells, such as the brain, lung, spleen, and / or liver. In some embodiments, such therapeutic levels of hSmpd1 can result in the alleviation of NPA disease-related symptoms, the improvement of NPA disease-related disease biomarkers, the reversal of certain NPA disease-related symptoms, and / or the prevention of the progression of NPA disease-related symptoms, or any combination thereof. Treatment with the vectors and compositions provided herein can provide one or more of behavioral relief (correction), reduction of cholesterol accumulation in the brain, and / or reduction or absence of lipid-filled macrophages in the liver, spleen, and lung.

[0013] As used herein, "disease," "disorder," and "condition" refer to NPA disease and / or hSMPD1 (ASMase) deficiency in a subject.

[0014] As used herein, "Niemann-Pick A symptoms" or "NPA" and "symptoms" refer to symptoms seen in patients with NPA disease and in animal models of NPA disease. Such symptoms include, but are not limited to, weight loss or cachexia, loss of motor function, lipid accumulation (e.g., in the spleen, liver, lungs, bone marrow, and / or brain), splenomegaly, liver enlargement, progressive deterioration of the nervous system, loss of cognitive function, and premature death.

[0015] The vectors provided herein are highly effective for administering an effective amount of a viral vector comprising a transgene encoding an acid sphingomyelinase polypeptide to the CNS of a mammal, followed by administration of an effective amount of the viral vector comprising the transgene to liver tissue of the mammal. In another embodiment, the region of the CNS to which the viral vector is delivered is the brain.

[0016] In a further aspect, a method is provided for treating Niemann-Pick Type A (NPA) in a mammal suffering from NPA by administering an AAV viral vector comprising a transgene encoding an effective amount of an acid sphingomyelinase polypeptide (e.g., hSMPD1) to liver tissue of the mammal, followed by delivery of the AAV vector comprising a transgene encoding an effective amount of an acid sphingomyelinase polypeptide to the CNS of the mammal, thereby treating NPA in the mammal. In another embodiment, the region of the CNS to which the viral vector is delivered is the brain.

[0017] 1. Human acidic sphingomyelin phosphodiesterase 1 (hSmpd1) The SMPD1 gene encodes the SMPD1 protein, which has enzymatic activity and is also known as acid sphingomyelinase (or ASMase). As used herein, a "functional SMPD1 protein" or "functional ASMase" (also called human SMPD1 or hSMPD1) can convert the lipid sphingomyelin to ceramide so that fat does not accumulate in cells. Loss of SMPD1 enzyme function is associated with cell loss and dysfunction in tissues and organs, including the brain, lung, spleen, and liver, in people with Niemann-Pick disease type A.

[0018] As used herein, the term "functional hSmpd1" refers to an SMPD1 enzyme that provides at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100%, of the biological activity level of native (wild-type) hSMPD1 from a patient without NPA.

[0019] hSMPD1 can be, for example, a full-length protein (including a signal peptide and a mature protein), a mature protein, a mature protein having a short native signal peptide (e.g., 629 amino acids) as described herein, a mature protein having a full-length native signal peptide (e.g., 631 amino acids), a mature protein having an exogenous signal peptide, or a functional fragment.

[0020] As used herein, "signal peptide" refers to a short peptide present at the N-terminus of a newly synthesized protein. Signal peptides, and in some cases, nucleic acid sequences encoding such peptides, may also be referred to as signal sequences, targeting signals, localization signals, localization sequences, transit peptides, leader sequences, or leader peptides.

[0021] As described herein, hSMPD1 may include a native signal peptide (i.e., amino acids 1-46 of SEQ ID NO:2 or SEQ ID NO:30) or, alternatively, an exogenous signal peptide. With reference to the numbering of hSMPD1 in SEQ ID NO:2, the signal peptide is present (from about amino acids 1-46 of SEQ ID NO:2 or SEQ ID NO:30) and the mature protein includes from about amino acids 47 to about 629 of SEQ ID NO:2 (or SEQ ID NO:31). The amino acid sequence of SEQ ID NO:2 is also reproduced in SEQ ID NO:23.

[0022] In certain embodiments, hSMPD1 includes a signal peptide from an exogenous source protein. Because certain NPAs are associated with longer signal peptides, the signal peptide selected will generally be selected to be the same length as provided in the constructs herein (e.g., about 46 amino acids, but may be selected to be shorter, e.g., about 10 to about 25 amino acids in length). In certain embodiments, such an exogenous signal peptide may be selected to be the same length as provided in the constructs herein (e.g., about 46 amino acids, but may be selected to be shorter, e.g., about 10 to about 25 amino acids in length). The peptide is preferably of human origin and may include, for example, the IL-2 signal peptide. Other signal / leader peptides may be found naturally in immunoglobulins (e.g., IgG), cytokines (e.g., IL-2, IL12, IL18, etc.), insulin, albumin, β-glucuronidase, alkaline protease, or fibronectin secretory signal peptides, among others. See also, for example, signalpeptide.de / index.php?m=listspdb_mammalia. Such chimeric hSmpd1 may have a foreign leader instead of the entire native signal peptide. Optionally, the N-terminal truncation of the hSmpd1 enzyme may lack only a portion of the signal peptide (e.g., a deletion of about 2 to about 25 amino acids, or any value therebetween), the entire signal peptide, or a fragment longer than the signal peptide (e.g., up to about amino acid 46). Optionally, such enzymes may contain a C-terminal truncation of about 5, 10, 15, or 20 amino acids in length.

[0023] In certain embodiments, a functional hSMPD1 can be selected that has a sequence that encodes a protein that is at least 95% identical, at least 97% identical, or at least 99% identical to the sequence of SEQ ID NO:2 (amino acids 1-629). SEQ ID NO:23 has the same amino acid sequence as SEQ ID NO:2, but has a different coding sequence. In certain embodiments, hSMPD1 sequences are provided that encode a protein that is at least 95%, at least 97%, or at least 99% identical to the mature protein (amino acids 47-629) of SEQ ID NO:2 or 23. For example, in certain embodiments, the hSMPD1 protein has an Ala substitution at position 36 and / or an R substitution at position 506 with respect to the numbering of SEQ ID NO:2 or 23.

[0024] In certain embodiments, a functional hSMPD1 may be selected that has a sequence that encodes a protein that is at least 95% identical, at least 97% identical, or at least 99% identical to the sequence of SEQ ID NO: 3 (amino acids 1-629). In certain embodiments, hSMPD1 sequences are provided that encode a protein that is at least 95%, at least 97%, or at least 99% identical to the mature protein of SEQ ID NO: 3 (amino acids 47-629). For example, in certain embodiments, the hSMPD1 protein has a Val at position 36 and / or an R substitution at position 506 with respect to the numbering of SEQ ID NO: 3.

[0025] As used herein, "conservative amino acid replacement" or "conservative amino acid substitution" refers to the modification, replacement, or substitution of an amino acid with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size) known by those skilled in the art. Also see, for example, FRENCH et al. What is a conservative substitution? Journal of Molecular Evolution, March 1983, Volume 19, Issue 2, pp 171-175 and YAMPOLSKY et al. The Exchangeability of Amino Acids in Proteins, Genetics. 2005 Aug;170(4):1459-1472, each of which is incorporated herein by reference in its entirety.

[0026] In one aspect, provided herein are nucleic acid sequences, including expression cassettes and vectors comprising same, that encode a functional hSMPD1 protein. In one embodiment, the nucleic acid sequence is an engineered hSMPD1 sequence of SEQ ID NO: 4, which encodes hSMDP1 with an A at position 36, with reference to the numbering of SEQ ID NO: 2 or 23, and a 629 amino acid hSMPD1 protein (with a short leader sequence) with an Ala (A) at position 36 and a Gly (G) at position 506. Specifically, the nucleic acid sequence is at least about 80% identical to the engineered hSMPD1 sequence of SEQ ID NO: 4, which encodes a functional hSMPD1 with an Ala at position 36, with reference to the numbering of SEQ ID NO: 2. In a particular embodiment, the encoded protein is a 629 amino acid hSMPD1 protein with a native signal peptide. In other embodiments, the encoded protein is a 631 protein with a longer native signal peptide. In certain embodiments, the encoded protein has an Arg(R) at position 506.

[0027] In another aspect, provided herein are nucleic acid sequences, including expression cassettes and vectors, encoding functional hSMPD1 proteins. In one embodiment, the nucleic acid sequence is an engineered hSMPD1 sequence (hMPDD1.CoV2 or CoV2) of SEQ ID NO: 22, which encodes hSMPD1 with an A at position 36, with reference to the numbering of SEQ ID NOs: 2 and 23, and a 629 amino acid hSMPD1 protein (with a short leader sequence) with an Ala (A) at position 36 and a Gly (G) at position 506. Specifically, the hSPMPD1.CoV2 nucleic acid sequence is at least about 80% identical to the engineered hSMPD1 sequence of SEQ ID NO: 22, which encodes a functional hSMPD1 with an Ala at position 36, with reference to the numbering of SEQ ID NOs: 2 and 23. In a particular embodiment, the encoded protein is a 629 amino acid hSMPD1 protein with a native signal peptide. In another embodiment, the encoded protein is a 631 protein with a longer native signal peptide. In a specific embodiment, the encoded protein has an Arg(R) at position 506.

[0028] In one aspect, provided herein are nucleic acid sequences encoding functional hSMPD1 proteins, including expression cassettes and vectors comprising the same. In one embodiment, the nucleic acid sequence is an engineered hSMPD1 sequence of SEQ ID NO: 5, which encodes hSMPD1 with a V at position 36, with reference to the numbering of SEQ ID NO: 3, a 629 amino acid hSMPD1 protein (with a short leader sequence) with a Val (V) at position 36 and a Gly (G) at position 506. In a particular embodiment, the nucleic acid sequence is at least about 80% identical to the engineered hSMPD1 sequence of SEQ ID NO: 5, which encodes a functional hSMPD1 with an Ala at position 36, with reference to the numbering of SEQ ID NO: 3. In a particular embodiment, the encoded protein is a 629 amino acid hSMPD1 protein with a native signal peptide. In other embodiments, the encoded protein is a 631 protein with a longer native signal peptide. In a particular embodiment, the encoded protein has an Arg® at position 506.

[0029] As used herein, "nucleic acid" refers to a polymeric form of nucleotides, including RNA, mRNA, cDNA, genomic DNA, peptide nucleic acid (PNA), as well as synthetic forms and mixed polymers of the above. Nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide (e.g., peptide nucleic acid oligomers). The term also includes DNA in single-stranded and double-stranded forms. Those skilled in the art will understand that functional variants of these nucleic acid molecules are described herein. Functional variants are nucleic acid sequences that can be directly translated using the standard genetic code to provide the same amino acid sequence as that translated from the parent nucleic acid molecule.

