Compositions and methods for the treatment of lysosomal acid lipase deficiency (LAL-d)

Abstract

The present disclosure relates to gene therapy compositions and methods for the preparation and use thereof, for the treatment of Lysosomal Acid Lipase Deficiency (LAL-D) disorders, such as Wolman Disease and cholesterol ester storage disease (CESD).

Description

[0001] COMPOSITIONS AND METHODS FOR THE TREATMENT OF LYSOSOMAL ACID LIPASE DEFICIENCY (LAL-D)

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 676,736 filed July 29, 2024, which is incorporated by reference herein in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 2, 2025, is named PAT059516-WO-PCT_ST26_SQL.xml and is 155,582 bytes in size.

[0006] BACKGROUND OF THE INVENTION

[0007] Lysosomal acid lipase (LAL) plays a central role in intracellular lipid metabolism (Kyriakides EC, et al., Pediatrics, 1970; 46:431-436; Yoshida H, etal. Lab Anim Sci. 1990; 40:486-489). LAL is a 399-amino-acid protein that is expressed by all cell types and encoded by the Lipase A (LIPA) gene on chromosome 10 (q23 ,2-q23.3) . LAL hydrolyses cholesteryl esters and triglycerides in human lysosomes, generating free cholesterol and free fatty acids (Pericleous etal., The Lancet Gastroenterology & Hepatology (2017)). Cholesteryl esters are derived from lipoproteins or stored in lipid droplets and are hydrolyzed mainly within the lysosomal compartment by LAL (Goldstein et al., J. of Biol. Chem (1975); Li & Zhang, Arterioscler Thromb Vase Biol (2019)). LAL-mediated lipid catabolism leads to the release of free cholesterol and free fatty acids. Hydrolysis of cholesteryl esters and the transfer of free cholesterol for cytosolic storage as lipid droplets contribute to the formation of foam cells. Hydrolysis of cholesteryl esters and triglycerides also releases free cholesterol and free fatty acids for membrane assembly and energy production. LAL-derived lipolytic products mediate macrophage M2 activation fueled by fatty acid oxidation, lipid mediator production and hepatic very low-density lipoprotein (VLDL) secretion. In hepatocytes and macrophage foam cells, LAL mediates degradation of lipid droplets through an autophagic process. LAL hydrolyzes aggregates of low-density lipoprotein (LDL) in extracellular, acidic and lytic compartments independent of endocytic mechanisms (Zhang, 2018) and potentially contributes to the immune -metabolic reprogramming of immune cells (Gomaraschi et al., Trends in Pharmacological Sciences (2019)). Lysosomal Acid Lipase Deficiency (LAL-D) is a rare, heterogenous, inborn error of metabolism caused by autosomal recessive mutations in the LIPA gene that result in a failure of the LAL protein to sufficiently hydrolyze cholesteryl esters and triglycerides into free fatty acids in the lysosome. LAL-D is characterized by accumulation of cholesteryl esters and triglycerides in the body including the liver, spleen, and macrophages. Affected individuals are either homozygous or compound heterozygous for LIPA mutations, and more than 100 LIPA mutations have been identified (Pisciotta L et a , Atherosclerosis 2017; 265: 124-132) with observed or predicted pathogenic impact (Carter et al., 2019), which mainly include missense and frameshift mutations as well as splicing errors and deletions. The most commonly inherited defect is a splice junction mutation in exon 8 (rsl 16928232: c.894G>A, p.S275_Q298del), which is referred to as E8SJM (Klima etal., 1993), resulting in a mutant enzyme that retains 2-5% of normal activity in patient cells (review in Pericleous et al., 2017). It is assumed that 50-70% of adults and children with LAL-D have E8SJM (Muntoni S, et al., Arteriosclerosis, Thrombosis, and Vascular Biology. 2007; 27: 1866-1868; Lohse P, et al., J. Lipid Res., 2000; 41:23-31).

[0008] The true prevalence of LAL-D is unknown, but is estimated to be between 1:40,000 to 1:300,000 (Pastores et al., Drug Des Devel Ther, 14: 591-601, 2020). LAL- D is presented across a clinical spectrum from infancy to adulthood. The extent of tissue deposition of cholesteryl esters and triglycerides appears to be directly proportional to the severity of the disease and inversely proportional to the age of presentation. There are two types, infantile LAL-D and late-onset LAL-D. Infantile LAL-D, also referred to as Wolman Disease, is the most severe form and patients have the lowest level of LAL activity. Wolman Disease affects infants within the first year of life and is generally fatal. Common presenting symptoms of Wolman Disease include massive hepatosplenomegaly, acute liver failure (typically at under 4 months of age), microvesicular steatosis, malabsorption, failure to thrive, adrenal calcification, and gastrointestinal disturbances.

[0009] Late-onset LAL-D, referred to as cholesteryl ester storage disease (CESD), affects both children and adults and is less severe than Wolman Disease but still causes severe clinical symptoms. Common presenting symptoms of CESD include dyslipidemia (elevated LDL-C, low HDL-C), elevated liver transaminases, hepatomegaly, hepatosplenomegaly, splenomegaly, accelerated atherosclerosis, abnormal hepatic function, hepatic fibrosis and cirrhosis, portal hypertension, decompensated liver disease, and gastrointestinal disturbances (Pericleous et a , 2017). Clinical features can be subtle, leading to misdiagnosis of patients as having Niemann- Pick disease, Gaucher disease, metabolic dysfunction associated steatotic liver disease (MASLD), metabolic dysfunction associated steatohepatitis (MASH), hereditary dyslipidaemia, or cryptogenic cirrhosis. Both infant and later onset LAL-D are difficult to diagnose due to the current lack of newborn screening, the rarity of LAL-D, and its similarity to other more common conditions. It is unclear how many fatalities are due to undiagnosed LAL-D and there is currently no consensus on routine testing for LAL activity in adults presenting with MASLD (Kohli R. et al., Molecular Genetics and Metabolism (2020)).

[0010] There is currently no curative therapy for LAL-D. Sebelipase alfa (Kanuma®), a recombinant human lysosomal acid lipase, was approved by the FDA in 2015 as an enzyme replacement therapy (ERT) and is the only FDA-approved treatment for LAL- D. However, ERT has limited efficacy and a high patient burden (Burton et al., N Engl J Med, 373: 1010-1020, 2015). In the phase 3 clinical trial for Sebelipase alfa, elevations in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, and low-density lipoprotein (LDL)-cholesterol, were, on average, reduced only by 50%, at best, with a 32% overall reduction in liver fat content and a 6.8% reduction in spleen volume (Burton et al., 2015). Further, the mortality rate of infants treated with sebelipase alpha was 4 / 9 (44.4%) at 24 months demonstrating that ERT alone is unable to ensure the survival of infants with LAL-D (Jones et al., Orphanet J. of Rare Diseases (2017)). Moreover, due to its short half-life in serum, ERT treatment must be administered through intravenous (IV) infusion every two weeks for children and adults, and up to twice a week in infants. Burdensome life-long treatment with enzyme replacement is recommended. Also, ERT alone does not address key aspects of the disease, such as systemic consequences including gastrointestinal and immune system complications that lead to high morbidity and contribute towards early mortality, particularly in patients with the infantile-onset form ofLALD (Jones etal., 2017). Prior to ERT, the only other treatment options available for individuals with LAL-D include dietary substrate reduction (DSR), lipid lowering medication, liver transplantation, and stem cell transplantation, none of which are able to correct the multisystem nature of the disorder. Even with the available treatments, death still occurs in patients with infantileonset Wolman Disease, and while CESD in later-onset patients is less severe, patients must endure strict lifelong dietary restriction and intestinal problems and the disease may still progress to liver failure and require liver transplantation. Gene therapies, such as AAV, is a favorable approach for the treatment of LAL- D and have the potential to provide liver-directed therapy with enzyme secretion that can address both hepatic and systemic disease. LAL-D is a monogenic disease known to be due to genetic abnormalities in the LIPA gene, leading to a deficiency of LAL protein. Therefore, there is a clear target for single-gene replacement, that is expected to be fundamentally disease modifying. Additionally, there is a wide variety of recurrent and de novo mutations that require a genotype agnostic approach such as gene replacement.

[0011] AAV shows a high propensity to enter tissues after intravenous (IV) delivery to the blood, allowing for systemic multi-organ perfusion of the designed gene therapy using a single dose scheme (Zygmunt et al., Mol Ther Methods Clin Dev, 15: 305-319, 2019). In theory, a single dose could last, at least in post-mitotic cells, for the lifetime of the animal (Chicoine et al., Mol Ther, 22: 713-724, 2014; Martin et al ., Am J Physiol Cell Physiol, 296: C476-488, 2009). AAV is unique in its safety profile, as the viral genome, once transduced into its carrier cell, remains stably expressed as an episomal DNA and only very rarely ever integrates into the host genome (Grieger et al., Adv Biochem Eng Biotechnol 99: 119-145, 2005; Xiao et al. J. Virol 'll'. 2224-2232, 1998). Liver and spleen are the most highly perfused organs when AAV serotypes, including AAV9, are delivered intravenously and typically receive logarithmically higher numbers of AAV DNA vector genomes (vg) than other organs when adult animals are dosed (Bish et al., Hum Gene Ther, 19: 1359-1368, 2008; Cunningham et al., Methods Mol Biol, 1937: 213-219., 2019; Palaschak et al., Methods Mol Biol, 1950: 333-360, 2019). Such features make AAV an ideal gene delivery method for treatment of genetic disorders such as LAL-D, where liver and spleen are the most affected organs (Burton etal., 2015). However, there is currently no gene therapy in development for LAL-D. Therefore, there is a high unmet need for additional therapies for the treatment of LAL-D.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention provides gene replacement therapy (e.g., polynucleotides, recombinant expression vectors, rAAV particles, etc.) for the treatment of diseases and disorders associated with impaired or deficient lysosomal acid lipase (LAL) expression, function and / or activity (e.g., LAL-D (e.g., Wolman Disease, CESD). Pharmaceutical compositions and methods of using the gene replacement therapy (e.g., polynucleotides, recombinant expression vectors, rAAV particles, etc.) are also provided. In one aspect, the invention provides polynucleotides comprising, optionally from 5’ to 3 ’ : a) an intron sequence, wherein the intron sequence is selected from a simian virus 40 (SV40) intron, a minute virus of mice (MVM) intron, or a RK intron sequence, or a functional variant or fragment thereof; and b) a lipase A (LIPA) transgene sequence, or a biologically active fragment thereof. In some embodiments, any of the polynucleotides described herein further include a promoter sequence. In some embodiments, the promoter sequence is a ubiquitous or a tissue specific promoter sequence. In some embodiments, the tissue specific promoter sequence is a liver tissue specific promoter sequence. In some embodiments, the promoter sequence is selected from an alpha- 1 antitrypsin (AAT) promoter or a chicken P-actin (CBA) promoter sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter).

[0014] In another aspect, the invention provides polynucleotides comprising, optionally from 5’ to 3’: a) a promoter sequence, wherein the promoter sequence is selected from an alpha- 1 antitrypsin (AAT) promoter or a chicken P-actin (CBA) promoter sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); b) an intron sequence, wherein the intron sequence is selected from a simian virus 40 (SV40) intron, a minute virus of mice (MVM) intron, or a RK intron sequence, or a functional variant or fragment thereof; and c) a lipase A (LIPA) transgene sequence, or a biologically active fragment thereof.

[0015] In some embodiments, the promoter sequence and the intron sequence are selected from any one of the following: a) a CBA promoter sequence and a SV40 intron sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g, retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); b) an AAT promoter sequence and a SV40 intron sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) an AAT1promoter sequence and a MVM intron sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); or d) an AAT promoter sequence and a RK intron sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter).

[0016] In some embodiments, the promoter sequence is an AAT promoter sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g, retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter). In some embodiments, the AAT promoter sequence is a human AAT (hAAT) promoter sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter). In some embodiments, the AAT promoter sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. In some embodiments, the AAT promoter sequence comprises or consists of SEQ ID NO: 9.

[0017] In some embodiments, the promoter sequence is CBA promoter sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g, retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter). In some embodiments, the CBA promoter sequence is a CB7 promoter sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter). In some embodiments, the CBA promoter sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the CBA promoter sequence comprises or consists of SEQ ID NO: 8.

[0018] In some embodiments, the intron sequence is a SV40 intron sequence. In some embodiments, the SV40 intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. In some embodiments, the SV40 intron sequence comprises or consists of SEQ ID NO: 10. In some embodiments, the SV40 intron sequence is a modified SV40 intron sequence. In some embodiments, the modified SV40 intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified SV40 intron sequence comprises at least one mutation in an ATG codon and / or the 19S acceptor site. In some embodiments, the modified SV40 intron sequence comprises at least one mutation selected from the following as compared to SEQ ID NO: 10: a) a mutation of A to T or A to G at position 79; b) a mutation of A to T at position 30; c) a mutation of G to C at position 31; and / or d) a mutation of AG to TC at positions 30 and 31. In some embodiments, the modified SV40 intron sequence comprises an A to T mutation at position 79 and AG to TC mutations at positions 30 and 31 as compared to SEQ ID NO: 10. In some embodiments, the modified SV40 intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the modified SV40 intron sequence comprises or consists of SEQ ID NO: 11. In some embodiments, the SV40 intron sequence comprises an A to T mutation at position 79 as compared to SEQ ID NO: 10. In some embodiments, the modified SV40 intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the modified SV40 intron sequence comprises or consists of SEQ ID NO: 12.

[0019] In some embodiments, the intron sequence is a MVM intron sequence, or a functional variant or fragment thereof. In some embodiments, the MVM intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the MVM intron sequence comprises or consists of SEQ ID NO: 13. In some embodiments, the MVM intron sequence is a modified MVM intron sequence. In some embodiments, the modified MVM intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified MVM intron sequence comprises at least one mutation in an ATG codon. In some embodiments, the modified MVM intron sequence comprises at least one mutation selected from the following as compared to SEQ ID NO: 13: a) a mutation of A to T, A to C, or A to G at position 16; and / or b) a mutation of G to C, G to A, or G to T at position 40. In some embodiments, the modified MVM intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the modified MVM intron sequence comprises or consists of SEQ ID NO: 14.

[0020] In some embodiments, the intron sequence is a RK intron sequence, or a functional variant or fragment thereof. In some embodiments, the RK intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the RK intron sequence comprises or consists of SEQ ID NO: 15. In some embodiments, the RK intron sequence is a modified RK intron sequence. In some embodiments, the modified RK intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified RK intron sequence comprises at least one mutation in an ATG codon. In some embodiments, the modified RK intron sequence comprises an A to G, A to C, or A to T mutation at position 78 as compared to SEQ ID NO: 15. In some embodiments, the modified RK intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the modified RK intron sequence comprises or consists of SEQ ID NO: 16.

[0021] In some embodiments, any of the polynucleotides described herein further include a splice donor site 5’ of the intron and a splice acceptor site 3’ of the intron. In some embodiments, the splice donor site and / or the splice acceptor site is modified. In some embodiments, the polynucleotide comprises a sequence 5’ (e.g., within the splice donor site) and / or 3’ (e.g., within the splice acceptor site) of the intron sequence that matches a human consensus sequence. In some embodiments, the sequence that matches the human consensus sequence at the 5’ end of the intron comprises GAG, CAG, AAC, or AAG. In some embodiments, the sequence that matches the human consensus sequence at the 3 ’ end of the intron comprises GTT, GTA, GCC, GAA, or GAT.

[0022] In some embodiments, the LIPA transgene sequence is a human LIPA transgene sequence. In some embodiments, the LIPA transgene sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the LIPA transgene sequence comprises or consists of SEQ ID NO: 6. In some embodiments, the LIPA transgene sequence is codon optimized (e.g., for enhanced LAL protein expression and / or human expression). In some embodiments, the codon optimized LIPA transgene sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the codon optimized LIPA transgene sequence comprises or consists of SEQ ID NO: 7.

[0023] In yet another aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3 ’ : a CB7 promoter nucleic acid sequence, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; and a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof.

[0024] In a further aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); a simian SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; and a. LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof. In yet a further aspect, the invention provides polynucleotide comprising, optionally from 5’ to 3’: a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; and a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof.

[0025] In one aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3’: a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; and a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof.

[0026] In another aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; and a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof.

[0027] In yet another aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); and a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; wherein the polynucleotide does not contain an intron nucleic acid sequence.

[0028] In a further aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3 ’ : a) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); b) a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon and 19S acceptor site (e.g., a mutation of A to T at position 79 and a mutation of A to T at position 30, G to C at position 31, or AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10); and c) a. LIPA transgene nucleic acid sequence, or a biologically active fragment thereof. In yet a further aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); b) a MVM intron nucleic acid sequence, or a functional variant or fragment thereof; and c) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof.

[0029] In one aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3’: a) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); b) a RK intron nucleic acid sequence, or a functional variant or fragment thereof; and c) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof.

[0030] In another aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3 ’ : a) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); b) a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon (e.g., a mutation of A to T at position 79 as compared to SEQ ID NO: 10); and c) a. LIP A transgene nucleic acid sequence, or a biologically active fragment thereof.

[0031] In yet another aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3 ’ : a) a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; b) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; c) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0032] In a further aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; b) a simian virus 40 (SV40) intron nucleic acid sequence comprising SEQ ID NO: 10; c) a lipase A (LIPA) codon optimized transgene nucleic acid sequence comprising SEQ ID NO: 7.

[0033] In yet a further aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; b) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; c) a LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0034] In one aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; b) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; and c) a LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7.

[0035] In another aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; b) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; and c) a. LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0036] In yet another aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; and b) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; wherein the polynucleotide does not contain an intron nucleic acid sequence.

[0037] In a further aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; b) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 11; and c) a LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0038] In yet a further aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; b) a MVM intron nucleic acid sequence comprising or consisting of SEQ ID NO: 14; and c) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0039] In one aspect, the invention provides a polynucleotide comprising, optionally from 5 ’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; b) a RK intron nucleic acid sequence comprising or consisting of SEQ ID NO: 16; and c) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6;

[0040] In another aspect, the invention provides a polynucleotide comprising, optionally from 5’ to 3’: a) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; b) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 12; and c) a LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6.

[0041] In some embodiments, any of the polynucleotides described herein further include a post-transcriptional regulatory element (PRE) (e.g., WPRE or HPRE) nucleic acid sequence, a polyadenylation (Poly A) signal (e.g., a PolyA signal in Table 8) nucleic acid sequence, and / or an enhancer (e.g. , CMV enhancer or Apo E HCR enhancer) nucleic acid sequence, or functional variants or fragments thereof.

[0042] In yet another aspect, the invention provides recombinant expression vectors comprising any of the polynucleotides as described herein. In a further aspect, the invention provides recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ inverted terminal repeat (ITR). b) a promoter, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter), wherein the promoter or fragment thereof is selected from an alpha- 1 antitrypsin (AA7) promoter or a chicken P-actin (CBA) promoter; c) an intron, wherein the intron is selected from a simian virus 40 (SV40) intron, a minute virus of mice (MVM) intron, or a RK intron, or a functional variant or fragment thereof; d) a lipase A (LIPA) transgene, or a biologically active fragment thereof; and 3) a 3’ ITR. In some embodiments, the promoter and the intron are selected from any one of the following: a) a CBA promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and a SV40 intron, or a functional variant or fragment thereof; b) an AAT promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and a SV40 intron, or a functional variant or fragment thereof; c) an AAT promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and a MVM intron, or a functional variant or fragment thereof; or d) an AAT promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and an RK intron, or a functional variant or fragment thereof.

[0043] In some embodiments, the promoter is an AAT promoter, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter). In some embodiments, the AAT promoter is a human AAT (hAAT) promoter, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter). In some embodiments, the AAT promoter sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. In some embodiments, the AAT promoter comprises or consists of SEQ ID NO: 9.