[0030] In certain embodiments, the nucleic acid molecules encoding functional hSmpd1 and other constructs described herein are useful for generating expression cassettes and vector genomes, which can be engineered for expression in yeast cells, insect cells, or mammalian cells (e.g., human cells). Methods are known and have been previously described (e.g., WO96 / 09378). A sequence is considered to be engineered if at least one non-preferred codon compared to the wild-type sequence is replaced by a more preferred codon. As used herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon that codes for the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The codon usage frequency of a particular organism can be found in a codon frequency table, for example, the codon frequency table at kazusa.jp / codon. Preferably, , more than one unpreferred codon, preferably most or all unpreferred codons, are replaced by more preferred codons. Preferably, the codons most frequently used in the organism are used in the engineered sequence. Replacement with preferred codons generally results in higher expression. It will also be understood by the skilled artisan that as a result of the degeneracy of the genetic code, many different nucleic acid molecules can code for the same polypeptide. It will also be understood that the skilled artisan may, using routine techniques, make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Thus, unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate and code for the same amino acid sequence. The nucleic acid sequence can be cloned using routine molecular biology techniques or generated de novo by DNA synthesis, which can be carried out using routine procedures by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins).

[0031] In certain embodiments, the nucleic acids, expression cassettes, and vector genomes described herein comprise an hSMPD1 coding sequence that is an engineered sequence. In certain embodiments, the engineered sequence is useful for improving production, transcription, expression, or safety in a subject. In certain embodiments, the engineered sequence is useful for increasing the efficacy of the resulting therapeutic composition or treatment. In further embodiments, the engineered sequence is useful for increasing the efficacy of the expressed functional hSMPD1 protein, and may also allow for lower doses of therapeutic reagents that deliver functional hSMPD1. In certain embodiments, the engineered hSMPD1 coding sequence is characterized by an improved translation rate compared to the wild-type hSMPD1 coding sequence.

[0032] Efficacy may be determined by a variety of suitable methods, including improvement of lethargic behavior, reduction or stabilization of gait abnormalities, and CNS / respiratory / cardiac involvement. For example, animals may be subjected to a rotarod performance test to assess motor coordination. Filipin immunostaining on brain sections may be used to detect cholesterol deposition. In certain embodiments, efficacy is determined by a reduction in the amount of accumulated cholesterol. Other assays for measuring efficacy may include sphingomyelin quantification (liver / brain / spleen) and chitinase assays (biomarkers of disease severity).

[0033] In certain embodiments, subjects provided herein are monitored for DRG pathology via SNAP pathology and / or through the use of biomarkers. See, e.g., U.S. Patent Application No. 63 / 279,561, filed November 15, 2021, which is incorporated herein by reference.

[0034] By "engineered" is meant that the nucleic acid sequence encoding the functional hSMPD1 enzyme described herein is assembled and placed in any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which introduces the hSmpd1 sequence carried thereon into a host cell, e.g., to generate a non-viral delivery system (e.g., RNA-based system, naked DNA, etc.), or to generate a viral vector in a packaging host cell, and / or for delivery to a host cell of interest. In certain embodiments, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. Methods used to generate such engineered constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0035] In the context of nucleic acid sequences, the terms "percent identity", "sequence identity", "percent sequence identity", or "percent identical" refer to residues in two sequences that are the same when aligned for correspondence. The length of sequence identity comparison may be over the entire length of the construct, the entire length of the gene coding sequence, or a fragment of at least about 500-1000 nucleotides. However, identity between smaller fragments, e.g., having at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 nucleotides or more, may also be desired.

[0036] Percent identity can be readily determined for amino acid sequences spanning the entire length of a protein, polypeptide, about 100 amino acids, about 300 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequence coding sequence. Suitable amino acid fragments can be at least about 8 amino acids in length and up to about 50 amino acids. In general, when referring to "identity", "homology", or "similarity" between two different sequences, the "identity", "homology", or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence.

[0037] Identity can be determined by preparing an alignment of sequences using various algorithms and / or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; Clustal Omega; FASTA; e.g., using the Needleman-Wunsch algorithm, Smith-Waterman algorithm). The alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. For example, sequence alignment programs such as "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs are available for amino acid sequences. Generally, any of these programs are used with default settings, but the skilled artisan may change these settings as necessary. Alternatively, the skilled artisan may utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, for example, JD Thomson et al, Nucl. Acids. Res., "A comprehensive See, for example, “comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0038] 2. Expression Cassette In certain embodiments, provided herein is an expression cassette having an engineered nucleic acid sequence encoding a functional hSmpd1 and regulatory sequences that direct its expression. In further embodiments, an expression cassette having an engineered nucleic acid sequence as described herein that encodes a functional hSmpd1 and regulatory sequences that direct its expression.

[0039] As used herein, the term "expression" or "gene expression" refers to the process by which information from a gene is used to synthesize a functional gene product. A gene product can be a protein, peptide, or nucleic acid polymer (such as RNA, DNA, or PNA).

[0040] As used herein, an "expression cassette" refers to a nucleic acid sequence that is biologically useful and that directs or contains a nucleic acid sequence (e.g., a gene cDNA that encodes a protein, enzyme, or other useful gene product, mRNA, etc.) and its gene product. "Registered" refers to a nucleic acid molecule that includes a regulatory sequence that regulates and is operably linked thereto. As used herein, "operably linked" includes both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis with the nucleic acid sequence. Such regulatory sequences typically include, for example, one or more of a promoter, enhancer, intron, Kozak sequence, polyadenylation sequence, and TATA signal. An expression cassette may contain, among other elements, regulatory sequences upstream (5') of the gene sequence, e.g., one or more of a promoter, enhancer, intron, etc., and enhancer, or regulatory sequences downstream (3') of the gene sequence, e.g., one or more of a 3' untranslated region (3'UTR) that includes a polyadenylation site. In certain embodiments, the regulatory sequence is operably linked to the nucleic acid sequence of the gene product, and the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5' untranslated region (5'UTR). In certain embodiments, an expression cassette comprises a nucleic acid sequence of one or more gene products. In some embodiments, the expression cassette can be a monocistronic or bicistronic expression cassette. In other embodiments, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell.

[0041] Typically, such expression cassettes can be used to generate viral vectors and contain coding sequences for the gene products described herein adjacent to packaging signals of the viral genome, as well as other expression control sequences such as those described herein. In certain embodiments, the vector genome may contain two or more expression cassettes.

[0042] In certain embodiments, an expression cassette refers to a nucleic acid polymer comprising a coding sequence of a functional hSMPD1 (including variants and fragments thereof) and a promoter. In further embodiments, the expression cassette comprises one or more regulatory sequences in addition to the promoter. In certain embodiments, the expression vector is a vector genome. In certain embodiments, the expression cassette or vector genome is packaged into a vector. In certain embodiments, a plasmid comprising an expression cassette as described herein is provided.

[0043] As used herein, the term "regulatory sequence" or "expression control sequence" refers to nucleic acid sequences, such as initiator sequences, enhancer sequences, and promoter sequences, that induce, repress, or otherwise control the transcription of protein-coding nucleic acid sequences to which they are operably linked.

[0044] As used herein, the term "operably linked" refers to both the nucleic acid sequence encoding hSmpd1 and adjacent expression control sequences, and / or expression control sequences acting in trans or at a distance to control its transcription and expression.

[0045] The term "heterologous" when used in reference to a protein or nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or nucleic acid is present in a distinct sequence or subsequence that is not found in the same relationship to each other in nature.

[0046] In certain embodiments, the expression cassette provided comprises a promoter that is a chicken β-actin promoter. Various chicken beta-actin promoters have been described alone or in combination with various enhancer elements (e.g., CB7 (also called CB7 hybrid promoter) is a chicken beta-actin promoter with a cytomegalovirus (CMV IE) enhancer element, the CAG promoter, which contains the first exon and first intron of chicken beta actin, and the splice acceptor of the rabbit beta globin gene) or the CBh promoter [SJ Gray et al, Hu Gene Ther, 2011 Sep;22 ( 9):1143-1153]. In certain embodiments, the expression cassette comprises a promoter that is the ubiquitin promoter, UbC.

[0047] In other embodiments, suitable promoters include the elongation factor 1 alpha (EF1 alpha) promoter (see, e.g., Kim DW et al, Use of the human elongation factor1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23), the synapsin 1 promoter (see, e.g., Kugler S et al, Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47), the neuron-specific enolase (NSE) promoter (see, e.g., Kim J et al, Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004 Feb;145(2):613-9. Epub 2003 Oct 16), or the CB6 promoter (see, e.g., Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol Biotechnol.2016 Jan;58(1):30-6. doi:10.1007 / s12033-015-9899-5).

[0048] Examples of promoters that are tissue specific include promoters that are specific for the liver and other tissues (albumin, Miyatake et al., (1997) J. Virol., 71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) Gene Ther., 3:1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503-14), bone osteocalcin (Stein et al., (1997) Mol. Biol. Rep., 24:185-96), bone sialoprotein (Chen et al., (1996) J. Bone Miner. Res., 11:654-64), lymphocytes (CD2, Hansal et al., (1996) J. Bone Miner. Res., 11:654-64), among others. al., (1998) J. Immunol., 161:1063-8, immunoglobulin heavy chain, T cell receptor chain), neural promoters such as the neuron-specific enolase (NSE) promoter (Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503-15), neurofilament light chain gene, Piccioli et al., (1991) Proc. Natl. Acad. Sci. USA, 88:5611-5), and neuron-specific vgf gene (Piccioli et al., (1995) Neuron, 15:373-84). In certain embodiments, the promoter is the human thyroxine-binding globulin (TBG) promoter. Alternatively, a regulatable promoter may be selected. See, for example, WO2011 / 126808B2, incorporated herein by reference.

[0049] In certain embodiments, the expression cassette comprises one or more expression enhancers. In certain embodiments, the expression cassette comprises two or more expression enhancers. These enhancers can be the same or different. For example, the enhancer can be alphamic / The expression cassette may include a bik enhancer or a CMV enhancer (e.g., a CMV IE enhancer). The enhancer may be present in two copies located adjacent to each other. Alternatively, the double copies of the enhancer are separated by one or more sequences. In yet further embodiments, the expression cassette further includes an intron, such as a chicken beta-actin intron, a human beta-globulin intron, an SV40 intron, and / or a commercially available Promega® intron (i.e., a Promega chimeric intron). Other suitable introns include those known in the art, such as those described in WO2011 / 126808.

[0050] The provided expression cassettes may include one or more expression enhancers, such as post-transcriptional regulatory elements from woodchuck (WPRE), human (HPRE), ground squirrel (GPRE), or arctic ground squirrel (AGSPRE) hepatitis virus, or synthetic post-transcriptional regulatory elements. These expression enhancing elements are particularly advantageous when placed in the 3'UTR and can significantly increase mRNA stability and / or protein yield. In certain embodiments, the provided expression cassettes include a regulatory sequence that is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., as described in U.S. Pat. Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated by reference). In certain embodiments, the WPRE is a modified WPRE sequence that can be engineered upstream of the polyA sequence and downstream of the coding sequence [see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16:605-619, incorporated herein by reference].

[0051] Additionally, the provided expression cassettes include a suitable polyadenylation signal. In a particular embodiment, the polyA sequence is rabbit globin polyA (also called rabbit beta globin polyA or RBG polyA). See, for example, WO2014 / 151341. In another embodiment, the polyA sequence is bovine growth hormone polyA. Alternatively, another polyA is included, such as the human growth hormone (hGH) polyadenylation sequence, SV50 polyA, or a synthetic polyA.

[0052] In certain embodiments, the expression cassette may contain one or more miRNA (also referred to as miR or microRNA) target sequences in the untranslated region. The miRNA target sequences are designed to be specifically recognized by miRNAs present in cells in which transgene expression is undesirable and / or where reduced levels of transgene expression are desired. In certain embodiments, the expression cassette contains a miRNA target sequence that specifically reduces the expression of hSMPD1 in dorsal root ganglion. In certain embodiments, the miRNA target sequence is located in the 3'UTR, the 5'UTR, and / or both the 3' and 5'UTR of the expression cassette. In certain embodiments, the expression cassette contains at least two tandem repeats of a dorsal root ganglion (DRG)-specific miRNA target sequence, the at least two tandem repeats containing at least a first miRNA target sequence and at least a second miRNA target sequence that may be the same or different. In certain embodiments, the first start of the at least two drg-specific miRNA tandem repeats is within 20 nucleotides of the 5' or 3' end of the hSmpd1 coding sequence. In certain embodiments, the start of the first of the at least two DRG-specific miRNA tandem repeats is at least 100 nucleotides from the 5' or 3' end of the hSMPD1 coding sequence. In certain embodiments, the miRNA tandem repeat comprises a length of 200-1200 nucleotides. An exemplary DRG targeting sequence (e.g., miR182) is provided in SEQ ID NO: 24, and a 4x tandem repeat is provided in SEQ ID NO: 28. In certain embodiments, the inclusion of the miR target does not modify the expression or efficacy of the therapeutic transgene in one or more target tissues compared to an expression cassette lacking the miR target sequence. In certain embodiments, the miR is miR183. Filed Dec. 20, 2019, filed Jun. 25, 2020 See International Patent Application No. PCT / US19 / 67872, published as WO202 / 132455, International Patent Application No. PCT / US2021 / 032003, filed May 12, 2021 (which claims priority to U.S. Provisional Patent Application No. 63 / 023,594, filed May 12, 2020, U.S. Provisional Patent Application No. 63 / 038,488, filed June 12, 2020, U.S. Provisional Patent Application No. 63 / 043,562, filed June 24, 2020, and U.S. Provisional Patent Application No. 63 / 079,299, filed September 16, 2020, all of which are incorporated by reference in their entireties), published herein as WO2021 / 231579, published November 18, 2021.

[0053] Examples of exemplary expression cassettes are provided in the Examples below, including, for example, CB7.CI.hSMPD1co(TY).RBG, which contains a CB7 hybrid promoter (including a CMV IE enhancer and a CB promoter), a chicken beta actin intron, hSMPD1.V36A.co (SEQ ID NO: 4), and a rabbit beta globin polyA (SEQ ID NO: 8). In another embodiment, the expression cassette contains a CB7 hybrid promoter, a chicken beta actin intron, the hSMPD1cov2 coding sequence of SEQ ID NO: 22), and a rabbit beta globin polyA [CB7.CI.hSMPD1cov2.RBG (SEQ ID NO: 21)]. In yet another embodiment, the expression cassette comprises a CB7 hybrid promoter, a chicken beta actin intron, an hSMPD1cov2 coding sequence of SEQ ID NO: 22, 4xmiR [SEQ ID NO: 28, containing four copies of SEQ ID NO: 24], and rabbit beta globin polyA [CB7.CI.hSMPD1coV2.4xmiR182.rBG (SEQ ID NO: 19)].

[0054] It is to be understood that the compositions in these and the described expression cassettes are intended to apply to other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0055] 3. Production of Delivery Vectors In one aspect, provided herein is a vector comprising a nucleic acid sequence encoding a functional hSMPD1. In certain embodiments, the vector comprises an expression cassette as described herein for delivery of the hSmpd1 coding sequence.

[0056] As used herein, a "vector" is a biological or chemical moiety that contains a nucleic acid sequence and can be introduced into a suitable target cell for replication or expression of the nucleic acid sequence. Examples of vectors include, but are not limited to, recombinant viruses, plasmids, lipoplexes, polymersomes, polyplexes, dendrimers, cell penetrating peptide (CPP) conjugates, magnetic particles, or nanoparticles. In certain embodiments, a vector is a nucleic acid molecule into which an engineered nucleic acid encoding a functional hSMPD1 can be inserted and then introduced into a suitable target cell. Such vectors preferably have one or more origins of replication and one or more sites into which recombinant DNA can be inserted. For example, vectors encoding drug resistance genes often have a means by which cells containing the vector can be selected from cells that do not. Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods for generating, producing, characterizing, or quantifying vectors are available to those skilled in the art.

[0057] In certain embodiments, the vector is a non-viral plasmid and may be combined with various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polyglycan compositions, and other polymer, lipid and / or cholesterol-based-nucleic acid conjugates, including, for example, "naked DNA," "naked plasmid DNA," RNA, and mRNA) and other constructs as described herein. See, for example, X. Su et al., Mol. Pharmaceutics, 2013, 113:131-135, 1999. 11, 8(3), pp774-787, web published: March 21, 2011, see WO2013 / 182683, WO2010 / 053572, and WO2012 / 170930, all of which are incorporated herein by reference.

[0058] In certain embodiments, the vectors described herein are "replication-defective viruses" or "viral vectors," which refers to synthetic or artificial viral particles in which an expression cassette containing a nucleic acid sequence encoding hSMPD1 is packaged in a viral capsid or envelope, and any viral genomic sequences packaged in the viral capsid or envelope are replication-defective (i.e., retain the ability to infect target cells but cannot generate progeny virions). In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only nucleic acid sequences encoding hSMPD1 flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. Thus, it is considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0059] As used herein, a recombinant viral vector is an adeno-associated virus (AAV), adenovirus, bocavirus, hybrid AAV / bocavirus, herpes simplex virus, or lentivirus.

[0060] In certain embodiments, a host cell is provided that comprises a nucleic acid that comprises an hSMPD1 coding sequence. In certain embodiments, the host cell comprises a plasmid that comprises the hSMPD1 coding sequence described herein.

[0061] As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV) is produced. Host cells may be prokaryotic or eukaryotic (e.g., human, insect, or yeast) cells, containing exogenous or heterologous DNA that is introduced into the cells by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Examples of host cells may include, but are not limited to, isolated cells, cell cultures, Escherichia coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK-293 cells, liver cells, kidney cells, central nervous system cells, neurons, glial cells, or stem cells.

[0062] In certain embodiments, the host cell comprises an expression cassette for the production of hSMPD1, whereby the protein is produced in sufficient quantities in vitro for isolation or purification. In certain embodiments, the host cell comprises an expression cassette encoding hSMPD1 (e.g., including a functional fragment thereof). As provided herein, the hSMPD1 polypeptide can be included in a pharmaceutical composition administered to a subject as a therapeutic agent (i.e., enzyme replacement therapy).

[0063] As used herein, the term "target cell" refers to any cell in which expression of functional hSmpd1 is desired. In certain embodiments, the term "target cell" is intended to refer to a cell of a subject undergoing treatment for NPA disease. Examples of target cells include, but are not limited to, liver cells, kidney cells, smooth muscle cells, and neurons. In certain embodiments, the vector is delivered to the target cell ex vivo. In certain embodiments, the vector is delivered to the target cell in vivo.

[0064] It is to be understood that the compositions in the vectors described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0065] 4. Recombinant adeno-associated virus (rAAV) In certain embodiments, provided herein is a rAAV comprising an AAV capsid and a vector genome packaged therein. The vector genome comprises an AAV5' inverted terminal repeat (ITR), a nucleic acid sequence encoding a functional hSMPD1 as described herein, a regulatory sequence directing the expression of hSMPD1 in a target cell, and an AAV3'ITR. In certain embodiments, the vector genome comprises an expression cassette as provided herein flanked by the AAV5'ITR and AAV3'ITR. Such a rAAV is suitable for use in the treatment of NPA disease.

[0066] As used herein, "rAAV.hSmpd1" refers to a rAAV having a vector genome that includes a hSMPD1 coding sequence. "rAAVhu68.hSMPD1" refers to a rAAV having an AAVhu68 capsid and a vector genome that includes a hSMPD1 coding sequence.

[0067] As used herein, "vector genome" refers to a nucleic acid sequence that is packaged into a vector. In one embodiment, vector genome refers to a nucleic acid sequence that is packaged into a rAAV capsid that forms a rAAV vector. Such a nucleic acid sequence contains AAV inverted terminal repeats (ITRs). In certain embodiments, the ITRs are from a different AAV than the one that supplies the capsid. In a preferred embodiment, the ITR sequence from AAV2, or a deleted version thereof (ΔITR), may be used for convenience and to facilitate regulatory approval. However, ITRs from other AAV sources may be selected. If the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. In certain embodiments, the vector genome comprises a truncated AAV2 ITR of 130 base pairs, with the external A element deleted. Without wishing to be bound by theory, it is believed that the shortened ITRs are restored to the wild-type length of 145 base pairs during vector DNA amplification using the internal (A') element as a template. In other embodiments, full-length AAV 5' and 3' ITRs are used. Typically, AAV vector genomes include AAV 5' ITR, regulatory sequences, hSMPD1 coding sequence, and AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, termed ΔITR, in which the D sequence and terminal separation sites (trs) are deleted, has been described. In other embodiments, full-length AAV 5' and 3' ITRs are used. In certain embodiments, the vector genome includes one or more miRNA target sequences.

[0068] In certain embodiments, rAAVs are provided having a vector genome comprising a 5'ITR, a promoter, a chicken beta-actin intron, an hSMPD1 coding sequence, a polyA sequence, and a 3'ITR. In certain embodiments, rAAVs are provided having a vector genome comprising a 5'ITR, a CB7 hybrid promoter, a chicken beta-actin intron, an hSMPD1 coding sequence, a rabbit globin polyA sequence, and a 3'ITR. In certain embodiments, rAAVs are provided having a vector genome comprising a 5'ITR, a TBG promoter, a chicken beta-actin intron, an hSMPD1 coding sequence, a WPRE, a bovine growth hormone polyA sequence, and a 3'ITR. In certain embodiments, rAAVs are provided having a vector genome comprising a 5'ITR, a UbC promoter, an hSMPD1 coding sequence, an SV40 polyA sequence, and a 3'ITR.