[0044] In some embodiments, the promoter is a CBA promoter, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter). In some embodiments, the CBA promoter sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the CBA promoter comprises or consists of SEQ ID NO: 8. In some embodiments, the intron is a SV40 intron. In some embodiments, the SV40 intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. In some embodiments, the SV40 intron comprises or consists of SEQ ID NO: 10. In some embodiments, the SV40 intron sequence is a modified SV40 intron sequence. In some embodiments, the modified SV40 intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified SV40 intron sequence comprises at least one mutation in an ATG codon and / or the 19S acceptor site. In some embodiments, the modified SV40 intron sequence comprises one or more mutations selected from the following as compared to SEQ ID NO: 10: a) A to T at position 79; b) A to T at position 30; c) a G to C at position 31; and / or d) AG to TC at positions 30 and 31. In some embodiments, the modified SV40 intron sequence comprises an A to T mutation at position 79 and AG to TC mutations at positions 30 and 31 as compared to SEQ ID NO: 10. In some embodiments, the modified SV40 intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the modified SV40 intron sequence comprises or consists of SEQ ID NO: 11. In some embodiments, the modified SV40 intron sequence comprises an A to T mutation at position 79 as compared to SEQ ID NO: 10. In some embodiments, the modified SV40 intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the modified SV40 intron comprises or consists of SEQ ID NO: 12.

[0045] In some embodiments, the intron is a MVM intron, or a functional variant or fragment thereof. In some embodiments, the MVM intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the MVM intron comprises or consists of SEQ ID NO: 13. In some embodiments, the MVM intron sequence is a modified MVM intron sequence. In some embodiments, the modified MVM intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified MVM intron sequence comprises at least one mutation in an ATG codon. In some embodiments, the modified MVM intron sequence comprises at least one mutation selected from the following as compared to SEQ ID NO: 13: a) a mutation of A to T, A to C, or A to G at position 16; and / or b) a mutation of G to C, G to A, or G to T at position 40. In some embodiments, the modified MVM intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the modified MVM intron comprises or consists of SEQ ID NO: 14. In some embodiments, the intron is a RK intron, or a functional variant or fragment thereof. In some embodiments, the RK intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the RK intron comprises or consists of SEQ ID NO: 15. In some embodiments, the RK intron sequence is a modified RK intron sequence. In some embodiments, the modified RK intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified RK intron sequence comprises at least one mutation in an ATG codon. In some embodiments, the modified RK intron sequence comprises an A to G, A to C, or A to T mutation at position 78 as compared to SEQ ID NO: 15. In some embodiments, the modified RK intron sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the modified RK intron comprises or consists of SEQ ID NO: 16.

[0046] In some embodiments, any of the recombinant expression vectors provided herein further include a splice donor site 5’ of the intron and a splice acceptor site 3’ of the intron. In some embodiments, the splice donor site and / or the splice acceptor site is modified. In some embodiments, the polynucleotide comprises a sequence 5’ (e.g., within the splice donor site) and / or 3’ (e.g., within the splice acceptor site) of the intron sequence that matches a human consensus sequence. In some embodiments, the sequence that matches the human consensus sequence at the 5’ end of the intron comprises GAG, CAG, AAC, or AAG. In some embodiments, the sequence that matches the human consensus sequence at the 3 ’ end of the intron comprises GTT, GTA, GCC, GAA, or GAT.

[0047] In some embodiments, the lipase A (LIPA) transgene comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the lipase A (LIPA) transgene comprises or consists of SEQ ID NO: 6. In some embodiments, the lipase A (LIPA) transgene is codon optimized (e.g., for enhanced LAL protein expression and / or human expression). In some embodiments, the codon optimized lipase A (LIPA) transgene comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the codon optimized lipase A (LIPA) transgene comprises or consists of SEQ ID NO: 7.

[0048] In some embodiments, any of the recombinant expression vectors provided herein further include a post-transcriptional regulatory element (PRE) (e.g, WPRE or HPRE), or a functional variant or fragment thereof. In some embodiments, any of the recombinant expression vectors provided herein further comprise a polyadenylation (Poly A) signal. In some embodiments, the PolyA signal is a bovine growth hormone polyadenylation (bgh-PolyA) signal, synthetic polyA signal, or an SV40 polyA signal. In some embodiments, the PolyA signal comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a functional fragment thereof. In some embodiments, the PolyA signal comprises or consists of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a functional fragment thereof. In some embodiments, the PolyA signal comprises or consists of SEQ ID NO: 23, or a functional fragment thereof.

[0049] In some embodiments, any of the recombinant expression vectors provided herein further comprise an enhancer. In some embodiments, the enhancer is selected from a CMV enhancer and an ApoE HCR enhancer. In some embodiments, the enhancer is an ApoE HCR1 enhancer. In some embodiments, the ApoE HCR1 enhancer comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 22. In some embodiments, the ApoE HCR1 enhancer comprises of consists of a polynucleotide sequence of SEQ ID NO: 22. In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the CMV enhancer comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21. In some embodiments, the CMV enhancer comprises of consists of a polynucleotide sequence of SEQ ID NO: 21.

[0050] In some embodiments, any of the recombinant expression vectors provided herein further comprise an untranslated region (UTR). In some embodiments, the UTR is a LIPA 3’UTR. In some embodiments, the UTR comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29. In some embodiments, the UTR comprises or consists of a polynucleotide sequence of SEQ ID NO: 29. In some embodiments, any of the recombinant expression vectors provided herein further comprise a Kozak sequence.

[0051] In some embodiments, any of the recombinant expression vectors provided herein further comprise a stuffer. In some embodiments, the stuffer is selected from one or more sequences in Table 12A or 12B. In some embodiments, the staffer comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34. In some embodiments, the stuffer comprises or consists of a polynucleotide sequence of SEQ ID NO: 34.

[0052] In some embodiments, the 5’ ITR and / or 3’ ITR is an AAV2 ITR. or a variant or fragment thereof. In some embodiments, the 5’ ITR comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, the 5’ ITR comprises or consists of a polynucleotide sequence of SEQ ID NO: 17. In some embodiments, the 5’ ITR is a 5’ ITR AAV2 R short and comprises or consists of a polynucleotide sequence of SEQ ID NO: 18 or a polynucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18. In some embodiments, the 3’ ITR is an unmodified AAV2 ITR. In some embodiments, the 3’ ITR comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19 or SEQ ID NO: 80. In some embodiments, the 3’ ITR comprises of consists of a polynucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 80. In some embodiments, the 3’ ITR is a 3’ ITR AAV2 R short and comprises or consists of a polynucleotide sequence of SEQ ID NO: 20 or a polynucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20.

[0053] In some embodiments, any of the recombinant expression vectors provided herein further comprise at least one antibiotic resistant gene. In some embodiments, the at least one antibiotic resistant gene is selected from kanamycin and / or ampicillin. In some embodiments, the at least one antibiotic resistant gene comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the at least one antibiotic resistant gene comprises or consists of a polynucleotide sequence selected from SEQ ID NO: 32 and SEQ ID NO: 33. In some embodiments, any of the recombinant expression vectors provided herein further comprise at least one antibiotic resistant promoter. In some embodiments, the at least one antibiotic resistant promoter is an AmpR (e.g., SEQ ID NO: 30) or pSyn E. Coli (e.g., SEQ ID NO: 31) promoter.

[0054] In some embodiments, any of the recombinant expression vectors provided herein further comprise at least one origin of replication polynucleotide sequence. In some embodiments, the at least one origin of replication polynucleotide sequence is selected from an Ori, M13 Ori, and / or Ori pUC sequence. In some embodiments, the at least one origin of replication polynucleotide sequence comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, SEQ ID NO: 27, and / or SEQ ID NO: 28. In some embodiments, the at least one origin of replication polynucleotide sequence comprises or consists of a polynucleotide sequence selected from SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

[0055] In some embodiments, any of the recombinant expression vectors provided herein further comprise at least one mutation in one or more adjacent nucleotides to the donor site and / or 16S acceptor site of the intron. In some embodiments, any of the recombinant expression vectors provided herein further comprise an AAC, CAG, or GAG sequence adjacent to the donor site of the intron and / or a GCC or GTA sequence adjacent to the 16S acceptor site of the intron. In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3’ ITR nucleic acid sequence.

[0056] In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d) a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3’ ITR nucleic acid sequence.

[0057] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3 ’ ITR nucleic acid sequence.

[0058] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d) a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3 ’ ITR nucleic acid sequence.

[0059] In yet a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR AAV2 R short nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3’ ITR AAV2 R short nucleic acid sequence.

[0060] In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and d) a 3’ ITR nucleic acid sequence; wherein the polynucleotide does not contain an intron nucleic acid sequence.

[0061] In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon and 19S acceptor site (e.g., a mutation of A to T at position 79 and a mutation of A to T at position 30, G to C at position 31, or AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10); d) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3 ’ ITR nucleic acid sequence.

[0062] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a MVM intron nucleic acid sequence, or a functional variant or fragment thereof; d) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3 ’ ITR nucleic acid sequence.

[0063] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a RK intron nucleic acid sequence, or a functional variant or fragment thereof; d) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3’ ITR nucleic acid sequence.

[0064] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c) a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon (e.g., a mutation of A to T at position 79 as compared to SEQ ID NO: 10); d) a. LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e) a 3’ ITR nucleic acid sequence.

[0065] In yet a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; c) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

[0066] In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; c) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d) a lipase A (LIPA) codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; and e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

[0067] In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

[0068] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) ahAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; and d) a LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

[0069] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 18; b) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d) a. LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e) a 3’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 20.

[0070] In yet a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) ahAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and d) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; wherein the recombinant expression vector does not contain an intron.

[0071] In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a SV40i intron nucleic acid sequence comprising or consisting of SEQ ID NO: 11; d) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80. In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a MVM intron nucleic acid sequence comprising or consisting of SEQ ID NO: 14; d) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

[0072] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) ahAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a RK intron nucleic acid sequence comprising or consisting of SEQ ID NO: 16; d) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

[0073] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 12; d) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

[0074] In some embodiments, any of the recombinant expression vectors described herein further include a post-transcriptional regulatory element (PRE) (e.g. , WPRE or HPRE) nucleic acid sequence, a polyadenylation (Poly A) signal (e.g., bovine growth hormone polyadenylation (bgh-PolyA) signal) nucleic acid sequence, and / or an enhancer (e.g., CMV enhancer or ApoE HCR enhancer) nucleic acid sequence, or functional variants or fragments thereof.

[0075] In yet a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a CMV enhancer nucleic acid sequence; c) a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a Ml 3 ori nucleic acid sequence.

[0076] In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) a CMV enhancer nucleic acid sequence; c) a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e) a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a Ml 3 ori nucleic acid sequence. In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a M13 ori nucleic acid sequence.

[0077] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e) a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a M13 ori nucleic acid sequence.

[0078] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR AAV2 R short nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e) a LIPA 3’ UTR nucleic acid sequence; f) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; g) a bGH poly(A) signal nucleic acid sequence; h) a staffer nucleic acid sequence; i) a 3’ ITR AAV2 R short nucleic acid sequence; j) a P syn E. coli nucleic acid sequence; k) a M KanR* nucleic acid sequence; and 1) an ori pUC nucleic acid sequence.

[0079] In yet a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; e) a bGH poly(A) signal nucleic acid sequence; f) a 3’ ITR nucleic acid sequence; g) an AmpR promoter nucleic acid sequence; h) a KanR nucleic acid sequence; i) an ori nucleic acid sequence; and j) a M13 ori nucleic acid sequence; wherein the polynucleotide does not contain an intron nucleic acid sequence.

[0080] In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon and 19S acceptor site (e.g., a mutation of A to T at position 79 and a mutation of A to T at position 30, G to C at position 31, or AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10); e) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a M13 ori nucleic acid sequence.

[0081] In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a MVM intron nucleic acid sequence, or a functional variant or fragment thereof; e) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a M13 ori nucleic acid sequence.

[0082] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a RK intron nucleic acid sequence, or a functional variant or fragment thereof; e) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a M13 ori nucleic acid sequence.

[0083] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence; b) an ApoE HCR1 enhancer nucleic acid sequence; c) a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d) a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon (e.g., a mutation of A to T at position 79 as compared to SEQ ID NO: 10); e) a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f) a bGH poly(A) signal nucleic acid sequence; g) a 3’ ITR nucleic acid sequence; h) an AmpR promoter nucleic acid sequence; i) a KanR nucleic acid sequence; j) an ori nucleic acid sequence; and k) a M13 ori nucleic acid sequence.

[0084] In yet a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a CMV enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 21; c) a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; d) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a Ml 3 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0085] In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) a CMV enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 21; c) a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; d) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e) a lipase A (LIPA) codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0086] In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e) a. LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a Ml 3 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0087] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e) a LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0088] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 18; b) an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e) a. LIPA 3’ UTR nucleic acid sequence comprising or consisting of SEQ ID NO: 29; f) aLIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; g) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; h) a staffer nucleic acid sequence comprising or consisting of SEQ ID NO: 34; i) a 3’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 20; j) a P syn E. coli nucleic acid sequence comprising or consisting of SEQ ID NO: 31; k) a M KanR* nucleic acid sequence comprising or consisting of SEQ ID NO: 33; and 1) an ori pUC nucleic acid sequence comprising or consisting of SEQ ID NO: 28.

[0089] In yet a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; e) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; f) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; g) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; h) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; i) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and j) a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0090] In one aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 11; e) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a Ml 3 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0091] In another aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a MVM intron nucleic acid sequence comprising or consisting of SEQ ID NO: 14; e) aLLPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a Ml 3 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0092] In yet another aspect, the invention provides a recombinant expression vector comprising, optionally from 5’ to 3’: a) a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) an ApoE HER! enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a RK intron nucleic acid sequence comprising or consisting of SEQ ID NO: 16; e) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a Ml 3 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

[0093] In a further aspect, the invention provides a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a) a 5 ’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b) an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c) a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d) a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 12; e) a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f) a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g) a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h) an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i) a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j) an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k) a Ml 3 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-47. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 46. In some embodiments, the recombinant expression vector comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47. In some embodiments, the recombinant expression vector comprises or consists of any one of SEQ ID NOs: 38-47. In some embodiments, the recombinant expression vector comprises or consists of any one of SEQ ID NO: 38. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 39. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 40. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 41. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 42. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 43. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 44. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 45. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 46. In some embodiments, the recombinant expression vector comprises or consists of SEQ ID NO: 47.

[0094] In some embodiments, the recombinant expression vector has at least about a 90% splice efficiency of LIPA mRNA in a cell or tissue. In some embodiments, the recombinant expression vector increases LAL activity in a cell, tissue, or serum. In some embodiments, LAL activity is measured through a Lalistat 2-based LAL activity assay. In some embodiments, LAL activity is increased to >10% of mean normal enzyme activity. In some embodiments, the recombinant expression vector increases LIPA mRNA levels and / or expression in a cell or tissue, optionally wherein LIPA mRNA levels and / or expression is measured through real-time reverse transcription polymerase chain reaction. In some embodiments, the recombinant expression vector increases Lysosomal Acid Lipase (LAL) protein levels and / or expression in a cell, tissue or serum, optionally where LAL protein levels and / or expression is measured through immunoblot or enzyme-linked immunosorbent assay and / or liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the cell is selected from any cell that expresses LIPA RNA or LAL protein, optionally epithelial cells, endocrine cells, neuronal cells, glial cells, germ cells, trophoblast cells, endothelial cells, muscle cells, adipocytes, pigment cells, mesenchymal cells, blood or immune cells, specifically a Kupffer cell, Hofbauer cell, macrophage, hepatocyte, B cell, T cell, enteroendocrine cell, fibroblast, Langerhans cell, enterocyte, monocyte, platelet, glia cell, glandular cell, luminal cell, basal respiratory cell, oligodendrocyte, osteoblast, hepatic stellate cell (HSC), myeloid cell, adipocyte, splenocyte, intestinal cell, skeletal muscle cell, red blood cell, and / or smooth muscle cell. In some embodiments, the tissue is selected from liver tissue, spleen tissue, skeletal muscle tissue, kidney tissue, brain tissue (e.g., cerebellum, cerebral cortex, retina), endocrine tissue (e.g., thyroid, parathyroid, adrenal gland), prostate / testis tissue, endometrium / ovarian tissue, respiratory tissue (e.g., lung, bronchus), myeloid tissue, gastrointestinal tissue (e.g, duodenum, small intestine, colon rectum), and / or lymphoid tissue (e.g., appendix, spleen, lymph node, tonsil, bone marrow). In some embodiments, the tissue is spleen and / or liver tissue. In some embodiments, the recombinant expression vector increases LIPA mRNA levels and / or expression in liver and / or spleen. In some embodiments, the recombinant expression vector increases Lysosomal Acid Lipase (LAL) protein levels and / or expression in liver and / or spleen. In some embodiments, the recombinant expression vector increases serum Lysosomal Acid Lipase (LAL) protein levels and / or expression.

[0095] In some embodiments, the recombinant expression vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex vector, or a retroviral vector. In some embodiments, the recombinant viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector has liver tropism. In some embodiments, the AAV vector is a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh, AAVrhlO, AAVrh74, AAV-DJ, AAV-DJ / 8, Anc80, and AAV7m8, or a derivative, hybrid or chimeric serotype thereof. In some embodiments, the AAV vector is an AAV9 serotype. In some embodiments, the AAV vector is a single stranded AAV9 (ssAAV9) vector or a self-complementary AAV9 (scAAV9) vector. In some embodiments, the AAV vector is a recombinant adeno-associated viral (rAAV) vector.

[0096] In yet a further aspect, the invention provides viral particles comprising any of the recombinant expression vectors as disclosed herein. In some embodiments, the viral particle is an AAV particle. In some embodiments, the AAV particle is an isolated AAV particle. In some embodiments, the AAV particle is a recombinant adeno-associated viral (rAAV) particle.

[0097] In one aspect, the invention provides host cells comprising any of the polynucleotides as disclosed herein. In another aspect, the invention provides host cells comprising any of the recombinant expression vectors as disclosed herein. In yet another aspect, the invention provides host cells comprising any of the viral particles as disclosed herein. In some embodiments, the host cell is a mammalian or an insect cell. In some embodiments, any of the host cells disclosed herein further include a helper construct. In some embodiments, the helper construct is a pHelp plasmid encoding at least one adenovirus regulatory element (e.g, E2A, E4, and / or VAI / II RNA genes). In some embodiments, any of the host cells disclosed herein further include a Rep2-Cap9 plasmid. In a further aspect, the invention provides a host cell comprising any of the polynucleotides as disclosed herein; a pHelp plasmid; and a Rep2-Cap9 plasmid. In yet a further aspect, the invention provides a host cell comprising any of the recombinant expression vectors as disclosed herein; a pHelp plasmid; and a Rep2-Cap9 plasmid. In one aspect, the invention provides a host cell comprising any of the viral particles as disclosed herein; a pHelp plasmid; and a Rep2-Cap9 plasmid. In some embodiments, the host cell is a HEK293 cell.

[0098] In one aspect, the invention provides pharmaceutical compositions comprising any of the polynucleotides as disclosed herein, and a pharmaceutically acceptable excipient. In another aspect, the invention provides pharmaceutical compositions comprising any of the recombinant expression vectors as disclosed herein, and a pharmaceutically acceptable excipient. In yet another aspect, the invention provides pharmaceutical compositions comprising any of the viral particles as disclosed herein, and a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated for intravenous (IV) delivery, portal vein delivery, or intramuscular delivery.

[0099] In a further aspect, the invention provides methods of treating a subject having or suspected of having a Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof. In some embodiments, the methods of treating a subject having or suspected of having a Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having a Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having a Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the viral particles disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having a Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0100] In yet a further aspect, the invention provides methods of treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject. In some embodiments, the methods of treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject include administering a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject include administering a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject include administering a therapeutically effective amount of any of the viral particles disclosed herein. In some embodiments, the methods of treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject include administering a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0101] In one aspect, the invention provides methods of decreasing triglycerides, cholesterol, and / or fatty acids in a subject. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a subject include administering a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a subject include administering a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a subject include administering a therapeutically effective amount of any of the viral particles disclosed herein. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a subject include administering a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0102] In another aspect, the invention provides methods of decreasing liver-to-body weight (LW / BW) and / or spleen-to-body weight (SPL / BW) ratio in a subject. In some embodiments, the methods of decreasing liver-to-body weight (LW / BW) and / or spleen-to-body weight (SPL / BW) ratio in a subject include administering a therapeutically effective amount of any of the polynucleotides as disclosed herein. In some embodiments, the methods of decreasing liver- to-body weight (LW / BW) and / or spleen-to-body weight (SPL / BW) ratio in a subject include administering a therapeutically effective amount of any of the recombinant expression vectors as disclosed herein. In some embodiments, the methods of decreasing liver-to-body weight (LW / BW) and / or spleen-to-body weight (SPL / BW) ratio in a subject include administering a therapeutically effective amount of any of the viral particles as disclosed herein. In some embodiments, the methods of decreasing liver-to-body weight (LW / BW) and / or spleen-to- body weight (SPL / BW) ratio in a subject include administering a therapeutically effective amount of any of the pharmaceutical compositions as disclosed herein.