[0069] In certain embodiments, the hSMPD1 coding sequence of SEQ ID NO: 22 or a sequence at least 80% identical to SEQ ID NO: 22 that encodes the functional hSMPD1 protein of SEQ ID NOs: 2 and 23. , at least 85% identical, at least 90% identical, at least 95% identical, or at least 99-100% identical to SEQ ID NO: 4. In certain embodiments, rAAVs are provided having a vector genome comprising an hSMPD1 coding sequence of SEQ ID NO: 4, or a sequence encoding a functional hSMPD1 protein that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99-100% identical to SEQ ID NO: 4. In certain embodiments, rAAVs are provided having a vector genome comprising an hSMPD1 coding sequence of SEQ ID NO: 5, or a sequence encoding a functional hSMPD1 protein that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99-100% identical to SEQ ID NO: 5. In certain embodiments, rAAVs are provided having a vector genome comprising an expression cassette of SEQ ID NO: 8, 10, or 14. In certain embodiments, rAAVs are provided having a vector genome of SEQ ID NO: 9, 11, 14, or 15. In certain embodiments, an rAAV is provided that comprises a vector genome of SEQ ID NO: 18. In certain embodiments, an rAAV is provided that comprises a vector genome of SEQ ID NO: 20.

[0070] Examples of exemplary vector genomes are provided in the Examples below, and include, for example, ITR.CB7.CI.hSMPD1co(TY).RBG.ITR, which includes a CB7 hybrid promoter (including a CMV IE enhancer and a CB promoter), a chicken beta actin intron, hSMPD1.V36A.co (SEQ ID NO: 4), and a rabbit beta globin polyA (SEQ ID NO: 8), flanked at the 5' end by a spacer sequence of the 5'AAV ITR, and at its 3' end by a spacer sequence and a 3'ITR. See, for example, SEQ ID NO: 9, and optionally further including full length 5' and 3' ITRs. In another embodiment, the vector genome includes a CB7 hybrid promoter, a chicken beta actin intron, hSMPD1cov2 coding sequence of SEQ ID NO: 22), and a rabbit beta globin polyA [ITR.CB7.CI.hSMPD1cov2.RBG.ITR (SEQ ID NO: 20)]. SEQ ID NO: 20 includes an expression cassette of SEQ ID NO: 21, flanked at the 5' end by a spacer sequence of the 5'AAV ITR, and at its 3' end by a spacer sequence and a 3'ITR. ITR, a spacer sequence, flanked at its 3' end by a spacer sequence and a 3'ITR. In yet another embodiment, the vector genome comprises a 5'ITR, a spacer sequence, an expression cassette of SEQ ID NO: 19 (CB7.CI.hSMPD1coV2.4xmiR182.rBG), a spacer sequence, and a 3'ITR. In certain embodiments, for example, when packaged into a rAAV capsid (viral particle), the 5'ITR and the 3'ITR are full-length sequences. The expression cassette of SEQ ID NO: 19 comprises a CB7 hybrid promoter, a chicken beta actin intron, an hSMPD1cov2 coding sequence of SEQ ID NO: 22, 4xmiR [SEQ ID NO: 28, including four copies of SEQ ID NO: 24], and a rabbit beta globin polyA [CB7.CI.hSMPD1coV2.4xmiR182.rBG (SEQ ID NO: 19)].

[0071] In certain embodiments, pharmaceutical compositions comprising these rAAV.hSMPD1 are provided. In certain embodiments, these rAAVs have an AAVhu68 capsid. The encoded (predicted) amino acid sequence of the AAVhu68 VP1 capsid is reproduced in SEQ ID NO: 17. AAVhu68 coding sequences useful for producing rAAVs with hu68 capsids are provided in SEQ ID NO: 17 and SEQ ID NO: 29 (AAVhu68M191). See, for example, International Patent Application Nos. PCT / US2021 / 055436, filed October 18, 2021, and WO2018 / 160582.

[0072] As used herein, the terms "rAAV" and "artificial AAV" are used interchangeably and refer to, but are not limited to, an AAV that includes a capsid protein and a vector genome packaged therein, the vector genome comprising a nucleic acid heterologous to the AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such an artificial capsid may incorporate a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) into the same AAV vector. The AAV may be generated by any suitable technique using heterologous sequences that may be obtained from a different selected AAV, that is a non-contiguous portion of the AAV, from a non-AAV viral source, or in combination with a heterologous sequence that may be obtained from a non-viral source. The artificial AAV may be, but is not limited to, a pseudotyped AAV capsid, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotyped vectors in which the capsid of one AAV is replaced with a heterologous capsid protein are useful in the present invention. In one embodiment, AAV2 / 5 and AAV2 / 8 are exemplary pseudotyped vectors. The selected genetic elements may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. The methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, see Green and Sambrook,Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0073] The term "AAV" as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or available in the context of the compositions and methods described herein, as well as engineered AAVs. Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles with AAV protein capsids, in which expression cassettes packaged are flanked by AAV inverted terminal repeats (ITRs) for delivery to target cells. AAV capsids are composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs can be selected as the source of capsids for the AAV viral vectors described above. See, e.g., U.S. Patent Application Publication No. 2007 / 0036760-A1, U.S. Patent Application Publication No. 2009 / 0197338-A1, EP1310571. See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), WO2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9), and WO2006 / 110689 and WO2003 / 042397 (rh.10), which also describe other AAVs that can be selected to generate AAVs and are incorporated by reference. Among the well-characterized AAVs isolated or engineered from humans or non-human primates (NHPs), human AAV2 was the first AAV developed as a gene transfer vector and has been widely used for efficient gene transfer experiments in various target tissues and animal models.Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein can be readily selected from among any AAV, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, AAVhu68, any variant of the known or mentioned AAVs, or any AAV or variant yet to be discovered, or mixtures thereof.AAV9 capsids include rAAVs having a capsid protein that comprises an amino acid sequence that is 99% identical to AAS99264. See also US7906111 and WO2005 / 033321. rAAVs having an AAVhu68 capsid are described, for example, in WO2018 / 160582, which are incorporated herein by reference. In certain embodiments, the capsid protein is designated in the name of the rAAV vector by a number or a combination of numbers and letters following the term "AAV". Also PCT / US19 / 19804 and PCT / US19 / 19861, each entitled "Novel Adeno-Associated Virus (AAV) Vectors, AAV Vectors Having Reduced Capsid Deamidation And Uses Therefor", filed February 27, 2019, which are incorporated herein by reference in their entireties. Please refer to.

[0074] As used herein, with respect to AAV, the term "variant" refers to any AAV sequence derived from a known AAV sequence, including AAV sequences with conservative amino acid substitutions and AAV sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or more sequence identity across the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes a variant that may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining percent identity of an AAV capsid, the comparison can be made across any of the variable proteins (e.g., vp1, vp2, or vp3). As used herein, "AAV9 variants" include, for example, those described in WO2016 / 049230, US8,927,514, US2015 / 0344911, and US8,734,809.

[0075] In certain embodiments, the AAV capsid is selected from among natural and engineered clade F adeno-associated viruses. In certain embodiments, the rAAV provided herein comprises an AAVhu68 capsid. See U.S. Published Patent Application No. 2020 / 0056159, incorporated herein by reference. AAVhu68 is within phylogenetic group F. AAVhu68 (SEQ ID NO:21) differs from another phylogenetic group F virus, AAV9, by two encoded amino acids at positions 67 and 157 in vp1. In contrast, other phylogenetic group F AAVs (AAV9, hu31, hu32) have an Ala at position 67 and an Ala at position 157. However, in other embodiments, the AAV capsid is selected from a different clade, e.g., clade A, B, C, D, or E, or from an AAV source outside any of these clades.

[0076] rAAVhu68 consists of the capsid and vector genome of AAVhu68. In one embodiment, a composition comprising rAAVhu68 comprises a collection of a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 proteins, and a heterogeneous population of vp3 proteins. As used herein, when used in reference to vp capsid proteins, the term "heterogeneous" or any grammatical variation thereof refers to a population of non-identical members, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 16 (and SEQ ID NO: 29, AAVhu68M191) encodes the amino acid sequence of the AAVhu68 vp1 protein (SEQ ID NO: 17). The AAVhu68 capsid includes subpopulations within the vp1, vp2, and vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO: 17. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in the asparagine-glycine pairs of SEQ ID NO: 17, and optionally further contain other deamidated amino acids, where the deamidation results in an amino acid change and other optional modifications. Various combinations of these and other modifications are described herein.

[0077] As used herein, a "subpopulation" of vp proteins, unless otherwise specified, refers to a group of vp proteins that have at least one defined common characteristic and that consist of at least one group member and fewer than all members of the reference group. For example, a "subpopulation" of vp1 proteins, unless otherwise specified, refers to at least one vp1 protein and fewer than all vp1 proteins in assembled AAV capsids. A "subpopulation" of vp3 may be one to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, in an assembled AAV capsid, vp1 proteins may be a subpopulation of vp proteins, vp2 proteins may be another subpopulation of vp proteins, and vp3 is yet a further subpopulation of vp proteins. In another example, vp1, vp2, and vp3 proteins may contain subpopulations with, for example, at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs.

[0078] In certain embodiments, the AAVhu68 capsid is further characterized by one or more of the following: the AAVhu68 capsid protein is an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:17, a vp1 protein produced from SEQ ID NO:16 (or SEQ ID NO:29), or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:16 (or SEQ ID NO:29) that encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO:17; vp2 protein, vp2 protein produced from a sequence that includes at least nucleotides 412-2211 of SEQ ID NO:16 or SEQ ID NO:29 (or SEQ ID NO:33 or 34), or vp2 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412-2211 of SEQ ID NO:16 or SEQ ID NO:29 (or SEQ ID NO:33 or 34) that encodes the predicted amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:17 (or SEQ ID NO:32), and / or AAVhu68 produced by expression from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:17 (or SEQ ID NO:35). vp3 protein, a vp3 protein produced from a sequence that includes at least nucleotides 607-2211 of SEQ ID NO:16 (or SEQ ID NO:35), or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2211 of SEQ ID NO:16 or SEQ ID NO:29 (or SEQ ID NO:36 or 37) that encodes a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:17 (or SEQ ID NO:35). In certain embodiments, the rAAVhu68 capsid comprises a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:17, wherein the vp1 proteins include a heterogeneous population of vp1 proteins that include a glutamic acid (Glu) at position 67 and / or a valine (Val) at position 157, optionally a heterogeneous population of vp2 proteins that include a valine (Val) at position 157, and a heterogeneous population of vp3 proteins.AAVhu68 capsids include at least one subpopulation in which at least 65% of the asparagine (N) of the asparagine-glycine pair located at position 57 of the vp1 protein and at least 70% of the asparagine (N) of the asparagine-glycine pairs at positions 329, 452, and / or 512 of the vp1, v2, and vp3 proteins are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:17, where deamidation results in an amino acid change.

[0079] As used herein, "encoded amino acid sequence" refers to predicted amino acids based on the translation of known DNA codons of a reference nucleic acid sequence that are translated into amino acids. As used herein, the term "clade" in relation to a group of AAVs refers to a group of AAVs that are systematically related to each other, as determined based on an alignment of AAV vp1 amino acid sequences using a Neighbor-Joining algorithm with a bootstrap value of at least 75% (out of at least 1000 replicates) and a Poisson-corrected distance measure of 0.05 or less. The Neighbor-Joining algorithm is described in the literature. See, for example, M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, 2009), 1999, 144:1311-1321, 2009, 19 ... See City Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of the AAV vp1 capsid protein, one of skill in the art can easily determine whether a selected AAV is in one of the clades identified herein, in another phylogenetic group, or outside these clades. See, for example, G Gao, et al, J Virol, 2004 Jun;78(10:6381-6388), which identifies phylogenetic groups A, B, C, D, E, and F (GenBank accession numbers AY530553-AY530629). See also WO2005 / 033321.

[0080] Methods for generating capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described, see, e.g., Gao, et al, Proc. Natl. Acad. Sci. USA 100(10), 6081-6086 (2003) and US 2013 / 0045186 A1.

[0081] ITRs or other AAV components can be easily isolated or engineered from AAV using techniques available to those of skill in the art. Such AAV can be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences can be engineered through synthesis or other suitable means by reference to published sequences such as those available in the literature or databases such as GenBank, PubMed, etc. AAV viruses can be engineered by conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to tailor stability and particle life span, for efficient degradation, for precise delivery to the nucleus, etc.

[0082] In certain embodiments, the rAAV is a self-complementary AAV. "Self-complementary AAV" or scAAV refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, e.g., DM McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene See Therapy, (August 2001), Vol. 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0083] In certain embodiments, the rAAV is nuclease-resistant. Such nucleases can be a single nuclease or a mixture of nucleases, which can be endonucleases or exonucleases. Nuclease-resistant rAAV indicates that the AAV capsid is fully assembled and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids that may be present from the production process. In many cases, the rAAV described herein is DNase-resistant.

[0084] The recombinant adeno-associated viruses (AAV) described herein can be produced using known techniques. AAV capsids can be produced. See, for example, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, US7588772B2. Such methods include culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid, a functional rep gene, an expression cassette as described herein flanked by AAV inverted terminal repeats (ITRs), and a suitable helper that functions to enable packaging of the expression cassette into AAV capsid proteins. Also provided herein is a host cell that contains a nucleic acid sequence encoding an AAV capsid, a functional rep gene, a vector genome as described, and a suitable helper that functions to enable packaging of the expression cassette into AAV capsid proteins. In one embodiment, the host cell is a HEK293 cell. These methods are described in further detail in WO2017 / 160360A2, which is incorporated herein by reference.

[0085] Other methods of producing rAAV available to those skilled in the art may be utilized. Suitable methods may include, but are not limited to, baculovirus expression systems or yeast-mediated production. See, for example, Robert M. Kotin, Large-scale recombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr 15; 20(R1):R2-R6. Published online 2011 Apr 29. doi:10.1093 / hmg / ddr141; Aucoin MG et al., Production of adeno-associated viral vectors in insect cells using triple infection: optimization of the rAAV vectors. of baculovirus concentration ratios.Biotechnol Bioeng.2006 Dec 20;95(6):1081-92, SAMI S.THAKUR,Production of Recombinant Adeno-associated viral vectors in yeast.Thesis presented to the Graduate School of the University of Florida,2012、Kondratov O et al.Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells,Mol Ther.2017 Aug 10.pii:S1525-0016(17)30362-3.doi:10.1016 / j.ymthe.2017.08.003.[Epub ahead of print]、Mietzsch M et al,OneBac2.0:Sf9 Cell Lines for Production of AAV1,AAV2,and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.Hum Gene Ther Methods.2017 Feb;28(1):15-22.doi:10.1089 / hgtb.2016.164、Li L et al.Production and characterization of novel recombinant adeno-associated virus replicative-form genomes:a eukaryotic source of DNA for gene transfer.PLoS One.2013 Aug 1;8(8):e69879.doi:10.1371 / journal.pone.0069879.Print2013、Galibert L et al,Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases.J Invertebr Pathol.2011 Jul;107 Suppl:S80-93.doi:10.1016 / j.jip.2011.05.008, and Kotin RM,Large-s See cale recombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr 15;20(R1):R2-6. doi:10.1093 / hmg / ddr141. Epub 2011 Apr 29.

[0086] A two-step affinity chromatography purification at high salt concentration, followed by anion exchange resin chromatography, is used to purify the vector drug product and remove empty capsids. These methods are described in "Scalable Purification Methods". This is described in more detail in WO2017 / 160360 entitled "AAV9-based rAAV9-based A ... In one example, for an affinity chromatography step, the diafiltered product may be applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which efficiently captures the AAV2 / 9 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, and the AAV particles are efficiently captured.

[0087] Conventional methods for characterization or quantification of rAAV are available to those skilled in the art. To calculate the content of empty and filled particles, the VP3 band volume for a selected sample (e.g., in the examples herein, a preparation purified on an iodixanol gradient, where the number of GC = the number of particles) is plotted against the GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test article peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL to GC / mL gives empty pt / mL. The percentage of empty particles is obtained by dividing empty pt / mL by pt / mL and multiplying by 100. In general, methods for assaying AAV vector particles containing empty capsids and packaged genomes are known in the art. For example, Grimm et al., Gene Therapy (1999) 6:1322-1330, Sommer et al. See Wobus et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting the cured AAV stock to SDS-polyacrylamide gel electrophoresis (e.g., gradient gels containing 3-8% Tris-acetate in buffer) of any gel capable of separating the three capsid proteins, then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used which binds to the primary antibody and includes a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently attached to the antibody, most preferably a sheep anti-mouse IgG antibody covalently attached to horseradish peroxidase. To semi-quantitatively determine binding between the primary and secondary antibodies, a method for detecting binding, preferably a radioisotope detector, is used. A detection method capable of detecting radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit, is used. For example, in SDS-PAGE, samples from the column fractions can be taken and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins were resolved in a precast gradient polyacrylamide gel (e.g., Novex). Silver staining can be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or other suitable staining methods, i.e., SYPRO Ruby or Coomassie staining. In one embodiment, the concentration of AAV vector genome (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the sample is further diluted and amplified using a TaqMan™ fluorogenic probe specific for the primers and the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in a Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR can also be used.

[0088] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serine protease, such as proteinase K (e.g., commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitably, the sample is diluted with an amount of proteinase K buffer equal to the sample size. The proteinase K buffer may be concentrated 2-fold or more. Typically, the proteinase K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally carried out at about 55° C. for about 15 minutes, but may be carried out at lower temperatures (e.g., about 37° C. to about 50° C.) for longer periods (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60° C.) for shorter periods (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95° C. for about 15 minutes, but may be performed at lower temperatures (e.g., about 70 to about 90° C.) and for longer periods (e.g., about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0089] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al., Hu Gene. See Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0090] Methods for determining the ratio of capsid proteins vp1, vp2, and vp3 are also available. See, for example, Vamseedhar Rayaprolu et al, Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics, J Virol. 2013 Dec; 87(24): 13150-13160, Buller RM, Rose JA. 1978. Characterization of adenovirus-associated virus-induced polypeptides See in KB cells. J. Virol. 25:331-338, and Rose JA, Maizel JV, Inman JK, Shatkin AJ. 1971. Structural proteins of adenovirus-associated viruses. J. Virol. 8:766-770.

[0091] As used herein, the term "treatment" or "treating" refers to compositions and / or methods for the purpose of alleviating one or more symptoms of NPA disease, restoring a desired function of hSmpd1, or improving a biomarker of the disease. In some embodiments, the term "treatment" or "treating" is defined to encompass administering to a subject one or more compositions described herein for the purposes set forth herein. Thus, "treatment" may include one or more of reducing the onset or progression of NPA disease, preventing the disease, reducing the severity of symptoms of the disease, slowing its progression, eliminating symptoms of the disease, slowing the progression of the disease, or increasing the effectiveness of a therapy in a given subject.

[0092] It is to be understood that the compositions in the rAAV described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout the specification.

[0093] 5. Pharmaceutical Compositions or Formulations In certain embodiments, provided herein is a pharmaceutical composition comprising a vector (e.g., rAAV) described herein in a formulation buffer. In certain embodiments, the pharmaceutical composition is suitable for co-administration with a functional hSmpd1 protein. In one embodiment, provided herein is a pharmaceutical composition comprising a rAAV described herein in a formulation buffer. In certain embodiments, the rAAV is at about 1×10 9 Genome copies (GC) / mL ~ approx. 1 x 10 14 In a further embodiment, the rAAV is formulated at about 3×10 9 GC / mL ~ approx. 3×10 13 In yet a further embodiment, the rAAV is formulated at about 1×10 9 GC / mL ~ approx. 1×10 13 In one embodiment, the rAAV is formulated at a concentration of at least about 1×10 11 Formulated in GC / mL.

[0094] In certain embodiments, the pharmaceutical composition comprises an expression cassette comprising the hSmpd1 coding sequence in a non-viral or viral vector system, which may include, for example, naked DNA, naked RNA, inorganic particles, lipid or lipid-like particles, chitosan-based formulations, and others known in the art and described, for example, by Ramamoorth and Narvekar, cited above. Such non-viral vector systems may include, for example, plasmids or non-viral genetic elements, or protein-based vectors.

[0095] In certain embodiments, the pharmaceutical composition comprises a non-replicating viral vector.Suitable viral vectors can include any suitable delivery vector, such as recombinant adenovirus, recombinant lentivirus, recombinant bocavirus, recombinant adeno-associated virus (AAV) or another recombinant parvovirus.In certain embodiments, the viral vector is recombinant AAV for delivering hSmpd1 to patients in need thereof.

[0096] In one embodiment, the pharmaceutical composition comprises a vector comprising an expression cassette comprising an hSmpd1 coding sequence and a formulation buffer suitable for delivery via intracerebroventricular (ICV), intrathecal (IT), intracisternal, or intravenous (IV) injection. In one embodiment, the expression cassette comprising the hSmpd1 coding sequence is packaged into a recombinant AAV.