[0103] In yet another aspect, the invention provides methods of increasing LIPA mRNA levels and / or expression in the liver and / or spleen of a subject. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in the liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any one of the polynucleotides disclosed herein. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in the liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any one of the recombinant expression vectors disclosed herein. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in the liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any one of the viral particles disclosed herein. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in the liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any one of the pharmaceutical compositions disclosed herein.

[0104] In a further aspect, the invention provides methods of increasing Lysosomal Acid Lipase (LAL) protein levels and / or expression in liver and / or spleen of a subject. In some embodiments, the methods of increasing Lysosomal Acid Lipase (LAL) protein levels and / or expression in liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of increasing Lysosomal Acid Lipase (LAL) protein levels and / or expression in liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of increasing Lysosomal Acid Lipase (LAL) protein levels and / or expression in liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any of the viral particles disclosed herein. In some embodiments, the methods of increasing Lysosomal Acid Lipase (LAL) protein levels and / or expression in liver and / or spleen of a subject include administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0105] In yet a further aspect, the invention provides methods of increasing serum Lysosomal Acid Lipase (LAL) protein levels and / or expression in a subject. In some embodiments, the methods of increasing serum Lysosomal Acid Lipase (LAL) protein levels and / or expression in a subject include administering to the subject a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of increasing serum Lysosomal Acid Lipase (LAL) protein levels and / or expression in a subject include administering to the subject a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of increasing serum Lysosomal Acid Lipase (LAL) protein levels and / or expression in a subject include administering to the subject a therapeutically effective amount of any of the viral particles disclosed herein. In some embodiments, the methods of increasing serum Lysosomal Acid Lipase (LAL) protein levels and / or expression in a subject include administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0106] In one aspect, the invention provides methods of decreasing mRNA levels and / or expression of genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a subject. In some embodiments, the methods of decreasing mRNA levels and / or expression of genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a subject include administering to the subject a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of decreasing mRNA levels and / or expression of genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a subject include administering to the subject a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of decreasing mRNA levels and / or expression of genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a subject include administering to the subject a therapeutically effective amount of any of the viral particles disclosed herein, a pharmaceutical compositions disclosed herein.

[0107] In some embodiments, the subject has or is suspected of having a Lysosomal Acid Lipase Deficiency (LAL-D) or one or more symptoms thereof. In some embodiments, the LAL-D is Wolman disease or cholesterol ester storage disease (CESD). In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is a human pediatric subject. In some embodiments, the human pediatric subject is less than about 12 years of age (e.g., between birth and 12 years of age, between 1-12 years of age, between 1-5 years of age, between, 6-12 years of age, less than about 1 year of age, or less than about 6 months of age). In some embodiments, the subject is a human adolescent subject. In some embodiments, the human adolescent subject is 12 to 18 years of age. In some embodiments, the subject is a human adult subject. In some embodiments, the human adult subject is 18 years of age or older.

[0108] In another aspect, the invention provides methods of treating a subject having or suspected of having Wolman disease or ameliorating one or more symptoms thereof. In some embodiments, the methods of treating a subject having or suspected of having Wolman disease or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having Wolman disease or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having Wolman disease or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the viral particles disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having Wolman disease or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the one or more symptoms thereof is selected from massive hepatosplenomegaly, acute liver failure, multi-organ failure, microvesicular steatosis, malabsorption, jaundice, portal hypertension, failure to thrive, adrenal calcification, gastrointestinal disturbances (e.g., diarrhea and vomiting with steatorrhea) that may lead to abdominal distension, and immune complications (increased susceptibility to infections and Hemophagocytic Lymphohistiocytosis (HLH)). In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is a human pediatric subject. In some embodiments, the subject is a human pediatric subject less than about 1 year of age (e.g., between birth and 1 year of age, or less than about 6 months of age).

[0109] In yet another aspect, the invention provides methods of treating a subject having or suspected of having cholesterol ester storage disease (CESD) or ameliorating one or more symptoms thereof. In some embodiments, the methods of treating a subject having or suspected of having cholesterol ester storage disease (CESD) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the polynucleotides disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having cholesterol ester storage disease (CESD) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having cholesterol ester storage disease (CESD) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the viral particles disclosed herein. In some embodiments, the methods of treating a subject having or suspected of having cholesterol ester storage disease (CESD) or ameliorating one or more symptoms thereof include administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the one or more symptoms thereof is selected from dyslipidemia (elevated LDL-C, low HDL-C), elevated liver transaminases, hepatomegaly, splenomegaly, accelerated atherosclerosis, abnormal hepatic function, hepatosplenomegaly, hepatic fibrosis and cirrhosis, portal hypertension, decompensated liver disease, and gastrointestinal disturbances (e.g., diarrhea and vomiting with steatorrhea). In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is a human pediatric subject. In some embodiments, the subject is a human pediatric subject less than about 12 years of age (e.g., between 1-12 years of age, between 1-5 years of age, or between 6-12 years of age). In some embodiments, the subject is a human pediatric subject less than about 5 years of age. In some embodiments, the subject is a human adolescent subject. In some embodiments, the human adolescent subject is 12 to 18 years of age. In some embodiments, the subject is a human adult subject. In some embodiments, the human adult subject is 18 years of age or older. In some embodiments, the step of administering is via portal vein delivery, intramuscular delivery, or intravenous (IV) injection. In some embodiments, the therapeutically effective amount is at a dose of at least about lei 3 vg / kg, at least about lel4 vg / kg, at least about lei 5 vg / kg, or at least about lei 6 vg / kg. In some embodiments, the subject has at least one mutation in the LIPA gene. In some embodiments, at least one additional therapeutic agent or therapy is administered to the subject. In some embodiments, the at least one additional therapeutic agent or therapy is selected from corticosteroids, enzyme replacement therapy (ERT), rapamycin, immunoadsorption, and rituximab.

[0110] In a further aspect, the invention provides methods of identifying a subject for treatment with any of the polynucleotides, a recombinant expression vectors, viral particles, or pharmaceutical compositions disclosed herein. In some embodiments, the method of identifying a subject for treatment with any of the polynucleotides, a recombinant expression vectors, viral particles, or pharmaceutical compositions disclosed herein include measuring LAL activity in the subject with a DBS-Lalistat 2 LAL activity assay, wherein a subject with <5% of mean normal LAL enzymatic activity is identified as a subject for treatment.

[0111] In yet a further aspect, the invention provides methods of monitoring the progression of treatment in a subject administered with any of the polynucleotides, a recombinant expression vectors, viral particles, or pharmaceutical compositions disclosed herein. In some embodiments, the methods of monitoring the progression of treatment in a subject administered with any of the polynucleotides, a recombinant expression vectors, viral particles, or pharmaceutical compositions disclosed herein include measuring LAL activity in the subject over time with a DBS-Lalistat 2 LAL activity assay.

[0112] In one aspect, the invention provides methods of decreasing triglycerides, cholesterol, and / or fatty acids in a cell or tissue. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a cell or tissue include contacting the cell or tissue with any of the polynucleotides disclosed herein. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a cell or tissue include contacting the cell or tissue with any of the recombinant expression vectors disclosed herein. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a cell or tissue include contacting the cell or tissue with any of the viral particles disclosed herein. In some embodiments, the methods of decreasing triglycerides, cholesterol, and / or fatty acids in a cell or tissue include contacting the cell or tissue with any of the pharmaceutical compositions disclosed herein.

[0113] In another aspect, the invention provides methods of increasing LAL activity in a cell, tissue, or serum. In some embodiments, the methods of increasing LAL activity in a cell, tissue, or serum include contacting the cell or tissue with any of the polynucleotides described herein. In some embodiments, the methods of increasing LAL activity in a cell, tissue, or serum include contacting the cell or tissue with any of the recombinant expression vectors described herein. In some embodiments, the methods of increasing LAL activity in a cell, tissue, or serum include contacting the cell or tissue with any of the viral particles described herein. In some embodiments, the methods of increasing LAL activity in a cell, tissue, or serum include contacting the cell or tissue with any of the pharmaceutical compositions described herein. In some embodiments, LAL activity is increased to >10% of mean normal enzyme activity.

[0114] In yet another aspect, the invention provides methods of increasing LIPA mRNA levels and / or expression in a cell or tissue. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in a cell or tissue include contacting the cell or tissue with any of the polynucleotides described herein. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in a cell or tissue include contacting the cell or tissue with any of the recombinant expression vectors described herein. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in a cell or tissue include contacting the cell or tissue with any of the viral particles described herein. In some embodiments, the methods of increasing LIPA mRNA levels and / or expression in a cell or tissue include contacting the cell or tissue with any of the pharmaceutical compositions described herein.

[0115] In a further aspect, the invention provides methods of increasing LAL protein levels and / or expression in a cell, tissue or serum. In some embodiments, the methods of increasing LAL protein levels and / or expression in a cell, tissue or serum include contacting the cell or tissue with any of the polynucleotides described herein. In some embodiments, the methods of increasing LAL protein levels and / or expression in a cell, tissue or serum include contacting the cell or tissue with any of the recombinant expression vectors described herein. In some embodiments, the methods of increasing LAL protein levels and / or expression in a cell, tissue or serum include contacting the cell or tissue with any of the viral particles described herein. In some embodiments, the methods of increasing LAL protein levels and / or expression in a cell, tissue or serum include contacting the cell or tissue with any of the pharmaceutical compositions described herein.

[0116] In yet a further aspect, the invention provides methods of decreasing mRNA levels and / or expression of one or more genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a cell or tissue. In some embodiments, the methods of decreasing mRNA levels and / or expression of one or more genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a cell or tissue include contacting the cell or tissue with any of the polynucleotides of described herein. In some embodiments, the methods of decreasing mRNA levels and / or expression of one or more genes relating to macrophage function and inflammation (e.g. , CXCL5 and TNF) in a cell or tissue include contacting the cell or tissue with any of the recombinant expression vectors described herein. In some embodiments, the methods of decreasing mRNA levels and / or expression of one or more genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a cell or tissue include contacting the cell or tissue with any of the viral particles described herein. In some embodiments, the methods of decreasing mRNA levels and / or expression of one or more genes relating to macrophage function and inflammation (e.g., CXCL5 and TNF) in a cell or tissue include contacting the cell or tissue with any of the pharmaceutical compositions described herein. In some embodiments, the one or more genes relating to macrophage function and inflammation include CXCL5 and / or TNF.

[0117] In some embodiments, any of the methods described herein are performed in vitro, in vivo, or ex vivo. In some embodiments, any of the cells described herein is selected from any cell that expresses LIPA RNA or LAL protein, optionally epithelial cells, endocrine cells, neuronal cells, glial cells, germ cells, trophoblast cells, endothelial cells, muscle cells, adipocytes, pigment cells, mesenchymal cells, blood or immune cells, specifically a Kupffer cell, Hofbauer cell, macrophage, hepatocyte, B cell, T cell, enteroendocrine cell, fibroblast, Langerhans cell, enterocyte, monocyte, platelet, glia cell, glandular cell, luminal cell, basal respiratory cell, oligodendrocyte, osteoblast, hepatic stellate cell (HSC), myeloid cell, adipocyte, splenocyte, intestinal cell, skeletal muscle cell, red blood cell, and / or smooth muscle cell. In some embodiments, any of the tissues described herein is selected from liver tissue, spleen tissue, skeletal muscle tissue, kidney tissue, brain tissue (e.g., cerebellum, cerebral cortex, retina), endocrine tissue (e.g., thyroid, parathyroid, adrenal gland), prostate / testis tissue, endometrium / ovarian tissue, respiratory tissue (e.g., lung, bronchus), myeloid tissue, gastrointestinal tissue (e.g, duodenum, small intestine, colon rectum), and / or lymphoid tissue (e.g., appendix, spleen, lymph node, tonsil, bone marrow). In some embodiments, the tissue is spleen and / or liver tissue.

[0118] In one aspect, the invention provides methods of making any of the recombinant expression vectors described herein. In some embodiments, the methods of making any of the recombinant expression vectors described herein include culturing any of the host cells described herein, lysing the cultured host cells, and extracting and purifying the recombinant expression vector from said lysed cultured host cells. In another aspect, the invention provides methods of making any of the viral particles described herein. In some embodiments, the methods of making any of the viral particles described herein include incubating any of the host cells described herein under conditions suitable to enclose a recombinant expression vector in a viral capsid, thereby making the viral particle.

[0119] In yet another aspect, the invention provides methods of making any of the AAV particles described herein. In some embodiments, the methods of making any of the AAV particles described herein include incubating any of the host cells disclosed herein under conditions suitable to enclose a recombinant expression vector in an AAV capsid, thereby making the AAV particle. In some embodiments, the methods of making any of the AAV particles described herein include introducing a nucleic acid comprising the recombinant expression vector into the host cell prior to incubating the host cell.

[0120] In a further aspect, the invention provides methods of making any of the pharmaceutical compositions disclosed herein. In some embodiments, the methods of making any of the pharmaceutical compositions disclosed herein include culturing any of the host cells disclosed herein, collecting the supernatant of the cultured host cells, concentrating and purifying recombinant viral vectors from the collected supernatant, and adding pharmaceutically acceptable excipients to the purified recombinant viral vectors.

[0121] In yet a further aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use as a medicament or in the manufacture of a medicament.

[0122] In one aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use as a medicament or in the manufacture of a medicament for treating Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof in a subject. In another aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use as a medicament or in the manufacture of a medicament for treating Wolman disease or ameliorating one or more symptoms thereof in a subject. In yet another aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use as a medicament or in the manufacture of a medicament for treating cholesterol ester storage disease (CESD) or ameliorating one or more symptoms thereof in a subject. In a further aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use as a medicament or in the manufacture of a medicament for treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject.

[0123] In yet a further aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in treating a subject having or suspected of having Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof.

[0124] In one aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject.

[0125] In another aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in decreasing triglycerides, cholesterol, and / or fatty acids in a subject.

[0126] In yet another aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in decreasing liver-to-body weight (LW / BW) and / or spleen-to-body weight (SPL / BW) ratio in a subject.

[0127] In a further aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in increasing LIPA mRNA levels and / or expression in the liver and / or spleen of a subject.

[0128] In yet a further aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in increasing Lysosomal Acid Lipase (LAL) protein levels and / or expression in the liver and / or spleen of a subject.

[0129] In one aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in increasing serum Lysosomal Acid Lipase (LAL) protein levels and / or expression in a subject.

[0130] In some embodiments, for any of the uses provided herein, the subject has or is suspected of having Lysosomal Acid Lipase Deficiency (LAL-D) or one or more symptoms thereof. In some embodiments, the LAL-D is Wolman disease or cholesterol ester storage disease (CESD). In some embodiments, the subject is a human subject. In some embodiments, the human subject is less than about 12 years of age (e.g., between birth and 12 years of age, between 1-12 years of age, between 1-5 years of age, between, 6-12 years of age, less than about 1 year of age, or less than about 6 months of age).

[0131] In another aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in treating a subject having or suspected of having Wolman disease. In some embodiments, the subject is a human subject less than about 1 year of age (e.g., between birth and 1 year of age, or less than about 6 months of age).

[0132] In yet another aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in treating a subject having or suspected of having cholesterol ester storage disease (CESD). In some embodiments, the subject is a human subject less than about 12 years of age (e.g., between 1-12 years of age, between 1-5 years of age, or between 6-12 years of age). In some embodiments, the subject has at least one mutation in the LIPA gene.

[0133] In a further aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in decreasing triglycerides, cholesterol, and / or fatty acids in a cell or tissue. In yet a further aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in increasing LAL activity in a cell, tissue or serum. In some embodiments, LAL activity is increased to >10% of mean normal enzyme activity. In one aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in increasing LIPA mRNA levels and / or expression in a cell of tissue. In another aspect, the invention provides any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein, for use in increasing LAL protein levels and / or expression in a cell, tissue or serum. In some embodiments, the cell is selected from any cell that expresses LIPA RNA or LAL protein, optionally epithelial cells, endocrine cells, neuronal cells, glial cells, germ cells, trophoblast cells, endothelial cells, muscle cells, adipocytes, pigment cells, mesenchymal cells, blood or immune cells, specifically a Kupffer cell, Hofbauer cell, macrophage, hepatocyte, B cell, T cell, enteroendocrine cell, fibroblast, Langerhans cell, enterocyte, monocyte, platelet, glia cell, glandular cell, luminal cell, basal respiratory cell, oligodendrocyte, osteoblast, hepatic stellate cell (HSC), myeloid cell, adipocyte, splenocyte, intestinal cell, skeletal muscle cell, red blood cell, and / or smooth muscle cell. In some embodiments, the tissue is selected from liver tissue, spleen tissue, skeletal muscle tissue, kidney tissue, brain tissue (e.g., cerebellum, cerebral cortex, retina), endocrine tissue (e.g., thyroid, parathyroid, adrenal gland), prostate / testis tissue, endometrium / ovarian tissue, respiratory tissue (e.g, lung, bronchus), myeloid tissue, gastrointestinal tissue (e.g., duodenum, small intestine, colon rectum), and / or lymphoid tissue (e.g., appendix, spleen, lymph node, tonsil, bone marrow). In some embodiments, the tissue is spleen and / or liver tissue.

[0134] In some embodiments, any of the uses disclosed herein is performed in vitro, in vivo, or ex vivo.

[0135] In yet another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in a medicament or in the manufacture of a medicament.

[0136] In a further aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof in a subject. In yet a further aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating Wolman disease or ameliorating one or more symptoms thereof in a subject. In one aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating cholesterol ester storage disease (CESD) or ameliorating one or more symptoms thereof in a subject. In another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject.

[0137] In another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in treating a subject having or suspected of having Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof.

[0138] In yet another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject.

[0139] In a further aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in decreasing triglycerides, cholesterol, and / or fatty acids in a subject in need thereof.

[0140] In yet a further aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in decreasing liver-to-body weight (LW / BW) and / or spleen-to-body weight (SPL / BW) ratio in a subject.

[0141] In one aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in increasing LIPA mRNA levels and / or expression in the liver and / or spleen of a subject.

[0142] In another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in increasing Lysosomal Acid Lipase (LAL) protein levels and / or expression in the liver and / or spleen of a subject.

[0143] In yet another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in increasing serum Lysosomal Acid Lipase (LAL) protein levels and / or expression in a subject.

[0144] In some embodiments, for any of the uses disclosed herein, the subject in need thereof has or is suspected of having Lysosomal Acid Lipase Deficiency (LAL-D) or one or more symptoms thereof. In some embodiments, the LAL-D is Wolman disease or cholesterol ester storage disease (CESD). In some embodiments, the subject is a human subject. In some embodiments, the human subject is less than about 12 years of age (e.g., between birth and 12 years of age, between 1-12 years of age, between 1-5 years of age, between, 6-12 years of age, less than about 1 year of age, or less than about 6 months of age). In a further aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in treating a subject having or suspected of having Wolman disease. In some embodiments, the subject is a human subject less than about 1 year of age (e.g., between birth and 1 year of age, or less than about 6 months of age).

[0145] In yet a further aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein in treating a subject having or suspected of having cholesterol ester storage disease (CESD). In some embodiments, the subject is a human subject less than about 12 years of age (e.g., between 1-12 years of age, between 1-5 years of age, or between 6-12 years of age). In some embodiments, the subject has at least one mutation in the LIPA gene.