[0097] In one embodiment, the pharmaceutical composition is for delivery to a subject as an enzyme replacement therapy (ERT). The pharmaceutical composition comprises a functional hSmpd1 polypeptide or a functional fragment thereof. Such pharmaceutical compositions are usually administered intravenously, but in some circumstances can also be administered intradermally, intramuscularly, or orally. The composition can be administered for prophylactic treatment of individuals suffering from or at risk of NPA disease. For therapeutic applications, the pharmaceutical composition is administered to patients suffering from established disease in an amount sufficient to reduce the concentration of accumulated metabolites and / or prevent or inhibit further accumulation of metabolites. For individuals at risk of lysosomal enzyme deficiency, the pharmaceutical composition is administered prophylactically in an amount sufficient to prevent or inhibit accumulation of metabolites. The pharmaceutical composition comprising the hSmpd1 protein described herein is administered in a therapeutically effective amount. Generally, the therapeutically effective amount can vary depending on the severity of the medical condition in the subject, as well as the age, general condition, and sex of the subject. The dosage can be determined by a physician and can be adjusted as necessary to suit the observed therapeutic effect. In one aspect, provided herein is a pharmaceutical composition for ERT formulated to contain a unit dose of hSmpd1 protein or a functional fragment thereof.

[0098] In certain embodiments, the formulation further comprises a surfactant, a preservative, an excipient, and / or a buffer dissolved in the aqueous suspension. In one embodiment, the buffer is PBS. In another embodiment, the buffer is an artificial cerebrospinal fluid (aCSF), such as Elliot's formulation buffer, or Harvard apparatus perfusion solution (artificial CSF with final ionic concentrations (mM): Na150, K3.0, Ca1.4, Mg0.8, P1.0, Cl155). A variety of suitable solutions are known, including those that include one or more of buffered saline, surfactants, physiologically compatible salts or mixtures of salts adjusted to an ionic strength equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or physiologically compatible salts adjusted to equivalent ionic concentrations.

[0099] Preferably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6 to 8, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desirable, while for intravenous delivery, a pH of 6.8 to about 7.2 may be desirable. However, for other delivery routes, other pHs within the broadest ranges, and subranges thereof, may be selected.

[0100] A suitable surfactant or combination of surfactants may be selected from among non-toxic non-ionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated in a primary hydroxyl group is selected, such as Pluronic® F68 [BASF] (also known as Poloxamer 188), which has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycapric acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named using the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0101] In one embodiment, the formulation may comprise, for example, sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., MgSO 4 7H2O), potassium chloride, salt in water. The suspension may contain a buffered saline solution, including one or more of calcium chloride (e.g., calcium chloride·2H2O), dibasic sodium phosphate, and mixtures thereof. Preferably, for intrathecal delivery, the osmolality is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290), see, e.g., emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent may be used as the suspending agent, or in combination with another suspending agent and other optional excipients. See, e.g., Elliotts B® solution [Lukare Medical].

[0102] In certain embodiments, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0103] In one embodiment, a frozen composition containing rAAV in a buffer solution as described herein is provided in frozen form. Optionally, one or more surfactants (e.g., Pluronic F68), stabilizers, or preservatives are present in the composition. Suitably, for use, the composition is thawed and titrated to the desired dose with a suitable diluent, e.g., sterile saline or buffered saline.

[0104] In certain embodiments, provided herein is a pharmaceutical composition comprising a vector (e.g., rAAV) described herein and a pharma- ceutically acceptable carrier. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to introduce the compositions of the present invention into suitable host cells. Specifically, rAAV vectors can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, or the like. In one embodiment, a therapeutically effective amount of the vector is included in the pharmaceutical composition. The choice of carrier is not a limitation of the present invention. Other conventional pharma- ceutical acceptable carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0105] The phrase "pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

[0106] As used herein, the term "dosage" or "amount" can refer to the total dosage or amount delivered to a subject over the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or divided dosage) administration.

[0107] Additionally, the replication-defective virus composition may be administered to a human patient in a dose range of about 1.0×10 (to treat an average subject weighing 70 kg), including all integers or fractions within the range. 9 GC~approx. 1.0×10 16 GC range, preferably 1.0 x 1012 GC~1.0×10 14 The composition can be formulated in dosage units containing an amount of replication-defective virus in the range of GC. In one embodiment, the composition contains at least 1 x 10 per dose, including all integers or decimals within the range. 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , Or 9 x 10 9 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9 x 10 10 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9 x 10 11 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9 x 10 12In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , or 9 x 10 13 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9 x 10 14 In another embodiment, the composition is formulated to contain at least 1 x 10 GC per dose, including all integers or decimals within the range. 15 , 2×10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9 x 10 15 In one embodiment, for human applications, the dose is formulated to contain 1×10 per dose, including all integers or fractions within the range. 10 ~Approx. 1×10 12 It may be in the GC range.

[0108] In certain embodiments, a pharmaceutical composition is provided comprising the rAAV described herein in a formulation buffer. In one embodiment, the rAAV is at about 1×10 9 Genome copies (GC) / mL ~ approx. 1 x 10 14 In a further embodiment, the rAAV is formulated at about 3×10 9 GC / mL ~ approx. 3×10 13In yet a further embodiment, the rAAV is formulated at about 1×10 9 GC / mL ~ approx. 1×10 13 In one embodiment, the rAAV is formulated at a concentration of at least about 1×10 11 In one embodiment, the pharmaceutical composition comprising the rAAV described herein is formulated at about 1×10 GC / mL per gram of brain mass. 9 GC ~ approximately 1 x 10 per gram of brain mass 14 It is administered in the dose of GC.

[0109] In certain embodiments, the compositions may be formulated in a suitable aqueous suspension medium (e.g., buffered saline) for delivery by any suitable route. The compositions provided herein are useful for systemic delivery of high doses of viral vectors. For rAAV, a high dose is at least 1×10 13 GC, or at least 1 × 10 14 However, for improved safety, the miRNA sequences provided herein can be included in expression cassettes and / or vector genomes delivered at other lower doses.

[0110] The aqueous suspension or pharmaceutical composition described herein is designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. In one embodiment, the pharmaceutical composition is formulated for delivery via intracerebroventricular (ICV), intrathecal (IT), or intracisternal injection. In one embodiment, the composition described herein is designed for delivery to a subject in need thereof by intravenous (IV) injection. Alternatively, other routes of administration (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, and other parenteral routes) may be selected. In certain embodiments, the composition is delivered by two different routes essentially simultaneously.

[0111] As used herein, the term "intracerebrovascular delivery" or "intracerebrovascular administration" refers to the route of administration of a drug via injection into the spinal canal, and more specifically into the subarachnoid space to reach the cerebrospinal fluid (CSF). Intrathecal delivery can be via lumbar puncture, intraventricular tap, suboccipital / intracisternal tap, or intrathecal administration. Methods for administering intrathecal delivery may include intrathecal puncture, intrathecal puncture, and / or C1-2 puncture. For example, the material may be introduced by lumbar puncture for diffusion throughout the subarachnoid space. Another example may be injection into the cisterna magna. Intracisternal delivery may increase vector diffusion and / or reduce toxicity and inflammation caused by administration. See, for example, Christian Hinderer et al. al,Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna,Mol Ther Methods Clin Dev.2014;1:14051.Published online 2014 Dec 10.doi:10.1038 / mtm.2014.51.

[0112] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to the route of administration of a drug directly into the cerebrospinal fluid of the ventricles of the brain or into the cerebellum-medullary cistern, more specifically, via a suboccipital puncture or via direct injection or permanently placed tube into the cisterna magna.

[0113] It is to be understood that the compositions in the pharmaceutical compositions described herein are intended to apply to other compositions, regimens, aspects, embodiments, and methods described throughout the specification.

[0114] 6.Treatment method Provided herein is a method for NPA disease, comprising delivering a therapeutically effective amount of a nucleic acid sequence or expression cassette comprising an hSmpd1 coding sequence, as provided herein. Specifically, the method comprises preventing, treating, and / or alleviating symptoms of NPA disease by delivering a therapeutically effective amount of rAAV.hSmpd1, or a composition comprising an hSmpd1 polypeptide as described herein, to a patient in need thereof. In certain embodiments, a composition comprising an expression cassette as described herein is administered to a subject in need thereof. In certain embodiments, the expression cassette is delivered via rAAV.

[0115] As used herein, a "therapeutically effective amount" refers to an amount of a composition that delivers an amount of hSmpd1 sufficient to alleviate or treat one or more of the NPA diseases. "Treatment" may include preventing the worsening of symptoms of NPA disease and possibly reversing one or more of those symptoms. A "therapeutically effective amount" for a human patient may be predicted based on animal models. See C. Hinderer et al, Molecular Therapy (2014); 22 12, 2018-2027, A. Bradbury, et al, Human Gene Therapy Clinical Development. March 2015, 26(1): 27-37, which are incorporated herein by reference.

[0116] In certain embodiments, treating includes preventing, treating, and / or alleviating one or more symptoms of NPA.

[0117] In certain embodiments, the treatment involves replacing or supplementing the patient's deficient SMPD1 via rAAV-based gene therapy. When expressed from the rAAV vectors described herein, expression levels of at least about 5% of the normal levels detected in the brain, spleen, liver, or other tissues or bodily fluids can provide a therapeutic effect. However, higher expression levels can be achieved. Such expression levels can be from about 5% to about 100% of normal functional human SMPD1 levels. In certain embodiments, higher than normal expression levels can be detected in serum, or another bodily fluid or tissue.

[0118] Suitable volumes for delivery of the provided compositions and concentrations thereof can be determined by one of skill in the art. For example, a volume of about 1 μL to 150 mL may be selected, with higher volumes being selected for adults. Typically, for newborns, a suitable volume may be selected from about 0.5 mL to about 10 mL, and for older infants, from about 0.5 mL to about 15 mL. For infants, a volume of about 0.5 mL to about 20 mL may be selected. For children, a volume of up to about 30 mL may be selected. For pre-teens and teenagers, a volume of up to about 50 mL may be selected. In yet other embodiments, patients may receive intrathecal administration in a volume of about 5 mL to about 15 mL, which may be selected, or may receive about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined. Dosages are adjusted to balance the therapeutic benefit against any side effects, and such dosages may vary depending on the therapeutic application for which the recombinant vector is used.

[0119] In certain embodiments, a composition comprising an rAAV described herein contains about 1×10 9 GC ~ approximately 1 x 10 per gram of brain mass 14 In certain embodiments, the rAAV is administered at a dose of about 1 x 10 GC / kg body weight. 9 GC ~ approx. 1 x 10 per kg of body weight 13 It is co-administered systemically at the GC dose.

[0120] In certain embodiments, the expression cassette is present in the vector genome at a concentration of about 1×10 per gram of brain mass, including all integers or decimals and endpoints within the range. 9 GC ~ approximately 1 x 10 per gram (g) of brain mass 13 In another embodiment, the dosage is delivered in an amount of 1×10 genome copies (GC) per gram of brain mass. 10 GC ~ approximately 1 x 10 per gram of brain mass 13 In certain embodiments, the dose of the vector administered to the patient is at least about 1.0x10 9 GC / g, approx. 1.5x10 9 GC / g, approx. 2.0x10 9 GC / g, approx. 2.5x10 9 GC / g, approx. 3.0x10 9 GC / g, approx. 3.5x10 9 GC / g, approx. 4.0x10 9 GC / g, approx. 4.5x10 9 GC / g, approx. 5.0x10 9 GC / g, approx. 5.5x10 9 GC / g, approx. 6.0x10 9 GC / g, approx. 6.5x10 9 GC / g, approx. 7.0x10 9 GC / g, approx. 7.5x10 9 GC / g, approx. 8.0x10 9 GC / g, approx. 8.5x10 9 GC / g, approx. 9.0x10 9 GC / g, approx. 9.5x10 9 GC / g, approx. 1.0x10 10 GC / g, approx. 1.5x10 10 GC / g, approx. 2.0x10 10 GC / g, approx. 2.5x10 10 GC / g, approx. 3.0x10 10 GC / g, approx. 3.5x10 10 GC / g, approx. 4.0x10 10 GC / g, approx. 4.5x10 10 GC / g, approx. 5.0x10 10 GC / g, approx. 5.5x10 10 GC / g, approx. 6.0x10 10 GC / g, approx. 6.5x10 10GC / g, about 7.0x10 10 GC / g, about 7.5x10 10 GC / g, about 8.0x10 10 GC / g, about 8.5x10 10 GC / g, about 9.0x10 10 GC / g, about 9.5x10 10 GC / g, about 1.0x10 11 GC / g, about 1.5x10 11 GC / g, about 2.0x10 11 GC / g, about 2.5x10 11 GC / g, about 3.0x10 11 GC / g, about 3.5x10 11 GC / g, about 4.0x10 11 GC / g, about 4.5x10 11 GC / g, about 5.0x10 11 GC / g, about 5.5x10 11 GC / g, about 6.0x10 11 GC / g, about 6.5x10 11 GC / g, about 7.0x10 11 GC / g, about 7.5x10 11 GC / g, about 8.0x10 11 GC / g, about 8.5x10 11 GC / g, about 9.0x10 11 GC / g, about 9.5x10 11 GC / g, about 1.0x10 12 GC / g, about 1.5x10 12 GC / g, about 2.0x10 12 GC / g, about 2.5x10 12 GC / g, about 3.0x10 12 GC / g, about 3.5x10 12 GC / g, about 4.0x10 12 GC / g, about 4.5x10 12 GC / g, about 5.0x10 12 GC / g, about 5.5x10 12 GC / g, about 6.0x10 12 GC / g, about 6.5x10 12 GC / g, about 7.0x10 12 GC / g, about 7.5x10 12 GC / g, about 8.0x10 12 GC / g, about 8.5x10 12GC / g, approximately 9.0x10 12 GC / g, approximately 9.5x10 12 GC / g, approximately 1.0x10 13 GC / g, approximately 1.5x10 13 GC / g, approximately 2.0x10 13 GC / g, approximately 2.5x10 13 GC / g, approximately 3.0x10 13 GC / g, approximately 3.5x10 13 GC / g, approximately 4.0x10 13 GC / g, approximately 4.5x10 13 GC / g, approximately 5.0x10 13 GC / g, approximately 5.5x10 13 GC / g, approximately 6.0x10 13 GC / g, approximately 6.5x10 13 GC / g, approximately 7.0x10 13 GC / g, approximately 7.5x10 13 GC / g, approximately 8.0x10 13 GC / g, approximately 8.5x10 13 GC / g, approximately 9.0x10 13 GC / g, approximately 9.5x10 13 GC / g, or approximately 1.0x10 14 GC / g brain mass.

[0121] In certain embodiments, the compositions provided herein are administered in combination with an immunosuppressant.Currently, immunosuppressants for such combination therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalogs), and cytostatic agents (including alkylating agents), antimetabolites, cytotoxic antibiotics, antibodies, and substances active against immunophilins.Immunosuppressants may include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25)-specific antibodies or CD3-specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-alpha) binding agents. In certain embodiments, immunosuppressive therapy may be initiated 0, 1, 2, 7 days, or earlier prior to gene therapy administration. Such therapy may involve co-administration of two or more drugs (e.g., prednisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued at the same dose or at an adjusted dose after gene therapy administration.

[0122] In certain embodiments, the rAAV provided herein is administered in combination with a therapy (combination therapy), such as enzyme replacement therapy (e.g., SMPD1 (also known as ASMase) enzyme replacement), chaperone therapy, substrate reduction therapy, and / or in combination with an antihistamine or other agent that reduces the likelihood of an infusion-related reaction. In certain embodiments, the combination therapy is a functional hSmpd1 protein. Administration can be by oral administration or intravenous infusion on an outpatient basis and can include dosages suitable for daily, every other day, weekly, biweekly (e.g., 0.2 mg / kg body weight), monthly, or bimonthly administration. Appropriate therapeutically effective dosages of the combination therapy are selected by the treating clinician and include from about 1 μg / kg to about 500 mg / kg, from about 10 mg / kg to about 100 mg / kg, from about 20 mg / kg to about 100 mg / kg, and from about 20 mg / kg to about 50 mg / kg. In some embodiments, a suitable therapeutic dose is selected from, for example, 0.5, 0.75, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 100, 150, 200, 250, 300, 400, or 500 mg / kg.

[0123] In certain embodiments, newborns (3 months of age or younger) are treated according to the methods described herein. In certain embodiments, infants between 3 months and 9 months of age are treated according to the methods described herein. In certain embodiments, children between 9 months and 36 months of age are treated according to the methods described herein. In certain embodiments, children between 3 and 12 years of age are treated according to the methods described herein. In certain embodiments, children between 12 and 18 years of age are treated according to the methods described herein. In certain embodiments, adults 18 years of age or older are treated according to the methods described herein.

[0124] In one embodiment, the patient with NPA disease is a male or female child at least about 3 months of age and under 12 months of age. In another embodiment, the patient with NPA disease is male or female and at least about 6 years of age and up to 18 years of age. In other embodiments, the subject may be older or younger, male or female.

[0125] In certain embodiments, the present invention provides for the use of the rAAV described herein in the preparation of a medicament for treating Niemann-Pick A and / or ameliorating symptoms thereof by ameliorating weight loss or cachexia, ameliorating loss of motor function, ameliorating loss of cognitive function, and prolonging survival. Provided in the fine print.

[0126] 7. Devices and Kits In one aspect, the vectors provided herein may be administered intrathecally, for example, via the methods and / or devices described in WO2017 / 136500 (incorporated herein by reference in its entirety). Alternatively, other devices and methods may be selected. In summary, the method includes the steps of advancing a spinal needle into the patient's cisterna magna, connecting a length of flexible tubing to the proximal hub of the spinal needle and connecting the outlet port of a valve to the proximal end of the flexible tubing, and after the advancing and connecting steps, allowing the tubing to self-prime with the patient's cerebrospinal fluid, connecting a first container containing a quantity of isotonic solution to the flush inlet port of the valve, and then connecting a second container containing a quantity of a pharmaceutical composition to the vector inlet port of the valve. After connecting the first and second containers to the valve, a passage for fluid flow is opened between the vector inlet port and the outlet port of the valve, and the pharmaceutical composition is injected into the patient through the spinal needle; after injection of the pharmaceutical composition, a passage for fluid flow is opened through the flush inlet port and the outlet port of the valve, and an isotonic solution is injected into the spinal needle to flush the pharmaceutical composition into the patient. This method and device can each optionally be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices can be used for such intrathecal delivery.

[0127] In certain embodiments, kits are provided that include concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intravenous, intrathecal, intraventricular, or intravesical administration. In one embodiment, the kit provides sufficient buffer to allow for injection. Such buffer may allow for about 1:1 to 1:5 dilution of the concentrated vector, or more. Such kits may include additional non-vector-based active ingredients with which combination therapy is utilized, and / or antihistamines, immunomodulators, and the like. In other embodiments, more or less buffer or sterile water is included to allow for dose titration and other adjustments by the treating physician. In still other embodiments, the kit includes one or more components of the device. Suitable dilution buffers, such as saline, phosphate buffered saline (PBS), or glycerol / PBS, are available.

[0128] It is to be understood that the compositions in the kits described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout the specification.

[0129] As used herein, the term "biological sample" refers to any cell, biological fluid, or tissue. Suitable samples for use in the present invention may include, but are not limited to, whole blood, white blood cells, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid, and skin cells. Such samples may be further diluted with saline, buffer, or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means.

[0130] With respect to these descriptions of the invention, each of the compositions described herein is contemplated, in separate embodiments, to be useful in the methods of the invention. In addition, each of the compositions described herein that are useful in the methods is also contemplated, in separate embodiments, to be an embodiment of the invention itself.

[0131] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published documents which provide such persons with general guidance to many of the terms used in this application.

[0132] As used herein, "patient" or "subject" refers to male or female humans and animal models used in clinical studies. In certain embodiments, the subject of these methods and compositions is a human diagnosed with NPA disease. In further embodiments, the human subject of these methods and compositions is a prenatal, neonatal, infant, toddler, preschooler, elementary school student, teen, young adult, or adult.

[0133] "Comprising" is a term meaning to include other components or method steps. When "comprising" is used, it should be understood that the relevant embodiment includes statements using the term "consisting of" to exclude other components or method steps, and the term "consisting essentially of" to exclude any components or method steps that materially change the nature of the embodiment or invention. Although various embodiments herein are described using the language "comprising," it should be understood that under various circumstances, the relevant embodiment may also be described using the language "consisting of" or "consisting essentially of."

[0134] References to "one embodiment," "another embodiment," or "a particular embodiment" when describing an embodiment do not imply that the referenced embodiment is mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment), unless expressly specified otherwise.

[0135] It should be noted that the terms "a" or "an" refer to one or more; for example, "an expression cassette" is understood to refer to one or more expression cassettes. Thus, "a" (or "an"), "one or more" The terms "one or more" and "at least one" are used interchangeably herein.

[0136] As used herein, the term "about" means a plus or minus 10% variation from a given reference, unless otherwise specified.