[0146] In one aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein for decreasing triglycerides, cholesterol, and / or fatty acids in a cell. In another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein for increasing LAL activity in a cell, tissue or serum. In some embodiments, LAL activity is increased to >10% of mean normal enzyme activity. In yet another aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein for increasing LIPA mRNA levels and / or expression in a cell or tissue. In a further aspect, the invention provides the use of any of the polynucleotides, the recombinant expression vectors, the viral particles, or the pharmaceutical compositions disclosed herein for increasing LAL protein levels and / or expression in a cell, tissue or serum. In some embodiments, the cell is selected from any cell that expresses LIPA RNA or LAL protein, optionally epithelial cells, endocrine cells, neuronal cells, glial cells, germ cells, trophoblast cells, endothelial cells, muscle cells, adipocytes, pigment cells, mesenchymal cells, blood or immune cells, specifically a Kupffer cell, Hofbauer cell, macrophage, hepatocyte, B cell, T cell, enteroendocrine cell, fibroblast, Langerhans cell, enterocyte, monocyte, platelet, glia cell, glandular cell, luminal cell, basal respiratory cell, oligodendrocyte, osteoblast, hepatic stellate cell (HSC), myeloid cell, adipocyte, splenocyte, intestinal cell, skeletal muscle cell, red blood cell, and / or smooth muscle cell. In some embodiments, the tissue is selected from liver tissue, spleen tissue, skeletal muscle tissue, kidney tissue, brain tissue (e.g., cerebellum, cerebral cortex, retina), endocrine tissue (e.g., thyroid, parathyroid, adrenal gland), prostate / testis tissue, endometrium / ovarian tissue, respiratory tissue (e.g. , lung, bronchus), myeloid tissue, gastrointestinal tissue (e.g. , duodenum, small intestine, colon rectum), and / or lymphoid tissue (e.g., appendix, spleen, lymph node, tonsil, bone marrow). In some embodiments, the tissue is spleen and / or liver tissue. In some embodiments, the use is performed in vitro, in vivo, or ex vivo.

[0147] In one aspect, the invention provides kits including any of the polynucleotides, recombinant expression vectors, viral particles, or pharmaceutical compositions disclosed herein. In some embodiments, any of the kits disclosed herein further include instructions and / or means for administering any of the polynucleotides, recombinant expression vectors, viral particles, or pharmaceutical compositions disclosed herein to a subject in need thereof.

[0148] Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

[0149] BRIEF DESCRIPTION OF THE DRAWINGS

[0150] FIG. 1 provides schematic diagrams depicting various cargo designs (p203, p204, p205, p206 and p207) for AAN9-LIPA vectors. All ITR o-ITR cassettes in the cargo plasmids contain a SV40 intron downstream of the promoter and a BGH polyA signal downstream of the LIPA transgene. The ITRAo-ITR cassettes in cargo plasmids p203, p204, p205 and p206 are in a self-complementary (sc) configuration whereas cargo plasmid p207 is in a singlestranded (ss) configuration. Cargo plasmids p203 and p204 contain a chicken beta-actin (CB) promoter to drive LIPA transgene expression. The LIPA transgene in p203 and p205 was derived from the native LIPA sequence whereas the LIPA transgene in p204 and p206 was codon optimized. Cargo plasmids p205 and p206 contain liver specific hAAT promoters. Cargo plasmid p207 contains additional sequences derived from a. LIPA 3’UTR and a fragment of inert staffer sequence to mimic native AAV genome length.

[0151] FIG. 2 is a Western blot depicting LAL protein expression in HepG2 cells. HepG2 cells were transfected with 203, 204, 205, 206, 207 plasmids, or control plasmid expressing fluorescent protein DsRED. Untransfected HepG2 served as control. LAL protein expression was detected in the cell lysates by Western Blot with a polyclonal rabbit anti-LAL antibody (ORIGENE, #TA309730) and shows as ~42kD, ~52kD and ~58kD bands. Western blot (bottom) to housekeeping protein with rabbit anti-GAPDH (Cell Signaling #5174) as a control. FIGs. 3A and 3B are Western blots depicting dose-dependent LAL protein expression in the liver of LIPA homozygous knockout (KO) mice. FIG. 3A depicts 5 -week-old male and female LIPA homozygous KO mice dosed with vehicle (Veh), AAV9-205 expressing LIPA from the AAT promoter (AAT) at 1E12 vg / kg and 5E13 vg / kg, or AAV9-203 expressing LIPA from the CBA promoter (CBA) at 1E12 vg / kg and 5E13 vg / kg. Samples were treated with PNGaseF prior to electrophoresis to remove glycosylation and LAL shows as a ~35kD and ~40kD band as detected with a monoclonal mouse anti -LAL antibody (Thermofisher, #MA5- 15278). Western blot (bottom) to housekeeping protein with rabbit anti-GAPDH (Cell Signaling #5174) was used as a control. FIG. 3B depicts 5-week-old male and female LIPA homozygous KO mice dosed with vehicle (Veh) or AAV9-205 at 2El l vg / kg, 1E12 vg / kg and 5E13 vg / kg. Samples were treated with PNGaseF prior to electrophoresis to remove glycosylation and LAL shows as a ~35kD and ~40kD band as detected with a monoclonal mouse anti-LAL antibody (Thermofisher, #MA5-15278). Western blot (bottom) to housekeeping protein with rabbit anti-GAPDH (Cell Signaling #5174) as a control.

[0152] FIG. 4 is a graph depicting liver cholesterol ester (CE) content in mice. 5-week-old male and female wildtype (WT) or LIPA homozygous knockout (KO) mice were dosed with vehicle, AAV9-205 expressing LIPA from the AAT promoter (AAT) at 5E10 vg / kg, 1E11 vg / kg, 2E11 vg / kg, 1E12 vg / kg or 5E13 vg / kg, or AAV9-203 expressing LIPA from the CBA promoter (CBA at 5E10 vg / kg, 1E11 vg / kg, 2E11 vg / kg, 1E12 vg / kg or 5E13 vg / kg. Total cholesterol and free cholesterol content were measured with an enzymatic assay, and CE content was inferred by subtracting the free cholesterol from the total cholesterol values. Each dot is from an individual mouse. ** p<0.01 and ****p<0.0001 (one-way ANOVA followed by Dunnett’s test).

[0153] FIG. 5 is a graph depicting spleen cholesterol ester (CE) content in mice. 5-week-old male and female wildtype (WT) or Lipa homozygous knockout (KO) mice were dosed with vehicle, AAV9-205 expressing LIPA from the AAT promoter (AAT) at 5E10 vg / kg, 1E11 vg / kg, 2E11 vg / kg, 1E12 vg / kg or 5E13 vg / kg, or AAV9-203 expressing LIPA from the CBA promoter (CBA) at 5E10 vg / kg, 1E11 vg / kg, 2E11 vg / kg, 1E12 vg / kg or 5E13 vg / kg. Total cholesterol and free cholesterol content were measured with an enzymatic assay, and CE content was inferred by subtracting the free cholesterol from the total cholesterol values. Each dot is from an individual mouse. ns=not significant. ** p<0.01 and ***p<0.001 (one-way ANOVA followed by Dunnett’s test).

[0154] FIG. 6 provides schematic diagrams depicting various cargo designs for AAN9-LIPA vectors with either intron replacement or modifications of the SV40 intron. All ITR-to-ITR cassetes in the cargo plasmids contain an hAAT promoter, LIPA transgene, and a BGH polyA signal as described for cargo plasmid p205 in FIG. 1. Cargo plasmid p333 contains no intron with an inert sequence between the hAAT promoter and LIPA transgene; cargo plasmid p334 contains a modified SV40 intron with mutations introduced in the 19S acceptor site and in ATG to abolish 19S-derived transcript and alternative translational start site; cargo plasmid p335 contains a modified minute virus of mice (MVM) small intron (GenBank accession no. NC_001510.1, nt 2312-2403) with mutations introduced in two ATG sites to abolish alternative translational start sites; cargo plasmid p336 contains a synthetic RK intron (W02009080720A1) with a mutation introduced in ATG to abolish alternative translational start site; and cargo plasmid p337 contains a SV40 intron with a mutation introduced in ATG to abolish alternative translational start site.

[0155] FIG. 7 is a graph depicting relative LIPA transgene mRNA expression in transfected HepG2 cells. LIPA expression was normalized using endogenous human GAPDH (hGAPDH) and calculation was performed using the AACt method. LIPA expression level of HepG2 cells transfected with p205 (set to value 1) was used as a reference for calculating relative expression. Dashed line indicates reference LIPA expression from p205. HepG2 cells transfected with a GFP expression plasmid and untransfected HepG2 cells of various cell passages were used as controls.

[0156] FIG. 8 is a Western blot depicting LAL protein expression in transfected HepG2 cells. HepG2 cells were transfected with p333, p334, p335, p336, p337 or p205 plasmids, or control plasmids expressing fluorescent proteins GFP or DsRED. Untransfected HepG2 cells served as a control. LAL protein expression was detected in the cell lysates by Western Blot with a polyclonal rabbit anti-LAL antibody (ORIGENE, #TA309730) and shows as ~42kD, ~52kD and ~58kD bands. Western blot (botom) to housekeeping protein with rabbit anti -GAPDH (Cell Signaling #5174) was used as a control.

[0157] FIGs. 9A-9C are graphs depicting LIPA mRNA splicing variants and efficiency of AAV9-205 (FIG. 9A), AAV9-335 (FIG. 9B), and AAV9-336 (FIG. 9C) in mouse liver tissue. Wildtype C57BL / 6J mice were transduced with 5E12 vg / kg and 2.5E13 vg / kg. Quantitative PCR was performed to evaluate splicing variants.

[0158] FIGs. 10A and 10B are Western blots depicting LAL protein expression in the liver (FIG. 10A) and serum (FIG. 10B). Adult male wildtype C57BL / 6J mice were dosed with vehicle (Veh), AAV9-205 produced by transient transfection in suspension culture in HEK293 cells followed by different purification steps (A) and (B) to achieve high-quality AAV, AAV9- 335 or AAV9-336 at 5E12 vg / kg or 2.5E13 vg / kg. LAL protein expression was detected with a monoclonal mouse anti-LAL antibody (Thermofisher, #MA5-15278). In FIG. 10A, liver samples were treated with PNGaseF (NEB, # P0709L) prior to electrophoresis to remove glycosylation and LAL shows as a ~35kD and ~40kD band. In FIG. 10B, serum samples were treated with PNGaseF prior to electrophoresis to remove glycosylation and LAL shows as a ~40kD band. The ~50kD and ~25kD bands are mouse IgG (light and heavy chain) in serum detected by a secondary anti-mouse antibody.

[0159] FIG. 11A-11C depict the production of LAL protein in Lipa knockout (KO) mice. FIG. 11A depicts a Western blot of liver tissue lysate from Lipa KO mice dosed with vehicle, AAV9-335 or AAV9-205 at the indicated dose. Each lane is a sample from an individual mouse. LAL protein is detected by a mouse monoclonal anti-LAL antibody (ThermoFisher #MA5-15278) at 1 : 1000 dilution and shows as a ~35kDa and a ~40kDa band. Samples were treated with PNGaseF prior to Western blotting to remove protein glycosylation. A protein ladder (first lane on the left) shows protein size in kDa. The last 7 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C- Myc / DDK tag. Western blot (bottom) depicting the amount of housekeeping protein GAPDH , was used as a control to normalize to the amount of LAL protein for each sample. FIG. 11B is a graph depicting a standard curve generated from the Western blot signal intensity from the recombinant LAL sample lanes in FIG. 11 A. FIG. 11C is a graph depicting the amount of LAL in the liver lysate based on the Western blot from FIG. 11 A. M = male, F = female, vg = vector genomes, kg = kilogram, ng = nanogram, kDa = kilodaltons.

[0160] FIG. 12 are graphs depicting blood neutrophil counts in male (left) and female (right) WT or Lipa KO mice dosed with vehicle (Veh) and Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose (vg / kg) injected. Each dot is from an individual mouse. Bars show the mean and error bars the SD.

[0161] FIGs. 13A and 13B are graphs depicting blood hemoglobin in mice. FIG. 13A is a graph depicting the levels of hemoglobin in 11 week old female homozygous Lipa KO mice after dose-dependent IV administration of scAAV9-335 or scAAV-205. Numbers under each bar are the dose (vg / kg) injected. Each dot is from an individual mouse. Bars show the mean and error bars the SD. FIG. 13B are graphs depicting male (left) and female (right) WT or

[0162] Lipa KO mice dosed with vehicle (Veh) and Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose (vg / kg) injected. Each dot is from an individual mouse. Bars show the mean and error bars the SD.

[0163] FIGs. 14A and 14B are graphs depicting serum alanine transaminase (ALT) in mice.

[0164] FIG. 14A is a graph depicting the levels of alanine transaminase (ALT) in 11 week old female homozygous Lipa KO mice after dose-dependent IV administration of scAAV9-335 or scAAV9-205. Numbers under each bar are the dose (vg / kg) injected. Each dot is from an individual mouse. Bars show the mean and error bars the SD. FIG. 14B are graphs depicting male (left) and female (right) WT or Lipa KO mice dosed with vehicle (Veh) and Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose (vg / kg) injected. Each dot is from an individual mouse. Bars show the mean and error bars the SD.

[0165] FIG. 15 are graphs depicting serum aspartate aminotransferase (AST) in male (left) and female (right) WT or Lipa KO mice dosed with vehicle (Veh) and Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose (vg / kg) injected. Each dot is from an individual mouse. Bars show the mean and error bars the SD.

[0166] FIGs. 16A and 16B depict liver to body weight (Liver / BW) and spleen to body weight (Spleen / BW) ratios in mice. FIG. 16A are graphs depicting Liver / BW (left) and Spleen / BW (right) ratios (expressed as %) of male (M) and female (F) wild-type (WT) and Lipa knockout (KO) 5 week old mice. Each dot is from an individual mouse. Bars show the mean and error bars the SD. *p<0.05, **p<0.01 and ***p<0.001 (one-way ANOVA followed by Tukey’s test). FIG. 16B are graphs depicting the Liver / BW (left) and Spleen / BW (right) ratios (expressed as %) of male (tope) and female (bottom) 5w and 1 Iw old homozygous wild-type (WT) and Lipa knockout (KO) mice. Liver / BW and Spleen / BW ratios were calculated by GraphPad software. Each dot represents the Liver / BW or Spleen / BW ratio from an individual mouse. The mean value with SD of each group is shown. WT = Organ from wild type mouse, KO = Organ from homozygous Lipa KO mouse, BW = body weight, SD = Standard deviation.

[0167] FIGs. 17A and 17B depict dose-dependent rescue of LAL-D phenotypes by AAV9- 335 in homozygous Lipa knockout (KO) mice. FIG. 17A provides representative images of dissected livers from 5 week old (left images) and 11 week old (right images) homozygous Lipa KO mice. 5 week old mice were IV dosed with either vehicle or AAV9-335. At six weeks post-dose, the liver morphology of 11 week old mice was analyzed. K0-N11: liver from 5w old female KO mouse Ni l; WT-N5: liver from 5w old female WT mouse N5; KO #35: liver from 1 Iw old female KO mouse dosed with scAAV-335 at 3.35E11 vg / kg; KO#11: liver from l lw old female KO mouse dosed with vehicle. The percentage of liver weight / body weight (BW) is shown under each image. FIG. 17B provides representative images of dissected spleens from 5 week old (left images) and 11 week old (right images) homozygous Lipa KO mice. 5 week old mice were IV dosed with either vehicle or AAV9-335. At six weeks postdose, the spleen morphology of l lw old mice were analyzed. K0-N11: spleen from female KO mouse N11; WT-N5: spleen from female WT mouse N5; KO #35: spleen from female KO mouse dosed with AAV9-335 at 3.35E11 vg / kg; KO#11: spleen from female KO mouse dosed with vehicle. The percentage of spleen weight / body weight (BW) is shown under each image. Each dot represents an individual mouse.

[0168] FIG. 18 are graphs depicting the ratio (expressed as %) of liver to body weight (Liver / BW) in 1 Iw old male (left) and female (right) Lipa KO mice after dose-dependent IV administration with AAV-335 or AAV-205 at 5 weeks of age. Wild-type (WT) mice and homozygous Lipa KO mice treated with vehicle (Veh) were used as controls. The mean value with standard deviation (SD) of each group is shown after analysis using GraphPad software. Each dot represents the Liver / BW ratio from an individual mouse, ns = not significant; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 (one-way ANOVA followed by Dunnett’s test).

[0169] FIG. 19 are graphs depicting the ratio (expressed as %) of spleen to body weight (Spleen / BW) in 1 Iw old male (left) and female (right) Lipa KO mice after dose-dependent IV administration with AAV-335 or AAV -205 at 5 weeks of age. Wild-type (WT) mice and homozygous Lipa KO mice treated with vehicle (Veh) were used as controls. The mean value with standard deviation (SD) of each group is shown after analysis using GraphPad software. Each dot represents the Spleen / BW ratio from an individual mouse, ns = not significant; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 (one-way ANOVA followed by Dunnett’s test).

[0170] FIGs. 20A and 20B are graphs depicting the accumulation score of foamy macrophage aggregates in the liver of mice. FIG. 20A arc graphs depicting male (left) and female (right)

[0171] WT or Lipa KO mice dosed with vehicle (Veh), and Lipa KO mice dosed with scAAV9-335.

[0172] Numbers under each bar are the dose vg / kg. Each dot is an individual mouse. Score of 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. FIG. 20B are graphs depicting male (left) and female (right) WT or Lipa KO mice dosed with vehicle (Veh), and Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose vg / kg. Each dot is an individual mouse. Score of 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

[0173] FIG. 21 are graphs depicting the accumulation score of foamy macrophage aggregates in the spleen of male (left) and female (right) WT or Lipa KO mice dosed with vehicle (Veh) and Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose vg / kg. Each dot is an individual mouse. Score of 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

[0174] FIG. 22 are graphs depicting the accumulation score of foamy macrophage aggregates in the small intestine (duodenum) of male (left) and female (right) WT or Lipa KO mice dosed with vehicle (Veh) and Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose vg / kg. Each dot is an individual mouse. Score of 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

[0175] FIG. 23 are graphs depicting liver total cholesterol content in male (left) and female (right) mice 6 weeks after IV dosing as measured by an enzymatic cholesterol assay kit. Shown are Lipa KO mice dosed with vehicle, naive 5 week old Lipa KO mice (baseline), naive 5wk old WT mice, WT mice dosed with vehicle, and each dosing group of Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose vg / kg. Each dot is the value from an individual mouse, ns, not significant and ****p<0.0001 (one-way ANOVA followed by Dunnett’s test).

[0176] FIG. 24 are graphs depicting inferred liver cholesterol ester in male (left) and female (right) mice as measured by an enzymatic cholesterol assay kit. Cholesterol ester (CE) values were calculated by subtracting the free cholesterol measurement from the total cholesterol measurement for each sample; negative CE values were assumed 0. Each dot is the liver CE value from an individual mouse. Shown are Lipa KO mice dosed with vehicle at 6 weeks after IV dosing, naive 5 week old Lipa KO mice (baseline), naive 5 week old WT mice, WT mice dosed with vehicle, and each dosing group oiLipa KO mice dosed with scAAV9-335. Numbers under each bar is the dose vg / kg. ns, not significant and ****p<0.0001 (one-way ANOVA followed by Dunnett’s test).

[0177] FIG. 25 are graphs depicting spleen total cholesterol content in male (left) and female (right) mice as by an enzymatic cholesterol assay kit. Each dot is the value from an individual mouse. Shown are Lipa KO mice dosed with vehicle at 6 weeks after IV dosing, naive 5 week old Lipa KO mice (baseline), naive 5wk old WT mice, WT mice dosed with vehicle, and each dosing group <Lt Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose vg / kg. ns, not significant; ** p<0.01 and ****p<0.0001 (one-way ANOVA followed by Dunnett’s test).