[0137] It is to be understood that the compositions in the devices described herein are intended to apply to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification. EXAMPLES

[0138] The invention will now be described with reference to the following examples, which are provided for illustrative purposes only and should in no way be construed as limiting the invention to these examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0139] Example 1: rAAV.hSMPD1 for treatment of NPC Cis-plasmids containing vector genomes were generated by cloning the hSMPD1V36A coding sequence of SEQ ID NO:4 or hSMPD1 with a V at position 36 of SEQ ID NO:5. The vector genomes were designed to have a truncated AAV2 (130 bp) 5'ITR at the extreme 5' end of the vector genome, an expression cassette, and a truncated AAV2 (130 bp) 3'ITR at the extreme 3' end of the vector genome. An expression cassette containing the CB7 promoter, chimeric intron, coding sequence, and rabbit beta globin polyA (CB7.CI.hSMPD1co.RBG, SEQ ID NO:8 or SEQ ID NO:9) was used to generate rAAV with the vector genome of SEQ ID NO:11 or SEQ ID NO:11. UbC An expression cassette containing the promoter, coding sequence, and SV40 polyA sequence (UbC.hSMPD1co.SV40, SEQ ID NO: 14) was used to generate rAAV with a vector genome of SEQ ID NO: 15. Recombinant AAV9 viral particles are produced by triple transfection of packaging 293 host cells with the cis plasmid described above, a trans plasmid containing the AA2 rep function and AAV capsid coding sequence, and one of the helper plasmids.

[0140] The resulting rAAV.hSMPD1 was used in the comparative studies in Example 2.

[0141] Example 2: Delivery of AAV.hSMPD1 via intracerebroventricular (ICV) injection for the treatment of NPA The Smpd1 KO mouse model was generated by CRISPR / Cas9 through the deletion of exon 2 of the mouse Smpd1 gene (Jackson Labs). The resulting animals are an excellent model of NPA disease. Similar to published models, mice are lethargic, have gait abnormalities, have CNS / respiratory / cardiac involvement, and untreated animals show significant accumulation of material in target tissues.

[0142] One-month-old Smpd1 KO mice were cultured at 6 × 10 10Smpd1 KO mice received 100 genomic copies of AAV. CB7.hSMPD1 or AAV.Ubc.hSMPD1 via ICV injection. Companion control mice received PBS. Mice were observed daily and weighed weekly. To assess motor coordination, mice were subjected to a rotarod performance test monthly. Smpd1 KO mice that received PBS showed a decrease in the latency to fall over time. This behavioral deficit was rescued by delivery of AAV. CB7.hSMPD1 or AAV.Ubc.hSMPD1 were administered to Smpd1 KO mice. Mice were euthanized 5.3 months after injection. Brain, liver, spleen, and lung tissues were harvested, fixed, and processed for histological analysis. Immunostaining on brain sections with filipin to detect cholesterol accumulation revealed abundant amounts of cholesterol in Smpd1 KO mice. The amount of accumulated cholesterol was significantly reduced in Smpd1 KO mice receiving AAV.CB7.hSMPD1 or AAV.Ubc.hSMPD1. Oil red staining was performed on liver, spleen, and lung tissues to detect the presence of lipid-filled macrophages. The presence of lipid-filled macrophages was seen in all three tissues from Smpd1 KO mice. The reduction or absence of lipid-filled macrophages was prominent in Smpd1 KO mice receiving AAV.CB7.hSMPD1 or AAV.Ubc.hSMPD1. Other measures of efficacy may include sphingomyelin quantification (liver / brain / spleen) and chitinase assays (biomarkers of disease severity).

[0143] These results are shown in Figures 4A to 4C.

[0144] Example 3: Delivery of AAV.CB76.hSMPD1 with and without DRG detargeting sequences in non-human primates An additional engineered sequence, SEQ ID NO:22, was generated (encoding SEQ ID NOs:2 and 23) and selected for use in generating additional recombinant AAV vectors. rAAVhu68.CB7.hSMPD1coV2 vectors were prepared with or without 4x miR182 targets (binding sequences). Cis plasmids were designed to contain AAV2-5'ITR, expression containing hSMPD1coV2 coding sequence (SEQ ID NO:22) with or without 4 copies (4x) miR182 targets (binding sites) (SEQ ID NO:24), and a vector genome containing AAV2-5'ITR. Cis plasmids were constructed to contain a trans plasmid encoding rep, a trans plasmid encoding the AAVhu68 VP1 capsid protein, and a trans plasmid encoding the AAVhu68 VP1 capsid protein, under the control of sequences directing their expression in 293 cells, as produced by conventional triple transfection. HEK293 packaging host cells were co-transfected with a cis plasmid containing adenovirus helper sequences for replicating and packaging the vector genome into hu68 capsids using the viable method. The resulting viral particles are referred to herein as AAVhu68.CB7.hSMPD1co (without miR binding sites) or AAV.CB7.hSMPD1co.miR-TS (with 4xmiR182 binding sites). The expression cassette for CB7.CI.hSMPDcoV2.RBG is provided in SEQ ID NO:21 and contains the CB7 promoter element (SEQ ID NO:26), hCMPD1coV2 coding sequence (SEQ ID NO:22), and rabbit globin polyA (SEQ ID NO:7). The vector genome for ITR.CB7.CI.hSMPDcoV2.RGB.ITR is provided in SEQ ID NO:20. The expression cassette for CB7.CI.hSMPDcoV2.miR-TS is provided in SEQ ID NO: 18 and contains the CB7 promoter element (SEQ ID NO: 26), hCMPD1coV2 coding sequence (SEQ ID NO: 22), 4xmiR182 binding sites (SEQ ID NO: 28), and rabbit globin polyA (SEQ ID NO: 7). The vector genome of ITR.CB7.CI.hSMPDcoV2.miR-TS contains SEQ ID NO: 18.

[0145] Rhesus monkey, 3x10 13GC AAVhu68.CB7.hSMPD1co or AAV.CB7.hSMPD1co.miR-TS was injected into the ICM. Animals were observed daily and DRG pathology was monitored by SNAP amplitude and biomarker detection via nerve conduction studies. Two months after injection, animals were necropsied and evaluated for biodistribution, ASMD expression, and DRG pathology.

[0146] (Sequence Listing Free Text) The following information is a numeric identifier <223> Below we provide an array containing free text. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0147] All patents and non-patent publications cited herein are incorporated by reference in their entirety, as well as U.S. Provisional Patent Application No. 63 / 144,103, filed February 1, 2021, U.S. Provisional Patent Application No. 63 / 286,939, filed December 7, 2022, and the sequence numbers referenced herein and depicted in the attached Sequence Listing, 21-9627PCT_NPA_ST25 (78kb), generated January 21, 2022, are incorporated by reference. While the invention has been described with reference to certain embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such variations are intended to be within the scope of the appended claims.

Claims

1. A recombinant AAV (rAAV), comprising an AAV capsid and a vector genome packaged therein, said vector genome comprising an engineered nucleic acid sequence encoding human acid sphingomyelinase (hSMPD1) operably linked to regulatory sequences directing expression of said hSMPD1, and an AAV 3' ITR, wherein said hSMPD1 coding sequence is SEQ ID NO:22, or a coding sequence at least 99% identical to SEQ ID NO:22 that encodes a functional hSMPD1 protein, SEQ ID NO:5, or a coding sequence at least 90% identical to SEQ ID NO:5 that encodes a functional hSMPD1 protein, or SEQ ID NO:4, or a coding sequence at least 90% identical to SEQ ID NO:4 that encodes a functional hSMPD1 protein.

2. The rAAV of claim 1, wherein the hSMPD1 coding sequence encodes the hSMPD1 protein having Ala or Val at position 36 with reference to SEQ ID NO:

2.

3. The hSMPD1 protein is (a) having the sequence of SEQ ID NO:2, SEQ ID NO:23, SEQ ID NO:3, or SEQ ID NO:1; or (b) comprising an exogenous leader sequence fused to a hSMPD1 protein comprising amino acids 47-631 of SEQ ID NO:2 (or SEQ ID NO:31), SEQ ID NO:23, SEQ ID NO:3, or SEQ ID NO:1, rAAV according to claim 1 or claim 2.

4. The regulatory sequence is (a) the UbC or CB7 promoter; or (b) the SV40 late or rabbit beta globin polyadenylation site; The rAAV of any one of claims 1 to 3, comprising: (a) the AAV vector genome comprises: (i) an expression cassette comprising a CB7 hybrid promoter, an intron, the hSMPD1 coding sequence, and a rabbit beta globin sequence, and optionally, four or more miR182 or miR183 binding sites; (ii) the sequence of SEQ ID NO:21, SEQ ID NO:19, SEQ ID NO:10, or SEQ ID NO:8; and / or (iii) the sequence of SEQ ID NO:20, SEQ ID NO:18, SEQ ID NO:9, or SEQ ID NO:11; contains; or (b) the AAV vector genome comprises a UbC promoter, the hSMPD1 coding sequence, and an SV40 late polyadenylation sequence; Optionally, the vector genome comprises the sequence of SEQ ID NO: 14 or 15. The rAAV according to any one of claims 1 to 4.

6. The rAAV of any one of claims 1 to 5, wherein the AAV vector genome further comprises a full-length AAV2 inverted terminal repeat sequence.

7. The rAAV of any one of claims 1 to 6, wherein the AAV capsid is an AAVhu68 capsid.

8. A pharmaceutical composition comprising a population of rAAV according to any one of claims 1 to 7 in a formulation buffer. (a) comprising the rAAV encoding the hSMPD1 having Ala at position 36, the rAAV encoding the hSMPD1 having Val at position 36, or a combination thereof; (b) suitable for co-administration with a functional hSMPD1 protein; and / or (c) formulated for delivery via intracerebroventricular (ICV), intrathecal (IT), intracisternal, or intravenous (IV) injection; 9. The pharmaceutical composition of claim 8.

10. 10. The rAAV of any one of claims 1 to 7, or the pharmaceutical composition of claim 8 or claim 9, for use in treating and / or ameliorating the symptoms of Niemann-Pick disease type A by ameliorating weight loss or cachexia, ameliorating loss of motor function, ameliorating loss of cognitive function, or prolonging survival.

11. A nucleic acid molecule comprising an engineered nucleic acid sequence encoding human acid sphingomyelinase (hSMPD1) operably linked to a sequence controlling expression of said hSMPD1, wherein the hSMPD1 coding sequence is SEQ ID NO:22, or a coding sequence at least 90% identical to SEQ ID NO:22 that encodes a functional hSMPD1, SEQ ID NO:4, or a coding sequence at least 90% identical to SEQ ID NO:4 that encodes a functional hSMPD1, or SEQ ID NO:5, or a coding sequence at least 90% identical to SEQ ID NO:5 that encodes a functional hSMPD1 protein.

12. A viral or non-viral vector comprising the nucleic acid molecule of claim 11.

13. A plasmid comprising the nucleic acid molecule of claim 11.

14. 14. The plasmid of claim 13, comprising a vector genome for packaging into an rAAV, the vector genome comprising an operational nucleic acid sequence, a regulatory sequence, and comprising a 5' inverted terminal repeat (ITR) and a 3' ITR at the extreme 5' and 3' ends of the vector genome, respectively.

15. 15. A packaging host cell comprising the plasmid of claim 13 or claim 14, AAV rep and cap coding sequences operably linked to regulatory sequences which direct their expression, and helper functions which enable replication and packaging of the vector genome into the AAV capsid.

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