[0178] FIG. 26 are graphs depicting inferred spleen cholesterol ester in male (left) and female (right) mice as by an enzymatic cholesterol assay kit. Cholesterol ester (CE) values were calculated by subtracting the free cholesterol measurement from the total cholesterol measurement for each sample; negative CE values were assumed 0. Each dot is the spleen CE value from an individual mouse. Shown are Lipa KO mice dosed with vehicle at 6 weeks after IV dosing, naive 5 week old Lipa KO mice (baseline), naive 5wk old WT mice, WT mice dosed with vehicle, and each dosing group of Lipa KO mice dosed with scAAV9-335. Numbers under each bar are the dose vg / kg. ns, not significant; ** p<0.01 and ****p<0.0001 (one-way ANOVA followed by Dunnett’s test). FIG. 27 depicts vector genome biodistribution in liver, skeletal muscle, small intestine and spleen of Lipa KO mice dosed with 6.7E12vg / kg, 3.35E13vg / kg and 6.7E13vg / kg of scAAV-335. Lipa KO mice dosed with vehicle was used as a control. Bar graph values were calculated as vector genomes per diploid genome and reported as median. Each dot represents value from an individual mouse.

[0179] FIG. 28 is a bar graph depicting LIPA mRNA expression in liver, skeletal muscle, small intestine and spleen oiLipa KO mice dosed with 6.7E12vg / kg, 3.35E13vg / kg and 6.7E13vg / kg of scAAV9-335. Lipa KO mice dosed with vehicle was used as a control. Bar graph values were calculated as cDNA copies per microgram of RNA and reported as median. Each dot represents value from an individual mouse.

[0180] FIG. 29 is a Western blot depicting dose-dependent expression of LAL protein in liver tissue lysate from Lipa KO mice 6 weeks after dosing with vehicle or scAAV9-335 at the indicated dose in vg / kg. Each lane is a sample from an individual mouse, which was treated with PNGaseF prior to Western blotting to remove protein glycosylation. ~20pg of total liver protein was loaded per lane. LAL protein was detected by a mouse monoclonal anti-LAL antibody (ThermoFisher #MA5-15278) at 1: 1000 dilution and shows as a ~35kDa and a ~40kDa band. ~25kD and ~50kD bands are mouse IgG in serum reacting to an anti-mouse secondary antibody. A protein ladder (first lane on the left) shows protein size in kDa. The last 6 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C-Myc / DDK tag. Blot with antibody detecting housekeeping protein GAPDH (Cell Signaling #5174) at 1: 1000 dilution is shown for each sample as a control. M = male mouse, F = female mouse, ng = nanogram, kDa = kilodaltons.

[0181] FIG. 30 is a Western blot depicting dose-dependent expression of LAL protein in liver tissue lysate from Lipa KO mice 6 weeks after dosing with vehicle or scAAV9-335 at the indicated dose in vg / kg. Each lane is a sample from an individual mouse, which was treated with PNGaseF prior to Western blotting to remove protein glycosylation. ~2pg of total liver protein was loaded per lane. LAL protein was detected by a mouse monoclonal anti-LAL antibody (ThermoFisher #MA5-15278) at 1: 1000 dilution and shows as a ~35kDa and a ~40kDa band. ~25kD and ~50kD bands are mouse IgG in serum reacting to an anti-mouse secondary antibody. A protein ladder (first lane on the left) shows protein size in kDa. The last 6 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C-Myc / DDK tag. Blot with antibody detecting housekeeping protein GAPDH (Cell Signaling #5174) at 1: 1000 dilution is shown for each sample as a control. M = male mouse, F = female mouse, ng = nanogram, kDa = kilodaltons. FIG. 31 are Western blots depicting dose-dependent expression of LAL protein in liver tissue lysate from Lipa KO mice 6 weeks after dosing with vehicle or scAAV9-335 at the indicated dose vg / kg. Each lane is a sample from an individual mouse, which was treated with PNGaseF prior to Western blotting to remove protein glycosylation. ~ 1 pg of total liver protein was loaded per lane. LAL protein was detected by a mouse monoclonal anti -LAL antibody (ThermoFisher #MA5-15278) at 1: 1000 dilution and shows as a ~35kDa and a ~40kDa band. A protein ladder (first lane on the left) shows protein size in kDa. The last 6 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C- Myc / DDK tag. Blot with antibody detecting housekeeping protein GAPDH (Cell Signaling #5174) at 1: 1000 dilution is shown for each sample as a control. M = male mouse, F = female mouse, ng = nanogram, kDa = kilodaltons.

[0182] FIG. 32 is a graph depicting LAL protein in Serum in 6 week old male (M) and female (F) mice dosed with vehicle (Veh), AAV9-205 at 6.70el3 vg / kg, or AAV9-335 at 8.58el3 vg / kg. Each dot represents an individual mouse, ns = not significant; *p<0.05, **p<0.01 and ****p<0.0001 (one-way ANOVA followed by Tukey’s test).

[0183] FIG. 33 are Western blots depicting dose -dependent expression of LAL protein in serum from Lipa KO mice 6 weeks after dosing with vehicle or scAAV9-335 at the indicated dose in vg / kg. Each lane is a sample from an individual mouse, which was treated with PNGase F prior to Western blotting to remove protein glycosylation. -0.25 pL of serum was loaded per lane. LAL protein was detected by a mouse monoclonal anti -LAL antibody (ThermoFisher #MA5-15278) at 1: 1000 dilution and shows as a ~35kDa and a ~40kDa band. ~25kD and ~50kD bands are mouse IgG in serum reacting to an anti-mouse secondary antibody. A protein ladder (first lane on the left) shows protein size in kDa. The last 5 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C-Myc / DDK tag. M = male mouse, F = female mouse, ng = nanogram, kDa = kilodaltons.

[0184] FIG. 34 is a Western blot depicting the expression of LAL protein in serum from Lipa KO mice at indicated timepoints after IV dosing with vehicle or scAAV9-335 at 6.70E13 vg / kg. Each lane is a sample from an individual mouse at each indicated timepoint, which was treated with PNGase F prior to Western blotting to remove protein glycosylation. ~0.15pL of serum was loaded per lane. LAL protein was detected by a mouse monoclonal anti -LAL antibody (ThermoFisher #MA5-15278) at 1: 1000 dilution and shows as a ~35kDa and a ~40kDa band. ~25kD and ~50kD bands are mouse IgG in serum reacting to an anti-mouse secondary antibody. A protein ladder (first lane on the left) shows protein size in kDa. The last 5 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C-Myc / DDK tag. Wk = week, M = male mouse, F = female mouse, ng = nanogram, kDa = kilodaltons.

[0185] FIG. 35 are Western blots depicting expression of LAL protein in liver, serum and spleen lysate from Lipa KO mice at 6 weeks after IV dosing with vehicle or scAAV9-335 at 6.70E13 vg / kg. Each lane is a sample from an individual mouse, which was treated with PNGase F prior to Western blotting to remove protein glycosylation. ~lpg of total liver or spleen protein or 0.5pL of serum was loaded per lane. LAL protein was detected by a mouse monoclonal anti-LAL antibody (ThermoFisher #MA5-15278) at 1: 1000 dilution and shows as a ~35kDa and a ~40kDa band. ~25kD and ~50kD bands are mouse IgG in serum reacting to an anti-mouse secondary antibody. A protein ladder (first lane on the left) shows protein size in kDa. The last 4 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C-Myc / DDK tag. Blot with antibody detecting housekeeping protein GAPDH (Cell Signaling #5174) at 1: 1000 dilution is shown sample as a control; note that serum does not contain GAPDH. Li = liver, Se = Serum, Sp = spleen, M = male mouse, F = female mouse, ng = nanogram, kDa = kilodaltons.

[0186] FIG. 36 are Western blots depicting the detection of LAL protein in spleen tissue lysate from Lipa KO mice 6 weeks after dosing with vehicle or scAAV9-335 at the indicated dose vg / kg. Each lane is a sample from an individual mouse, which was treated with PNGase F prior to Western blotting to remove protein glycosylation. ~5pg of total spleen protein was loaded per lane. LAL protein was detected by a mouse monoclonal anti-LAL antibody (ThermoFisher #MA5-15278) at 1: 1000 dilution and shows as a ~35kDa and a ~40kDa band. ~25kD and ~50kD bands are mouse IgG in serum reacting to an anti-mouse secondary antibody. A protein ladder (first lane on the left) shows protein size in kDa. The last 6 lanes are different amounts per lane of recombinant LAL protein (ORIGENE #TP301637) with an attached C-Myc / DDK tag. Blot with antibody detecting housekeeping protein GAPDH (Cell Signaling #5174) at 1 : 1000 dilution is shown for each sample as a control. M = male mouse, F = female mouse, ng = nanogram, kDa = kilodaltons.

[0187] FIGs. 37A and 37B are graphs depicting mRNA expression using qRT-PCR results of Cxcl5 (FIG. 37A) and Tnf (FIG. 37B) genes in the liver of Lipa KO mice dosed with scAAV9- 335 and scAAV9-205 at the indicated amounts. In each liver sample, the fold change of Cxcl5 mRNA or Tnf mRNA level (relative to Hprt mRNA level) was calculated compared to the corresponding value in vehicle-treated wild-type liver (WT-veh). The average fold change of Cxcl5 or Tnf mRNA level in vehicle-treated Lipa KO liver (KO-veh) was considered as 100%, and the ratio between the fold change in each liver sample was compared to the average fold change in the KO-veh liver. Number under each bar is the dose (vg / kg) injected. Each dot represents an individual mouse. Bars show the mean and SD error bars. ns=not significant, *p<0.05, ** p<0.01, and ***p<0.001 (one-way ANOVA followed by Dunnett’s test).

[0188] DETAILED DESCRIPTION OF THE INVENTION

[0189] The present disclosure relates to gene replacement therapy compounds comprising a LIPA transgene encoding lysosomal acid lipase (LAL), such as polynucleotides, recombinant expression vectors (e.g., AAV vectors), viral particles (e.g., rAAV particles), and pharmaceutical compositions thereof. The present disclosure also relates to methods for treating Lysosomal Acid Lipase Deficiency (LAL-D) disorders caused by mutations in the LIPA gene, such as Wolman Disease and cholesterol ester storage disease (CESD), and non-genetic disorders that are associated with lipid accumulation and storage, through the administration of a gene replacement therapy as provided herein. Upon administration, the LIPA transgene is expressed in target cells or tissue to generate the LAL protein or gene product to produce a therapeutic effect.

[0190] Several aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. One having ordinary skill in the relevant art, however, will readily recognize that the features described herein can be practiced without one or more of the specific details or with other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts can occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein. The terminology of the present disclosure is for the purpose of describing particular cases only and is not intended to be limiting of companions, methods and compositions of this disclosure.

[0191] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, . (Sambrook, et al., 2nd Ed, 1989) Cold Spring Harbor Press; Molecular cloning: a laboratory manual (Sambrook and Green, 4th Ed, 2012); Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, etal. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985); Polynucleotide Hybridization (B. D. Hames & S. J. Higgins eds. (1985; Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984; Animal Cell Culture (R. I. Freshney, ed. (1986; Immobilized Cells and Enzymes (IRL Press, (1986; and B. Perbal, A practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), all of which are herein incorporated by reference in their entirety for all purposes. These techniques are applicable to the production of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0192] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0193] Definitions

[0194] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention pertains or relates. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[0195] Throughout this disclosure, various aspects can be presented in a range format. Ranges provided herein are understood to be shorthand for all of the values within the range, inclusive of the first and last stated values. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96- 99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range. All specified ranges also include the endpoints unless otherwise stated.

[0196] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely examples and that equivalents of such are known in the art.

[0197] In this application, the use of the singular includes the plural unless specifically stated otherwise. The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. Unless specifically stated or obvious from context, as used herein, the terms “a,” “an,” and “the” are understood to be singular or plural and refer to one or more than one (at least one) of the grammatical object of the article. Similarly, unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Thus, the term “or” means “and / or” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B . It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

[0198] As used herein, the terms “about” or “approximately” typically refer to the value that immediately follows. For example, “about 15 or more contiguous nucleotides” typically refers to 15 or more contiguous nucleotides. Unless specifically stated or obvious from context, as used herein, the terms “about” or “approximately” mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within one (1) or more than one (1) standard deviation, per the practice in the art. In some aspects, the terms “about” or “approximately” embrace values that are within one (1), two (2), or three (3) standard deviations (SD) of the mean. For example, “about 15 or more contiguous nucleotides” may refer to 15 + / - 3 contiguous nucleotides, e.g. 12, 13, 14, 15, 16, 17 or 18 contiguous nucleotides. Alternatively, “about” or “approximately” can mean a range within 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Alternatively, particularly with respect to biological systems or processes, the terms can mean within an order of magnitude, e.g., within 5-fold, within 2-fold, etc., of a value. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 0.1. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about” or “approximately.”

[0199] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes,” “include,” and “included”) or “containing” (and any form of containing, such as “contains,” “contained,” and “contain”) have the meaning ascribed to them in U.S. Patent law, are not limiting, and are meant to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0200] The terms “consist”, “consists”, “consisting” or the like likewise have the meaning ascribed in U.S. Patent law and limits the scope of a claim to the specified elements, materials or steps and does not materially affect the basic and novel characteristics of the claimed invention. It excludes any additional elements, materials or steps regardless of their obviousness or practicality. In all cases where the term “comprise”, “comprises”, “comprising” or the like are used in reference to a sequence (e.g., an amino acid sequence), it shall be understood that said sequence may also be limited by the term “consist”, “consists”, “consisting” or the like.

[0201] “Consisting essentially of’ or “consists essentially” likewise have the meaning ascribed in U.S. Patent law and are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. As used herein, the phrase “consisting essentially of’ refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any excipients inactive for the intended purpose of the methods or compositions. In some aspects, the phrase “consisting essentially of’ expressly excludes the inclusion of one or more additional active agents other than a polynucleotide, recombinant expression vector, viral particle, or pharmaceutical composition thereof as provided herein. In some aspects, the phrase “consisting essentially of’ expressly excludes the inclusion of one or more additional active agents other than a polynucleotide, recombinant expression vector, viral particle, or pharmaceutical composition thereof as provided herein and a second co-administered agent.

[0202] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0203] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0204] The term “adeno-associated virus” or “AAV” refers to members of the Dependovirus genus or a variant, e.g., a functional variant, thereof. In some embodiments, the AAV is wildtype, or naturally occurring. In some embodiments, the AAV is recombinant. In some embodiments, the AAV is selected from a specific AAV serotype, which includes, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh, AAVrhlO, AAVrh74, AAV-DJ, AAV-DJ / 8, Anc80, AAV7m8, or any derivative, hybrid or chimeric AAV thereof. In some embodiments, the AAV has a particular tropism (e.g., liver tropism). In some embodiments, the AAV has a liver tropism. In some embodiments, the AAV serotype is an AAV9 serotype.

[0205] The nucleotide sequences of the genomes of the AAV serotypes are known in the art. For example, the complete genome of AAV1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV2 is provided in GenBank Accession No. NC 001401 and is presented in Srivastava et al., J Viral, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Viral, 75; the complete genome of AAV3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV4 is provided in GenBank Accession No. NC_001829; the AAV5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 relating to AAV-8); the AAV9 genome is provided in Gao etal., (2004) J. Virol., 78: 6381-6388; the AAV10 genome is provided in Williams, (2006) Mol. Ther., 13(1): 67-76; and the AAV11 genome is provided in Mori et al., (2004) Virology, 330(2): 375-383. Cloning of the AAVrh74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007).

[0206] The term "AAV helper construct" refers generally to a nucleic acid molecule that includes nucleotide sequences providing or encoding proteins or nucleic acids that provide AAV functions deleted from an AAV vector, e.g. a vector for delivery of a nucleotide sequence of interest to a target cell or tissue. AAV helper constructs are commonly used to provide transient expression of AAV rep and / or cap genes to provide adenovirus-derived regulatory functions for AAV replication. Typically, helper constructs lack AAV ITRs and can neither replicate nor package themselves. AAV helper constructs may be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been disclosed, such as the commonly used plasmids pAAV / Ad and plM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al., (1989) J. Virol., 63:3822-3828; McCarty etal., (1991) J. Virol., 65:2936-2945. A number of other vectors have been disclosed which encode Rep and / or Cap expression products. See, e.g., U.S. Pat. Nos. 5,139,941 and 6,376,237. Embodiments of these vector constructs and methods of preparing and purifying them are provided, e.g., in WO / 2019 / 094253 (PCT / US2018 / 058744), which is incorporated herein by reference in its entirety. In some embodiments, the AAV helper construct is a pHELP helper plasmid. In some embodiments, the AAV helper construct (e.g., pHelp) encodes adenovirus regulatory elements (e.g., E2A, E4, and / or VAI / II RNA genes). In some embodiments, the AAV helper construct is an AAV plasmid containing the AAV rep2 and cap9 wildtype genes (Rep2-Cap9 plasmid).

[0207] The term “AAV helper function" refers to an AAV-derived coding sequences which can be expressed to provide AAV gene products, e.g. , those that function in trans for productive AAV replication. For instance, AAV helper functions may include both of the major AAV open reading frames (ORFs), rep and cap. The Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. The Cap expression products supply necessary packaging functions. AAV helper functions may be used herein to complement AAV functions in trans that are missing from AAV vectors.

[0208] An “AAV vector” or “rAAV vector” as used herein refers to an adeno-associated virus (AAV) vector or a recombinant AAV (rAAV) vector comprising one or more nucleic acid sequences not of AAV origin (i.e., a polynucleotide heterologous to AAV such as a polynucleotide sequence of interest or that encodes a therapeutic transgene, e.g., LIPA), typically a sequence of interest for the genetic transformation of a cell. AAV vector or rAAV vector nucleic acid sequences may be derived from an adeno-associated virus serotype, including without limitation, an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh, AAVrhlO, AAVrh74, AAV-DJ, AAV-DJ / 8, Anc80, AAV7m8, or any derivative, hybrid or chimeric AAV or rAAV viral vector thereof. In some embodiments, the AAV serotype has a particular tropism (e.g., liver tropism). In some embodiments, the AAV serotype has a liver tropism. In some embodiments, the serotype is an AAV9 serotype, or any derivative, hybrid or chimeric AAV9 thereof. AAV vectors may have one or more of the AAV wild-type genes deleted in whole or part, e.g., the rep and / or cap genes, while retaining, e.g., functional flanking inverted terminal repeat (ITR) sequences. In some embodiments, an AAV or rAAV vector may be packaged in a protein shell or capsid, e.g., comprising one or more AAV or rAAV capsid proteins, which may provide a vehicle for delivery of vector nucleic acid to the nucleus of target cells. In some embodiments, an AAV or rAAV vector comprises one or more AAV ITR sequences (e.g., AAV2 ITR sequences). In some embodiments, an AAV or rAAV vector comprises one or more AAV ITR sequences (e.g. , AAV2 ITR sequences) and one or more additional nucleic acid sequences of AAV origin. In some embodiments, an AAV or rAAV vector comprises one or more AAV ITR sequences (e.g, AAV2 ITR sequences) but does not contain any additional viral nucleic acid sequence. Embodiments of these vector constructs are provided, e.g., in WO / 2019 / 094253 (PCT / US2018 / 058744), which is incorporated herein by reference in its entirety. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. A rAAV vector may either be single -stranded (ssAAV) or self-complementary (scAAV). In some embodiments, the rAAV vector is a ssAAV vector. In some embodiments, the rAAV vector is a scAAV vector.

[0209] A “single-stranded viral particle” is a particle comprised of at least two components, a protein capsid and a single-stranded viral genome. In some embodiments, a “single-stranded viral particle” is a single-stranded adeno-associated virus (ssAAV). For ssAAV vectors, the coding sequence and complementary sequence of the transgene expression cassette are on separate strands and are packaged in separate viral capsids. In some embodiments, a viral vector is engineered from a naturally occurring AAV to provide an ssAAV for use in gene therapy.

[0210] A “self-complementary viral particle” is a particle comprised of at least two components, a protein capsid and a self-complementary viral genome enclosed within the capsid. In some embodiments, a “self-complementary viral particle” is a self-complementary adeno-associated virus (scAAV). For scAAV vectors, at least a portion of the vector (e.g, at least a portion of the coding region) of the scAAV forms an intra-molecular double -stranded DNA. In some embodiments, a viral vector is engineered from a naturally occurring AAV to provide an scAAV for use in gene therapy. Embodiments of these vector constructs and methods of preparing and purifying them are provided, e.g., in WO / 2019 / 094253 (PCT / US2018 / 058744), which is incorporated herein by reference in its entirety.

[0211] The term “AAV particle” or “AAV viral particle” as used herein is intended to refer to a non-wild-type and / or artificially produced viral particle or virion composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV or an AAV capsid variant) (e.g., a parvovirus, adenovirus, lentivirus or adeno-associated virus, etc.) that comprises a gene or other heterologous nucleic acid (e.g., encoding a payload) and is capable of delivering the gene or other heterologous nucleic acid to cells, typically, mammalian, e.g., human, cells. In some embodiments, an AAV particle of the present disclosure may be produced recombinantly. In some embodiments, an AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary). In some embodiments, the AAV particle may be replication defective and / or targeted. The AAV particle may comprise a recombinant viral genome (e.g., comprising a nucleic acid encoding the gene of interest (e.g. , LIPA) packaged within a viral (e.g. , AAV) capsid. In some embodiments, an AAV particle may be a “recombinant adeno-associated virus particle” or “recombinant AAV (rAAV) particle.” The terms “viral genome” or “vector genome” refer to the nucleic acid sequence(s) encapsulated in an AAV particle. A viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.

[0212] In some embodiments, terms such as “virus,” “virion,” “viral particle,” “AAV virus,” “AAV virion,” “recombinant AAV virion,” “AAV viral particle,” “AAV vector particle,” “full capsids,” “full particles,” and the like refer to a viral particle, comprising a viral vector, e.g., alone or in combination with one or more additional components such as one or more viral capsids. In some embodiments, a viral particle is composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (z.e., a polynucleotide other than a wild-type AAV genome such as a transgene (e.g., LIPA) to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector". Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle. In some embodiments, an AAV virus may comprise, e.g., a linear, single -stranded AAV nucleic acid genome associated with an AAV capsid protein coat. In some embodiments, an AAV virus may comprise an infectious, replication-defective virus, e.g., those comprising an AAV protein shell encapsidating a heterologous nucleotide sequence of interest, e.g., in a viral vector which is flanked on one or both sides by AAV ITRs. A recombinant AAV virion may be produced in a suitable host cell which comprises sequences, e.g. , one or more plasmids, specifying an AAV vector, alone or in combination with nucleic acids encoding AAV helper functions and accessory functions (such as cap genes), e.g., on the same or additional plasmids. In some embodiments, the host cell is rendered capable of encoding AAV polypeptides that provide for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0213] By “administer” or “administering” or “administration” is meant providing, giving, supplying, or dispensing one or more compositions, agents, therapeutics and the like to a subject or patient, or applying or bringing the composition and the like into contact with the subject or patient. Administering or administration may be accomplished by any of a number of routes, such as, for example, without limitation, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous (IV), injection, intrathecal, intramuscular, dermal, intradermal, intracranial, inhalation, rectal, intravaginal, or intraocular. In some embodiments, administration is intravenous (IV). In some embodiments, administration is via the portal vein. In some embodiments, administration is intramuscular. By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. In some embodiments, an agent is a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein. The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect.

[0214] The term “ameliorate” means to decrease, reduce, delay, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or pathological condition (e.g., LAL-D (e.g., Wolman Disease or CESD)).

[0215] The term “amplicon” may refer to any piece of RNA or DNA formed as the product of amplification events, e.g., PCR. In some embodiments, full-length capsid amplicons may be used as templates for next generation sequencing (NGS) library generation. Full-length capsid amplicons may be used for cloning into a DNA library for any number of additional rounds of AAV selection as described herein.

[0216] The term “capsid” refers to the exterior, e.g. , a protein shell, of a virus particle, e.g. , an AAV particle, that is substantially (e.g., >50%, >60%, >70%, >80%, >90%, >95%, >99%, or 100%) protein. In some embodiments, the capsid is an AAV capsid comprising an AAV capsid protein described herein. The AAV capsid protein can be a wild-type AAV capsid protein or a variant, e.g., a structural and / or functional variant from a wild-type or a reference capsid protein, referred to herein as an “AAV capsid variant.” In some embodiments, the AAV capsid protein is a hybrid or chimeric capsid protein, e.g. , having at least one CAP protein from AAV9 (e.g., VP1, VP2, orVP3) or another AAV with liver tropism and at least one CAP protein from another serotype. In some embodiments, the AAV capsid variant described herein has the ability to enclose, e.g., encapsulate, a viral genome and / or is capable of entry into a cell, e.g., a mammalian cell. In some embodiments, the AAV capsid variant described herein may have modified tropism compared to that of a wild-type AAV capsid, e.g., the corresponding wildtype capsid.

[0217] The term “complementary” refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can form base pairs in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenine. However, when a U is denoted in the context of the present disclosure, the ability to substitute a T is implied, unless otherwise stated. Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can form a hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can form a hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can form hydrogen bonds with each other, the polynucleotide strands exhibit 90% complementarity. The term “complementary” as used herein can encompass fully complementary, partially complementary, or substantially complementary. As used herein, the term “substantially complementary” means that the siRNA has a sequence (e.g. , in the antisense strand) which is sufficient to bind the desired target mRNA, and to trigger the RNA silencing of the target mRNA. “Fully complementary”, “perfect complementarity”, or “100% complementarity” refers to the situation in which each nucleotide unit of one polynucleotide or oligonucleotide strand can base-pair with a nucleotide unit of a second polynucleotide or oligonucleotide strand.

[0218] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. In certain circumstances, spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0219] By “codon optimization” is meant gene engineering approaches that use synonymous codon changes to improve gene expression, improve RNA stability, and / or increase the translational efficiency of a gene of interest (e.g. , LIPA). Synonymous codon changes typically do not alter the primary amino acid sequence and have no effect on protein structure and function. In some embodiments, the gene of interest (e.g. , LIPA) is codon optimized. In some embodiments, the gene of interest (e.g, LIPA) is codon optimized to enhance LAL protein expression and / or human expression. In some embodiments, a codon-optimized sequence shows at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more increase in gene expression, transcription, RNA stability, and / or translation compared to the original, not codon-optimized sequence.

[0220] The term “combination” as used herein refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present invention and a combination partner (e.g. another drug, also referred to as “therapeutic agent” or “co- agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non-fixed combinations of the therapeutic agents. The term “fixed combination” means that the therapeutic agents, e.g. a compound of the present invention and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the therapeutic agents, e.g., a compound of the present invention and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more therapeutic agents. The term “pharmaceutical combination” as used herein refers to either a fixed combination in one dosage unit form, or non-fixed combination or a kit of parts for the combined administration where two or more therapeutic agents may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect.

[0221] The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g., tablets, capsules, powders, and liquids) for each active ingredient. Powders and / or liquids may be reconstituted or diluted to a desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.

[0222] The term “delivery” refers to the act or manner of delivering an AAV particle, a compound, substance, entity, moiety, cargo or payload.

[0223] The terms “DNA regulatory sequences”, “control elements,” “regulatory control elements,” “regulatory sequences,” and “regulatory elements,” used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, internal ribosome entry sites (IRES), transcription termination sequences, upstream regulatory domains, protein degradation signals, origin of replication, selection cassettes, introns, 5' and 3' untranslated regions (UTRs) and the like, which provide for and / or regulate the replication, transcription and / or translation of a non-coding sequence or a coding sequence and / or regulate translation of an encoded polypeptide in a cell. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters, tissue-specific promoters (e.g. , liver, spleen), and inducible promoters may be used. Not all of these control elements need always be present as long as the selected coding sequence is capable of being replicated, transcribed and / or translated in an appropriate host cell (e.g., hepatocyte, macrophage, spleen cell). Such elements may be operably linked to the sequence they are regulating.

[0224] By “disease” is meant any condition, disorder, or pathology that damages or interferes with the normal function of a cell, tissue, or organ. As used herein, the terms “condition,” “disease,” and “disorder” are used interchangeably. A “disease” refers to a state of being or health status of a patient or subject capable of being treated with the compounds, compositions, or methods provided herein. In some embodiments, the disease is a Lysosomal Acid Lipase Deficiency (LAL-D). In some embodiments, the disease is Wolman Disease. In some embodiments, the disease is cholesterol ester storage disease (CESD).

[0225] By “effective amount” is meant the amount of an active therapeutic agent, composition, compound, or biologic (e.g., AAV, vaccine, polypeptide, or polynucleotide) required to achieve a desired therapeutic effect. An effective amount can be administered in one or more administrations, applications, or dosages. In some embodiments, an effective amount is the amount required to ameliorate, reduce, delay, improve, abrogate, diminish, or eliminate the symptoms and / or effects of a disease, condition, or pathology (e.g., LAL-D (e.g., Wolman Disease or CESD)) relative to an untreated subject or a subject without disease, z.e., a healthy individual, or is the amount of the antibody or antigen-binding fragment thereof sufficient to elicit a desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount used to practice the present invention for therapeutic treatment of a disease (e.g., LAL-D (e.g., Wolman Disease or CESD)) varies depending upon the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the manner of administration, and the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.

[0226] An effective amount encompasses therapeutic and prophylactic treatment. The terms “therapeutically acceptable amount,” “therapeutically effective amount,” and “therapeutically effective dose” interchangeably refer to an amount sufficient to effect the desired result (e.g., a reduction in disease activity, inhibition of disease progression, etc.). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A “prophylactically effective dosage,” and a “therapeutically effective dosage,” of the molecules of the present disclosure can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with LAL-D (e.g., Wolman Disease or CESD).

[0227] In some embodiments, an effective amount is the amount required to achieve a therapeutic effect in a subject or patient. In some embodiments, the subject or patient is a human subject or patient. In one embodiment, an effective amount is sufficient to ameliorate one or more symptoms of a disease (e.g., LAL-D (e.g., Wolman Disease or CESD)). In some embodiments, an effective amount is the amount required to increase LIPA expression in a subject. In some embodiments, an effective amount of a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein is the amount required to ameliorate, reduce, delay, improve, abrogate, diminish, or eliminate the symptoms and / or effects of LAL-D (e.g. , Wolman Disease or CESD) in a subject that has been administered with the polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof. In some embodiments, an effective amount of a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein is the amount required to ameliorate, reduce, delay, improve, abrogate, diminish, or eliminate the symptoms and / or effects of Wolman Disease in a subject that has been administered with the polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof. In some embodiments, symptoms associated with Wolman Disease include, but are not limited to, massive hepatosplenomegaly, acute liver failure (typically at under 4 months of age), multi-organ failure, microvesicular steatosis, malabsorption, jaundice, portal hypertension, failure to thrive, adrenal calcification, gastrointestinal disturbances (e.g. , diarrhea and vomiting with steatorrhea) that may lead to abdominal distension, and immune complications (increased susceptibility to infections and Hemophagocytic Lymphohistiocytosis (HLH)). In some embodiments, an effective amount of a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein is the amount required to ameliorate, reduce, delay, improve, abrogate, diminish, or eliminate the symptoms and / or effects of CESD in a subject that has been administered with the polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof. In some embodiments, symptoms associated with CESD include, but are not limited to, dyslipidemia (elevated LDL-C, low HDL-C), elevated liver transaminases, hepatomegaly, splenomegaly, accelerated atherosclerosis, abnormal hepatic function, hepatosplenomegaly, hepatic fibrosis and cirrhosis, portal hypertension, decompensated liver disease, and gastrointestinal disturbances (e.g., diarrhea and vomiting with steatorrhea) (Pericleous et al., 2017). Clinical features can be subtle, leading to misdiagnosis of patients as having Niemann-Pick disease, Gaucher disease, metabolic dysfunction associated steatotic liver disease (MASLD), metabolic dysfunction associated steatohepatitis (MASH), hereditary dyslipidaemia, or cryptogenic cirrhosis. Symptoms, and thus also a decrease in symptoms, can be assessed using a variety of methods known in the art, such as the same methods as used in the diagnosis of a disease (e.g., LAL-D (e.g., Wolman Disease or CESD), including but not limited to clinical examination and routine laboratory tests, macroscopic and microscopic methods, molecular methods, radiographic methods, and the like.

[0228] In some embodiments, an effective amount of a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein is the amount required to decrease triglycerides, cholesterol, and / or fatty acids in a subject that has been administered with the polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof.

[0229] The term “encapsulate” means to enclose, surround or encase. As an example, a capsid protein, e.g., an AAV capsid variant, often encapsulates a viral genome. In some embodiments, encapsulate within a capsid, e.g., an AAV capsid variant, encompasses 100% coverage by a capsid, as well as less than 100% coverage, e.g., 95%, 90%, 85%, 80%, 70%, 60% or less. For example, gaps or discontinuities may be present in the capsid so long as the viral genome is retained in the capsid, e.g., prior to entry into a cell.

[0230] The term “enhancer” refers to a segment of DNA, which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. In some embodiments, an enhancer is of mammalian or viral origin. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. In some embodiments, the enhancer is operably linted to a promoter or other element. In some embodiments, the enhancer is a CMV enhancer (e.g., SEQ ID NO: 21). In some embodiments, the enhancer is a hepatic control region enhancer . In some embodiments, the hepatic control region enhancer is a hepatic control region 1 HCRl) enhancer of the Apolipoprotein E gene (ApoE HCRE) (e.g. , SEQ ID NO: 22).

[0231] As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post- translational modification of a polypeptide or protein.

[0232] The term “expression vector” or “expression construct” or “cassette” or “plasmid” or simply “vector” can include any type of genetic construct, including AAV or rAAV vectors, containing a polynucleotide coding for a gene product in which part or all of the polynucleotide encoding sequence is capable of being transcribed and is adapted for gene therapy. The transcript can be translated into a protein. In some cases, it may be partially translated or not translated. In certain aspects, expression includes both transcription of a gene and translation of mRNA into a gene product. In other aspects, expression only includes transcription of the polynucleotide encoding genes of interest.

[0233] The term “plasmid” refers to a nonchromosomal (and typically double -stranded) DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. A plasmid may be a circular nucleic acid. When the plasmid is placed within a unicellular organism, the characteristics of that organism are changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (TcR) transforms a cell previously sensitive to tetracycline into one which is resistant to it.

[0234] The term “vector” refers to any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule. In some embodiments, a "vector" is any genetic element (e.g., DNA, RNA, or a mixture thereof) that contains a nucleic acid of interest that is capable of being expressed in a host cell, e.g., a nucleic acid of interest within a larger nucleic acid sequence or structure suitable for delivery to a cell, tissue, and / or organism, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. For instance, a vector may comprise an insert (e.g., a heterologous nucleic acid encoding a gene to be expressed or an open reading frame of that gene) and one or more additional elements, e.g. , elements suitable for delivering or controlling expression of the insert. The vector may be capable of replication and / or expression, e.g., when associated with the proper control elements, and it may be capable of transferring genetic information between cells. In some embodiments, a vector may be a vector suitable for expression in a host cell, e.g., an AAV vector. In some embodiments, vectors may be viruses. In some embodiments, a vector may be a plasmid suitable for expression and / or replication, e.g. , in a cell or bioreactor, i.e. , the vector is a recombinant AAV vector. In some embodiments, vectors designed specifically for the expression of a heterologous nucleic acid sequence, e.g., a heterologous nucleic acid encoding a protein of interest, shRNA, and the like, in the target cell may be referred to as expression vectors, and generally have a promoter sequence that induces expression of the heterologous nucleic acid sequence. In other embodiments, vectors, e.g., transcription vectors, may be capable of being transcribed but not translated: they can be replicated in a target cell but not expressed. Transcription vectors may be used to amplify their insert. Vectors of the present disclosure may be produced recombinantly and may be based on and / or may comprise adeno-associated virus (AAV) parent or reference sequences. The heterologous molecule may be a polynucleotide and / or a polypeptide.

[0235] The term “expression vector” refers to a vector comprising a polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence encoding region to facilitate expression of the protein in target cells. An expression vector may comprise sufficient cis-acting elements for expression, alone or in combination with other elements for expression supplied by the host cell or in an in vitro expression system. The combination of control elements and a gene or genes to which they are operably linked for expression can sometimes be referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art. Expression vectors include, e.g., cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0236] The term “formulation” includes at least one AAV particle (active ingredient) and an excipient, and / or an inactive ingredient. By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. In some embodiments, this portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. A portion or fragment of a polypeptide may be a peptide. In some embodiments, the portion or fragment is a functional portion or fragment (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the entire polypeptide or nucleic acid molecule).

[0237] The term “heterologous” can refer to an entity that is genotypically distinct from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species can be a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked can be a heterologous promoter.

[0238] The term “homology” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

[0239] The term "host cell" denotes a cell comprising an exogenous nucleic acid of interest, for example, one or more microorganism, yeast cell, insect cell, or mammalian cell. For instance, the host cell may comprise an AAV helper construct, an AAV vector plasmid, an accessory function vector, and / or other transfer DNA. The term includes the progeny of the original cell which has been transfected. The progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

[0240] By " increase" or " increases" i s meant a positive alteration of at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In the context of a particular disease or disorder (e.g., LAL- D (e.g., Wolman Disease or CESD)), " increase" or " increases" means a measurable positive alteration in a level or expression (e.g., mRNA or protein) to a level considered in the literature as above the normal range of a subject with such a disease or disorder, to a level considered to be in the normal range in a subject without such a disease or disorder, or to a level that reduces or ameliorates symptoms of the disease (e.g., LAL-D (e.g., Wolman Disease or CESD)). The increase can be, for example, 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 80%, at least about 90%, at least about 95%, or more e.g. 100%. in some embodiments , " increase" or " increases" refers to a measurable level, activity and / or expression (e.g., mRNA, protein) (e.g., in serum, a cell (e.g, hepatocyte), tissue (e.g., liver tissue), organ (e.g., liver), subject (e.g., human subject)) as a result of administration of an agent, composition, compound, or biologic (e.g, a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein) . in some embodiments , " increase" or increases" means at least about 10 % , 20 % , 30 % , 40% , 50% , 60 % , 70 % , 80% , 90 % , or 100 % increase in a level, activity and / or expression (e.g., mRNA, protein) (e.g, in serum, a cell (e.g., hepatocyte), tissue (e.g., liver tissue), organ (e.g., liver), subject (e.g, human subject)) as a result of administration of an agent, composition, compound, or biologic (e.g., a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein).

[0241] In some embodiments, " increase" or increases" refers to any statistically significant measurable increase in enzyme (e.g, lysosomal acid lipase (LAL)) activity (e.g., in serum, a cell (e.g., hepatocyte), tissue (e.g., liver tissue), organ (e.g., liver), subject (e.g., human subject)) as a result of administration of an agent, composition, compound, or biologic (e.g., a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein). In some embodiments, increase" or increases" refers to any measurable statistically significant increase in LAL enzyme activity (e.g., in serum, a cell (e.g., hepatocyte), tissue (e.g., liver tissue), organ (e.g., liver), subject (e.g., human subject)) as a result of administration of a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein.

[0242] In some embodiments, " increase" or increases" refers to any measurable statistically significant increase in protein (e.g., lysosomal acid lipase (LAL)) level, activity and / or expression (e.g., in serum, a cell (e.g., hepatocyte), tissue (e.g., liver tissue), organ (e.g., liver), subject (e.g., human subject)) as a result of administration of an agent, composition, compound, or biologic (e.g, a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein). In some embodiments, increase" or increases" refers to any measurable statistically significant increase in LAL protein level, activity and / or expression (e.g., in serum, a cell (e.g., hepatocyte), tissue (e.g., liver tissue), organ (e.g., liver), subject (e.g., human subject)) as a result of administration of a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein.

[0243] In some embodiments, " increase" or increases" refers to any measurable statistically significant increase in mRNA (e.g., LIPA mRNA) level, activity and / or expression (e.g., in serum, a cell (e.g., hepatocyte), tissue (e.g, liver tissue), organ (e.g., liver), subject (e.g., human subject)) as a result of administration of an agent, composition, compound, or biologic (e.g., a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein). In some embodiments, increase" or increases" refers to any measurable statistically significant increase in LIPA mRNA level, activity, and / or expression (e.g., in serum, a cell (e.g., hepatocyte), tissue (e.g., liver tissue), organ (e.g., liver), subject (e.g., human subject)) as a result of administration of a polynucleotide, recombinant expression vector, rAAV particle, or pharmaceutical composition thereof as provided herein. Measuring an increase in a level, activity, and / or expression as provided herein is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the aspects and embodiments described herein.

[0244] The term “intron” refers to nucleic acid sequence(s), e.g., those within an open reading frame, that are noncoding for one or more amino acids of a protein expressed from the nucleic acid. Intronic sequences may be transcribed from DNA into RNA, but may be removed before the protein is expressed, e.g., through splicing. In some embodiments, intron sequences are added to a heterologous nucleic acid sequence to increase overall efficiency and output of gene expression. Nonlimiting examples of introns that may be used with the viral vectors disclosed herein include a simian virus 40 (SV40) intron, minute virus of mice (MVM) intron, RK intron, betaglobin intron, chicken beta-actin intron, etc., including functional variants and fragments thereof. In some embodiments, the intron is a SV40 intron. In some embodiments, the intron is a modified SV40 intron (e.g., SEQ ID NO: 10). In some embodiments, the SV40 intron includes at least one mutation in an ATG codon and / or the 19S acceptor site (e.g., SEQ ID NO: 11 or SEQ ID NO: 12). In some embodiments, the intron is a MVM intron (e.g. , SEQ ID NO: 13). In some embodiments, the intron is a modified MVM intron (e.g., SEQ ID NO: 14). In some embodiments, the intron is a RK intron (e.g., SEQ ID NO: 15). In some embodiments, the intron is a modified RK intron (e.g, SEQ ID NO: 16).

[0245] The terms “inverted terminal repeat” or “ITR” refer to a cis-regulatory element for the packaging of polynucleotide sequences into viral capsids. An ITR refers to a single stranded sequence of nucleotides followed downstream by its reverse complement, which can form a T- shaped palindromic structure, e.g., in adeno-associated viruses (AAVs) and / or recombinant adeno-associated viral vectors (rAAVs). Muzyczka et al.. (2001) Fields Virology, Chapter 29, Lippincott Williams & Wilkins. ITRs flank the 5’ and 3’ ends of the single stranded DNA genome of AAV in the native virus. In an AAV vector plasmid (or rAAV), they flank the gene of interest. Therefore, an ITR flanking the sequence on the 5’ end is termed the “5’ ITR ” and the ITR flanking the sequence on the 3’ end is termed the “3’ ITR.” In rAAV vectors, these sequences may play a functional role in genome packaging and in second-strand synthesis. In some embodiments, the ITR sequences are selected from particular AAV serotypes. In some embodiments, the 5’ ITR and / or the 3’ ITR sequence is selected from the AAV2 serotype. In some embodiments, the 5’ ITR sequence is a modified AAV2 ITR sequence. Modifications of an AAV2 ITR sequence may be introduced to reduce the resolution process by the Rep protein to enable self-complementary configuration (McCarty 2001). In some embodiments, the modification is the deletion of the terminal resolution site (trs) and D region. In some embodiments, the 5’ ITR sequence is a AAV2 5’ ITR sequence (e.g., SEQ ID NO: 17). In some embodiments, the 3’ ITR sequence is a AAV2 3’ ITR (e.g., SEQ ID NO: 19 or SEQ ID NO: 80). In some embodiments, the 5’ ITR sequence is a 5’ ITR AAV2 R short sequence (e.g, SEQ ID NO: 18). In some embodiments, the 3’ ITR sequence is a 3’ ITR AAV2 R short sequence (e.g., SEQ ID NO: 20).

[0246] As used herein, processes conducted “in vitro” refer to processes which are performed outside of the normal biological environment, for example, studies performed in a test tube, a flask, a petri dish, in artificial culture medium. Processes conducted “in vivo” refer to processes performed within living organisms or cells, for example, studies performed in cell cultures or in mice. Studies performed “ex vivo” refer to studies done in or on tissue from an organism in an external environment, e.g., with minimal alteration of natural conditions, e.g., allowing for manipulation of an organism's cells or tissues under more controlled conditions than may be possible in in vivo experiments.

[0247] The term “isolated” refers to a substance or entity that is altered or removed from the natural state, e.g., altered or removed from at least some of component with which it is associated in the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Such polynucleotides could be part of a vector and / or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature. In some embodiments, an isolated nucleic acid is recombinant, e.g., incorporated into a vector.

[0248] As used herein, an isolated nucleic acid comprising a “heterologous nucleic acid sequence” refers to an isolated nucleic acid comprising a portion (i. e. , the heterologous nucleic acid portion) that is not normally found operably linked to the rest of the isolated nucleic acid in a natural context. For instance, the heterologous nucleic acid may comprise a nucleic acid sequence not originally found in a cell, bacterial cell, virus, or organism from which other components of the isolated nucleic acid (e.g. , the promoter) naturally derive or where the other components of the isolated nucleic acid (e.g. , the promoter) are not naturally found operatively linked with the heterologous nucleic acid in the cell, bacterial cell, virus, or organism. In some embodiments, the heterologous nucleic acid sequence encodes a human protein.

[0249] A DNA sequence or DNA polynucleotide sequence that “encodes” a particular RNA is a sequence of DNA that is capable of being transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein, or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, or a guide RNA; also called “noncoding” RNA or “ncRNA”). A DNA sequence or DNA polynucleotide sequence may also “encode” a particular polypeptide or protein sequence, wherein, for example, the DNA directly encodes an mRNA that can be translated into the polypeptide or protein sequence. A “protein coding sequence” or a sequence that encodes a particular protein or polypeptide is a nucleic acid sequence that is capable of being transcribed into mRNA (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence may be determined by a start codon at the 5' terminus (N-terminus) and a translation stop nonsense codon at the 3' terminus (C-terminus). A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence will usually be located 3' to the coding sequence.

[0250] The term “library” refers to a diverse collection of linear polypeptides, polynucleotides, viral particles, or viral vectors. As examples, a library may be a DNA library or an AAV capsid library.

[0251] The term “lipase A” or “LIPA” refers to the homo sapiens LIPA gene located on human chromosome 10q23.2-23.3, which consists of 10 exons spread over approximately 38 kb. The LIPA gene has 3 transcript variants (Table 1). Variant 2 (NM 000235.4, SEQ ID NO: 2) lacks an internal segment in the 5’ UTR compared with variant 1 (NM 001127605.3, SEQ ID NO: 1). The two variants encode the same protein isoform in size of 399 amino acids (AAs), which has been experimentally validated by cDNA cloning (Baratta et al, World J Gastroenterol 25: 4172-4180). The annotated variant 3 (NM 001288979.2; SEQ ID NO: 3) lacks two consecutive exons in the 5’ region, which results in translation initiation at a downstream AUG and presumably a shorter protein isoform consists of 283 AAs. (Li and Zhang, Arterioscler Thromb Vase Biol. 39(5): 850-856, 2019). Loss-of-function mutations of human LIPA are the causes of rare autosomal recessive lysosomal disorders, Wolman disease and cholesteryl ester storage disease (CESD).

[0252] Table 1. LIPA Gene Transcript Variants

[0253]

[0254] The term “lysosomal acid lipase” or “LAL” refers to the protein encoded by the homo sapiens LIPA gene. LAL is an essential lysosomal enzyme that hydrolyzes cholesteryl ester and triglyceride delivered to the lysosome. As a result of alternative splicing of the LIPA gene, there are two isoforms of the LAL protein (Table 2). LIPA gene variants 1 and 2 encode the same 399 amino acid LAL protein isoform 1 (NP_001121077.1, NM 000235.4; SEQ ID NO: 4), and variant 3 encodes a 283 amino acid LAL protein isoform 2 (NP_001275908. 1; SEQ ID NO: 5), which has a shorter N-terminus compared to isoform 1.

[0255] Table 2. LAL Protein Isoforms

[0256] A "modification" or “mutation” as used herein, refers to a change of a primary amino acid or nucleic acid sequence as compared to a starting amino acid or nucleic acid sequence (e.g., a wild-type sequence), wherein the change results from a sequence or chemical alteration involving said amino acid or nucleic acid residue / positions. For example, typical modifications of include substitution of an amino acid residue or nucleic acid (e.g., at a particular position) with another amino acid (e.g. , a conservative or non-conservative substitution) or nucleic acid, insertion of one or more amino acids or nucleic acids adjacent to said residue / position, and deletion of said residue / position. Generally and preferably, the modification results in alteration in at least one physic-biochemical activity of the variant polypeptide or polynucleotide compared to a polypeptide or polynucleotide comprising the starting (or "wild type") amino acid or nucleic acid sequence.

[0257] A “molecular scaffold” is a framework or starting molecule that forms the sequence or structural basis against which to design or make a subsequent molecule. The term “naturally-occurring” or “unmodified” as used herein as applied to, e.g., a nucleic acid, a polypeptide, a cell, or an organism, is one found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (such as a virus) is naturally occurring whether present in that organism or isolated from one or more components of the organism.

[0258] As used herein, “open reading frame” or “ORF” refers to a sequence which does not contain a stop codon in a given reading frame.

[0259] The terms “operatively linked,” "operably linked,” or “coupled” are refer to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, the terms refer to the functional relationship of a transcriptional regulatory sequence and a sequence to be transcribed. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it, e.g., stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a sequence are contiguous to that sequence or are separated by short spacer sequences, z.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0260] The term “origin of replication” or “ori” refers to one of more sequences of DNA at which replication is initiated on a chromosome, plasmid or virus. In some embodiments, a recombinant expression vector of the present disclosure comprising at least one origin of replication sequence. In some embodiments, the at least one origin of replication is selected from Ori (e.g., SEQ ID NO: 26), M13 Ori (e.g., SEQ ID NO: 27), or Ori pUC (e.g., SEQ ID NO: 28) sequence. In some embodiments, a recombinant expression vector of the present disclosure comprises an Ori pUC sequence (SEQ ID NO: 28). In some embodiments, a recombinant expression vector of the present disclosure comprises an Ori sequence (SEQ ID NO: 26) and a M13 Ori sequence (SEQ ID NO: 27).

[0261] The term “orthogonal evolution” refers to a method wherein AAV particles are administered for a first round of AAV selection as described herein across a set of any number of cell- and / or subject-types that may be from different species and / or strains, and wherein any number of additional, z.e., subsequent, AAV selection rounds are performed either across a set of any number of cell- and / or subject-types that may be from different species and / or strains, or across a set of any number of cell- and / or subject-types that may be from the same species and / or strain. The term “packaging” as used herein can refer to a series of intracellular events that can result in the assembly and encapsidation of a rAAV particle. AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno- associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”

[0262] A “payload region” is any nucleic acid sequence (e.g, within the viral genome) which encodes one or more “payloads” of the disclosure. As non-limiting examples, a payload region may be a nucleic acid sequence within the viral genome of an AAV particle, which encodes a payload, wherein the payload is a polypeptide. Payloads of the present disclosure may be, but are not limited to, peptides, polypeptides, proteins, antibodies, RNAi agents, etc.

[0263] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials (e.g., carrier or diluent), compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio (i.e., when the pharmaceutically acceptable material is administered to an individual it does not cause undesirable biological effects nor does it interact in a deleterious manner with any of the components of the composition in which it is contained), and which does not abrogate the biological activity or properties of a compound disclosed herein.

[0264] The term “pharmaceutical composition” or simply “composition” as used herein, can refer to a biologically active compound, optionally mixed with at least one pharmaceutically acceptable chemical component, such as, though not limited to carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and the like.

[0265] The terms “polyadenylation (poly A) signal sequence,” “polyadenylation (poly A) signal and “polyadenylation (polyA) sequence” refer to a regulatory element that provides a signal for transcription termination and addition of an adenosine homopolymeric chain to the 3 ’ end of an RNA transcript. The polyA signal may comprise a termination signal (e.g. , an AAUAAA sequence or other non-canonical sequences) and optionally flanking auxiliary elements (e.g., a GU-rich element) and / or other elements associated with efficient cleavage and polyadenylation. The polyA sequence may comprise a series of adenosines attached by polyadenylation to the 3’ end of an mRNA. In some embodiments, DNA regulatory sequences or control elements are tissue-specific regulatory sequences. In some embodiments, the polyA signal sequence is a bovine growth hormone (BGH) polyA signal (e.g., SEQ ID NO: 23), a synthetic polyA signal (e.g., SEQ ID NO: 24), or SV40 polyA signal (e.g., SV40L133) ((e.g., SEQ ID NO: 25). The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length. They may include one or more of ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single- , double-, or multi-stranded DNA or RNA, genomic DNA (gDNA), messenger RNA (mRNA), complementary DNA (cDNA), recombinant DNA, DNA-RNA hybrids, plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, e.g. locked nucleic acids (LNA), peptide nucleic acids (PNA). Preferably, polynucleotides include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the variant maintains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, and compositions, and cells comprising the same.

[0266] In particular embodiments, polynucleotides are provided by this invention that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more contiguous amino acid residues of a polypeptide, as well as all intermediate lengths. It will be readily understood that "intermediate lengths," in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc , 101, 102, 103, etc:, 151, 152, 153, etc. 201, 202, 203, etc.

[0267] The term “post-transcriptional regulatory element” (“PRE”) refers to one or more regulatory elements that, when transcribed into mRNA, regulate gene expression at the level of the mRNA transcript. In some embodiments, a PRE is inserted downstream of a transgene (e.g., LIPA transgene). Examples of such post-transcriptional regulatory elements may include sequences that encode micro-RNA binding sites, RNA binding protein binding sites, etc. Examples of post-transcriptional regulatory element that may be used with the viral vectors disclosed herein include the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and the hepatitis post-transcriptional regulatory element (HPRE).

[0268] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues (natural or unnatural) covalently linked by peptide bonds. A protein or peptide typically contains at least two amino acids or amino acid variants, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids or variants joined to each other by peptide bonds. The terms include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof. In some instances, the polypeptide encoded is less than or equal to about 50 amino acids, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long, and the polypeptide is then termed a “peptide.” Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides and may be associated or linked. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0269] The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. In some embodiments, a variant comprises a sequence having at least about 50%, at least about 80%, or at least about 90%, identical (homologous) to a native or a reference sequence.

[0270] The term “preventing” or “prevention” refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0271] The term “prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease.

[0272] A “prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease.

[0273] The term “promoter” or “promoter sequence” as used herein is a DNA regulatory sequence capable of facilitating transcription (e.g., capable of causing detectable levels of transcription and / or increasing the detectable level of transcription over the level provided in the absence of the promoter) of an operatively linked coding or non-coding sequence, e.g., of a downstream (3' direction) coding or non-coding sequence, e.g., through binding RNA polymerase. In some embodiments, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5 ' direction) to include the minimum number of bases or elements to initiate transcription at levels detectable above background. In some embodiments, a promoter sequence may comprise a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. In addition to sequences sufficient to initiate transcription, a promoter may also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Various promoters, including inducible promoters and constitutive promoters, may be used to induce the vectors disclosed herein. Examples of promoters known in the art that may be used in some embodiments, e.g. , in viral vectors disclosed herein, include the cytomegalovirus (CMV) promoter, chicken P-actin (CBA) promoter, alpha- 1 antitrypsin (hAAT promoter, and those promoters derived from an immunoglobulin gene, SV40, or other tissue specific genes. In some embodiments, the promoter is an AAT promoter. In some embodiments, the AAT promoter is a human AAT1(hAAT) promoter (e.g., SEQ ID NO: 9). In some embodiments, the promoter is a CBA promoter. In some embodiments, the CBA promoter is a CB7 promoter (e.g., SEQ ID NO: 8). In addition, standard techniques are known in the art for creating functional promoters by mixing and matching known regulatory elements. Fragments of promoters, e.g., those that retain at least minimum number of bases or elements to initiate transcription at levels detectable above background, may also be used.

[0274] In some embodiments, a promoter can be a constitutively active promoter (i.e., a promoter that constitutively induces expression in any cell type and / or under any conditions). In other embodiments, a promoter can be a constitutively active promoter in a particular tissue context, e.g., in liver cells, etc. (i.e., a tissue-specific promoter). In other embodiments, a promoter can be an inducible promoter (i.e., a promoter whose activity is controlled by an external stimulus, e.g. , the presence of a particular temperature, compound, or protein). In some embodiments, a promoter may be a spatially restricted promoter that can induce activity or not depending on the physical context in which the promoter is found. Non-limiting examples of spatially restricted promoters include tissue specific promoter, cell type specific promoter, etc. In some embodiments, a promoter may be a temporally restricted promoter that induces expression depending on the temporal context in which the promoter is found. For example, a temporally restricted promoter may induce expression only at specific stages of embryonic development or during specific stages of a biological process. In some embodiments, the promoter is tissue-specific such that, in a multi-cellular organism, the promoter preferentially induces expression in a subset of specific cells (e.g, hepatocytes, macrophages).

[0275] As used herein, “recombinant” can refer to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such polynucleotides. Thus, for example, a protein synthesized by a microorganism is recombinant, for example, if it is synthesized from an mRNA synthesized from a recombinant gene present in the cell.

[0276] The term “sample” or “biological sample” refers to a subset of its tissues, cells, nucleic acids, or component parts (e.g. body fluids, including but not limited to blood, serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).

[0277] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “identity,” “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” The term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules.

[0278] The recitations “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide -bynucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison (e.g. , gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes), determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (z.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0279] By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods.

[0280] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; the contents of each of which are incorporated herein by reference in their entirety. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0281] By “reduces,” “decreases,” or “lowers” is meant a negative alteration of at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In the context of a particular disease (e.g., LAL-D (e.g., Wolman Disease or CESD)), “reduces,” “decreases,” or “lowers” means a reduction in a level (e.g., level of triglycerides, cholesterol, and / or fatty acids; liver-to-body weight (LW / BW); spleen- to-body weight (SPL / BW) ratio) or expression (e.g., mRNA or protein) to a level considered in the literature as below the normal range of a subject with such a disease or disorder, to a level considered to be in the normal range in a subject without such a disease or disorder, or to a level that reduces or ameliorates symptoms of the disease (e.g., LAL-D (e.g., Wolman Disease or CESD)). The decrease can be, for example, 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 80%, at least about 90%, at least about 95%, or more e.g. 100%.

[0282] By “reference” is meant a standard or control condition. The “reference level known in the art” refers to an experimentally determined control or threshold level identified or known in the prior art, or to the level identified in a sample from a control patient or in samples from a control patient population. In one embodiment, a reference is an untreated subject, or a subject administered with a placebo or normal saline, medium, buffer, and / or a control. In one embodiment, a reference is a healthy subject.

[0283] A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.

[0284] A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul etal., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel el al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0285] Terms that describe the orientation of polynucleotides include: 5' (normally the end of the polynucleotide having a free phosphate group) and 3' (normally the end of the polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the 5' to 3' orientation or the 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the “sense,” “plus,” or “coding” strand because its sequence is identical to the sequence of the premessenger (premRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA], For DNA and mRNA, the complementary 3' to 5' strand which is the strand transcribed by the RNA polymerase is designated as “template,” “antisense,” “minus,” or “non-coding” strand. As used herein, the term “reverse orientation” refers to a 5' to 3' sequence written in the 3' to 5' orientation or a 3' to 5' sequence written in the 5' to 3' orientation.

[0286] The term “region” refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area, an epitope and / or a cluster of epitopes. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and / or C-termini. In some embodiments, when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to polynucleotides, terminal regions may comprise 5’ and / or 3’ termini.

[0287] The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides; the term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized. DNA and RNA can be single-stranded (z.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double stranded, z.e., dsRNA and dsDNA, respectively). The term “mRNA” or “messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.

[0288] The term “silencer” refers to one or more DNA regulatory sequence(s) capable of reducing or preventing transcription of a coding or non-coding sequence to which it is operatively linked, e.g., by interacting with transcription regulation factors or repressors. For instance, in some embodiments, when the silencer is bound by a repressor, RNA polymerase may be prevented from transcribing the DNA into RNA, thus preventing gene expression. The silencer region may be upstream (e.g., 5’ to), downstream, or within the coding or non-coding sequence. Examples of silencers useful in any of the embodiments disclosed herein are known in the art. In addition, functional expression regulatory regions may be prepared by mixing and matching known regulatory elements, such as promoters and silencers. Fragments of silencers, e.g., those that retain at least minimum number of bases or elements to detectably repress transcription (as compared to transcription in the absence of a silencer), either partially or fully, may also be used. In some embodiments, a silencer may be a spatially restricted silencer that can repress transcription depending on the physical context in which the silencer is found. Nonlimiting examples of spatially restricted silencers include tissue specific silencer, cell type specific silencer, etc. In some embodiments, the silencer is a liver specific silencer. In some embodiments, a silencer may be a temporally restricted silencer that represses transcription depending on the temporal context in which the silencer is found. For example, a temporally restricted silencer may induce expression only at specific stages of embryonic development or during specific stages of a biological process.

[0289] The term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.

[0290] The terms “stuffer,” “stuffer sequence,” “spacer” or “spacer sequence,” “filler” or “filler sequence” are used interchangeably to refer to a nucleic acid sequence for insertion into AAV vectors to ensure efficient and reliable vector packaging by adjusting the size of the vector. Ideally, the staffer sequence is biologically neutral and does not share any homology with prokaryotic, viral DNA and / or human DNA and is devoid of cryptic splice site, polyadenylation peptide, promoter or regulatory elements that could negatively impact the transgene expression for vector genome integrity. In some embodiments, the staffer sequence is located upstream or downstream of the transgene expression cassette and / or between regulatory elements. In some embodiments, the staffer sequence is designed to avoid transcription factor binding sites that could interfere with transgene expression or promote transcription of antisense RNA; target sequences for microRNA expressed in the target cells or tissues that would promote transgene extinction; splice acceptor and donor sites that may interfere with accurate transgene splicing, strong secondary structures that would impede vector DNA replication and packaging; ATG or alternative translation initiation codon not followed by an in-frame stop codon, to prevent expression of any unwanted peptide; CpG motifs that could promote immune response; cryptic splice sites; polyadenylation signals; and / or promoter elements that could interfere with the expression of the therapeutic gene or the overall stability of the vector genome. Nonlimiting examples of staffer sequences include bacteriophage lambda DNA sequences and human derived DNA sequences (see e.g., W02008134720A2). In some embodiments, the recombinant expression vector of the present disclosure includes a staffer sequence. In some embodiments, the staffer sequence comprises one or more of the staffer sequences selected from Table 14A. In some embodiments, the staffer sequence comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34. In some embodiments, the staffer sequence comprises or consists of SEQ ID NO: 34.

[0291] The term “subject,” “patient,” or “individual” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. In some embodiments, the subject is a pediatric, an adolescent or an adult subject. In preferred embodiments, the subject is a human subject or a human patient. In some embodiments, the subject is a human pediatric subject. In some embodiments, the human pediatric subject is less than about 12 years of age (e.g., between birth and 12 years of age, between 1-12 years of age, between 1-5 years of age, between, 6-12 years of age, less than about 1 year of age, or less than about 6 months of age). In some embodiments, the human pediatric subject ranges in age between birth and 12 years of age. In some embodiments, the human pediatric subject ranges between 1-12 years of age. In some embodiments, the human pediatric subject ranges between 1-5 years of age. In some embodiments, the human pediatric subject ranges between 6-12 years of age. In some embodiments, the human pediatric subject is an infant. In some embodiments, the human pediatric subject is an infant less than about 1 year or 12 months of age (e.g., between birth and 1 year or 12 months of age, or less than about 6 months of age). In some embodiments, the human pediatric subject is an infant less than about six (6) months of age. In some embodiments, the human pediatric subject is an infant ranging from birth to 12 months or 1 year of age. In some embodiments, the human subject is 4 to 15 years of age. In some embodiments, the subject is a human adolescent subject. In some embodiments, the human adolescent subject ranges from 12 to 18 years of age. In other embodiments, the subject is a human adult subject. In some embodiments, the human adult subject is 18 years of age or older.

[0292] The term “substantially” refers to the qualitative condition of exhibiting total or neartotal extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0293] As used herein, “target cells” or “target tissue” refers to any one or more cells or tissues of interest. A target cell may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. A target tissue may be found in vitro, in vivo, in situ or in an organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient. In some embodiments, the target cell is any cell that expresses LIPA RNA or LAL protein. In some embodiments, the target cell is selected from epithelial cells, endocrine cells, neuronal cells, glial cells, germ cells, trophoblast cells, endothelial cells, muscle cells, adipocytes, pigment cells, mesenchymal cells, blood or immune cells. In some embodiments, the target cell is selected from a Kupffer cell, Hofbauer cell, macrophage, hepatocyte, B cell, T cell, enteroendocrine cell, fibroblast, Langerhans cell, enterocyte, monocyte, platelet, glia cell, glandular cell, luminal cell, basal respiratory cell, oligodendrocyte, osteoblast, hepatic stellate cell (HSC), myeloid cell, adipocyte, splenocyte, intestinal cell, skeletal muscle cell, red blood cell, and / or smooth muscle cell. In some embodiments, the target cell is a hepatocyte. In some embodiments, the target cell is a macrophage. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an intestinal cell (e.g., small intestinal cell). In some embodiments, the target cell is a skeletal muscle cell. In some embodiments, the target tissue is any tissue type that expresses LIPA RNA or LAL protein. In some embodiments, the target tissue is selected from liver tissue, spleen tissue, skeletal muscle tissue, kidney tissue, brain tissue (e.g., cerebellum, cerebral cortex, retina), endocrine tissue (e.g., thyroid, parathyroid, adrenal gland), prostate / testis tissue, endometrium / ovarian tissue, respiratory tissue (e.g, lung, bronchus), myeloid tissue, gastrointestinal tissue (e.g., duodenum, small intestine, colon rectum), and lymphoid tissues (e.g., appendix, spleen, lymph node, tonsil, bone marrow). In some embodiments, the target tissue is liver tissue. In some embodiments, the target tissue is spleen tissue. In some embodiments, the tissue is small intestine. In some embodiments, the tissue is skeletal muscle. Reference to protein or gene expression in a target cell or tissue does not exclude expression in another cell type, but is typically higher than expression in non-target tissue.

[0294] The term “transduction” is used to refer to the uptake of foreign DNA by a cell, where the foreign DNA is provided by a virus or a viral vector. Consequently, a cell has been “transduced” when exogenous DNA has been introduced inside the cell membrane. In some embodiments, the term “transduction” refers to the administration / delivery of the coding region of the LIPA gene to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the disclosure resulting in expression of LAL the recipient cell.

[0295] The term "transfection" is used to refer to the uptake of foreign DNA by a cell, such that the cell has been "transfected" once the exogenous DNA has been introduced inside the cell membrane. See, e.g., Graham et a , (1973) Virology, 52:456; Sambrook et a , (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al., (1986) Basic Methods in Molecular Biology, Elsevier; Chu et al., (1981) Gene, 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0296] The term “transformation” is used to refer to the uptake of foreign DNA by bacterial cells.

[0297] The term “transgene” refers to a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism through gene therapy (e.g., rAAV). This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In some embodiments, the transgene is a LIPA transgene. In some embodiments, a. LIP A transgene (e.g., SEQ ID NO: 6 or a sequence that is at least 80% identical to SEQ ID NO: 6) is used in any of the polynucleotides, recombinant expression vectors, rAAV particles, or pharmaceutical compositions of the present disclosure. In some embodiments, the LIPA transgene comprises SEQ ID NO: 6 or a sequence that is at least 80% identical to SEQ ID NO: 6. In some embodiments, a codon optimized LIPA transgene (e.g., SEQ ID NO: 7 or a sequence that is at least 80% identical to SEQ ID NO: 7) is used in any of the polynucleotides, recombinant expression vectors, rAAV particles, or pharmaceutical compositions of the present disclosure. In some embodiments, the codon optimized LIPA transgene comprises SEQ ID NO: 7 or a sequence that is at least 80% identical to SEQ ID NO: 7.

[0298] The terms “treat,” “treating,” “treatment,” “ameliorate,” or “ameliorating” and other grammatical equivalents as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms further include achieving a therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disease being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease such that an improvement is observed in the patient, notwithstanding that, in some embodiments, the patient is still afflicted with the underlying disease. In certain aspects, for prophylactic benefit, the pharmaceutical compositions are administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even if a diagnosis of the disease has not been made.

[0299] By an “untranslated region” or “UTR” is meant a nonprotein coding sequence adjacent to a coding section of an mRNA sequence. A 5' UTR is the nonprotein coding section of the mRNA that is located 5' to the coding section of mRNA used in translation. The 3' UTR is the nonprotein coding section of the mRNA that is located 3' to the coding region used in translation. In some embodiments, a recombinant expression vector of the present disclosure comprises one or more UTRs. In some embodiments, a recombinant expression vector of the present disclosure lacks a 5 ’ UTR. In some embodiments, a recombinant expression vector of the present disclosure comprises a LIP A 3’ UTR (e.g., SEQ ID NO: 29).

[0300] The term “variant” refers to a polypeptide or polynucleotide that has an amino acid or a nucleotide sequence that is substantially identical, e.g., having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to a reference sequence . In some embodiments, the variant is a functional variant. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0301] Polynucleotides

[0302] In some embodiments, the present invention provides polynucleotides for the treatment of diseases and disorders associated with impaired or deficient lysosomal acid lipase (LAL) function and / or activity (e.g., LAL-D (e.g., Wolman Disease, CESD). In some embodiments, the polynucleotides comprise a lipase A (LIPA) transgene, or a biologically active fragment thereof, encoding functional lysosomal acid lipase (LAL). The LIPA gene is located on human chromosome 10q23.2-23.3 and consists of 10 exons spread over approximately 38 kb. LIPA has 3 transcript variants as provided in Table 1. Variant 2 (NM_000235.4; SEQ ID NO: 2) lacks an internal segment in the 5' UTR compared with variant 1 (NM_001127605.3; SEQ ID NO: 1). The two variants encode the same protein isoform in size of 399 amino acids (AAs), which has been experimentally validated by cDNA cloning (Baratta et a , World J Gastroenterol, 25: 4172-4180). The annotated variant 3 (NM_001288979.2; SEQ ID NO: 3) lacks two consecutive exons in the 5' region, which results in translation initiation at a downstream AUG and presumably a shorter protein isoform consists of 283 AAs. (Li and Zhang, Arterioscler Thromb Vase Biol. 39(5): 850- 856, 2019). More than I 00 / J / L4 loss-of-function mutations have been identified (Pisciotta L et a , Atherosclerosis, 2017; 265: 124-132). The most commonly inherited defect is a splice junction mutation in exon 8, E8SJM (c.894G>A).

[0303] In some embodiments, a gene replacement therapy suitable for or adapted for administration to a subject (e.g., human subject) is used to deliver the LIPA transgene. Upon administration of any of the polynucleotides as provided herein, the LIPA transgene is expressed in a target cell or tissue to generate a functional LAL protein or gene product to produce a therapeutic effect. In some embodiments, the LIPA transgene refers to a functional fragment or a variant thereof. In some embodiments, the polynucleotide sequence of the LIPA transgene is further modified to enhance its activity, expression, stability, and / or solubility in vivo. Exemplary LIPA transgene nucleic acid sequences are provided in Table 3. In an embodiment, the LIPA transgene is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to a nucleic acid sequence comprising or consisting of SEQ ID NO: 6, wherein the protein retains LAL activity, such as the activity to hydrolyze cholesterol esters into free cholesterol and triglycerides into free fatty acids in the lysosome. For example, the nucleotide sequence that encodes a functional LAL protein may comprise one or more base pair substitutions, deletions or insertions which do affect the function of the LIPA protein. Furthermore, the nucleotide sequence that encodes a functional LIPA protein may comprise one or more base pair substitutions, deletions or insertions may increase or reduce expression of the LAL protein, and this change in expression pattern may be desired for treatment of the LAL-Dor the disorder related to lipid storage and accumulation. In an embodiment, the LIPA transgene comprises or consists of a nucleic acid sequence of SEQ ID NO: 6, or a functional variant or fragment thereof.

[0304] In some embodiments, the LIPA transgene sequence is codon-optimized. In some embodiments, the LIPA transgene is a codon-optimized polynucleotide sequence that increases expression of LAL protein, for example, relative to expression of LAL encoded by a polynucleotide sequence that is not codon-optimized. In a specific embodiment, the codon- optimized LIPA transgene comprises or consists of a nucleic acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 7. In another specific embodiment, the codon-optimized LIPA transgene comprises or consists of a nucleic acid sequence of SEQ ID NO: 7.

[0305] Table 3. LIPA Transgene Sequences

[0306] Polynucleotides of the present invention may further comprise at least one additional element to enhance target specificity and expression (See e.g., Powell et al. Discov Med. 2015 Jan; 19(102): 49-57). In some embodiments, the polynucleotides of the present invention further comprise a promoter, including variants or fragments thereof which retain regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter (e.g., as measured by qPCR)), operably linked to the LIPA transgene.

[0307] In some embodiments, the promoter is a species specific promoter, inducible promoter, tissue-specific promoter, ubiquitous promotor, or cell cycle-specific promoter. In some embodiments, the promoter may be naturally occurring or non-naturally occurring (e.g., synthetic promoter). Non-limiting examples of promoters include those derived from viruses, plants, mammals, or humans. In some embodiments, promoters may be derived from human cells or systems. In some embodiments, the promoter may be truncated or mutated, e.g., a promoter variant. In some embodiments, the promoter is a ubiquitous promoter, e.g., capable of expression in multiple tissues. In particular embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription.

[0308] In some embodiments, the promoter is an organ, tissue, or cell specific promoter. In some embodiments, promoter is an organ, tissue, or cell specific promoter that targets any organ, tissue, or cell type expressing LIPA RNA or LAL protein. In some embodiments, the promoter is capable of expression in the following nonlimiting cell types: epithelial cells, endocrine cells, neuronal cells, glial cells, germ cells, trophoblast cells, endothelial cells, muscle cells, adipocytes, pigment cells, mesenchymal cells, blood or immune cells, specifically a Kupffer cell, Hofbauer cell, macrophage, hepatocyte, B cell, T cell, enteroendocrine cell, fibroblast, Langerhans cell, enterocyte, monocyte, platelet, glia cell, glandular cell, luminal cell, basal respiratory cell, oligodendrocyte, osteoblast, hepatic stellate cell (HSC), myeloid cell, adipocyte, splenocyte, intestinal cell, skeletal muscle cell, red blood cell, and / or smooth muscle cell. In some embodiments, the promoter is a liver specific promoter (e.g., hAA T). macrophage promoter, spleen specific promoter, endothelial cell promoter, intestinal promoter, skeletal muscle promoter, and / or smooth muscle promoter. In some embodiments, the promoter is capable of expression in spleen tissues or cells, targeted regions within, and / or subsets of cells therein (e.g., macrophages).

[0309] In some embodiments, the promoter is capable of expression in liver tissues or cells, targeted regions within, and / or sub-sets of cells therein (e.g., hepatocytes). Exemplary liver specific promoters include but are not limited to, human serum albumin (HSA) promoter, alpha- 1 antitrypsin (AAT) promoter, thyroxine-binding globulin (TBG) promoter, and / or hybrid liverspecific promoter (HLP), including variants or fragments thereof which retain regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter (e.g., as measured by qPCR)). In some embodiments, the liver specific promoter is an alpha- 1 antitrypsin (AAT) promoter, including variants or fragments thereof which retain regulatory control or promoter activity. In some embodiments, the AAT promoter is a human AAT promoter (hAAT), including variants or fragments thereof which retain regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter (e.g., as measured by qPCR)).

[0310] In some embodiments, the promoter is a synthetic promoter. For example, synthetic minimal promoters are described in Chai S, et al. Strong ubiquitous micro-promoters for recombinant adeno-associated viral vectors. Mol Ther Methods Clin Dev. 2023, which is incorporated herein by reference in its entirety.

[0311] In some embodiments the promoter is a ubiquitous promoter. Exemplary ubiquitous promoters include, but are not limited to, cytomegalovirus (CMV) immediate-early enhancer / promoter, elongation factor- 1 alpha (EF-la) promoter, minimal CMV promoter (miniCMV), phosphoglycerate kinase (PGK) promoter, chicken P-actin (CBA) promoter, CB7 (aka CAG; a CBA-derived CMV early enhancer with the CBA promoter and a truncated SV40 late 16S intron), Ubiquitin C promoter (UbC), SRa promoter, GUSB (hGPp), UCOE (HNRPA2B1-CBX3), and / or CBh (a CMV / CBA-derivative CMV early enhancer with the CBA promoter with a chimeric CBA minute virus of mice (MVM) viral capsid protein (VP1) intron, including variants or fragments thereof which retain regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter (e.g., as measured by qPCR)). In some embodiments, the promoter is a CB7 promoter, including variants or fragments thereof which retain regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter (e.g., as measured by qPCR)).

[0312] Additional promoters are contemplated herein including, but not limited to, the P546 promoter, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter, including variants or fragments thereof which retain regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter (e.g., as measured by qPCR)).

[0313] In some embodiments, a promoter described herein (e.g., comprised within a polynucleotide described herein) is selected from promoter provided in Table 4, including variants or fragments thereof that retain regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter (e.g., as measured by qPCR)). In some embodiments, the promoter is a CB7 promoter (aka CAG; a CBA-derived CMV early enhancer with the CBA promoter and a truncated SV40 late 16S intron) as provided in Table 4, including variants or fragments thereof. In some embodiments, the CB7 promoter comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the CB7 promoter comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the CB7 promoter comprises or consists of the nucleic acid sequence of SEQ ID NO: 8.

[0314] In some embodiments, the promoter is a hAAT promoter as provided in Table 4, including functional variants or fragments thereof. In some embodiments, the hAAT promoter comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the hAAT promoter comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the hAAT promoter comprises or consists of the nucleic acid sequence of SEQ ID NO: 9.

[0315] Table 4. Promoter Sequences

[0316] In some embodiments, a polynucleotide as described herein further comprises an intron to increase overall efficiency and output of gene expression. Preferably, the intron is positioned between the promoter and transgene (e.g. , LIPA transgene) in a 5 ’ to 3 ’ orientation. Exemplary introns include, but are not limited to, a simian virus 40 (SV40) intron, minute virus of mice (MVM) intron, RK intron, P-globin intron, and / or a chicken P-actin (( BA) intron, including functional variants or fragments thereof. Additional introns contemplated herein include, but are not limited to, F.IX truncated intron 1 (300 bps), P-globin SD / immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor / immunoglobin splice acceptor (500 bps), SV40 late splice donor / splice acceptor (19S / 16S) (180 bps), and hybrid adenovirus splice donor / IgG splice acceptor (230 bps). In some embodiments, an intron described herein (e.g., comprised within a polynucleotide described herein) is selected from an intron provided in Table 5, including functional variants or fragments thereof.

[0317] In some embodiments, the intron is a SV40 intron, including functional variants or fragments thereof. In some embodiments, the SV40 intron comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. In some embodiments, the SV40 intron comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the SV40 intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 10.

[0318] In some embodiments, the intron is a modified SV40 intron (e.g., ATG mutant or double mutant). In some embodiments, the modified SV40 intron comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified SV40 intron comprises at least one mutation in an ATG codon. In some embodiments, the modified SV40 intron comprises a mutation of A to T or A to G at position 79 as compared to SEQ ID NO: 10. In some embodiments, the modified SV40 intron comprises a mutation of A to T at position 79 as compared to SEQ ID NO: 10 (underlined in Table 5), which is also referred to herein as the “SV40 ATG mutant” (SEQ ID NO: 12). In some embodiments, the SV40 intron comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the SV40 intron comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, additional modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the SV40 intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 12.

[0319] In some embodiments, the modified SV40 intron comprises at least one mutation in the 19S acceptor site. In some embodiments, the at least one mutation in the 19S acceptor site is a mutation of A to T at position 30 as compared to SEQ ID NO: 10. In some embodiments, the at least one mutation in the 19S acceptor site is a mutation of G to C at position 31 as compared to SEQ ID NO: 10. In some embodiments, the at least one mutation in the 19S acceptor site is a mutation of AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10. In some embodiments, the modified SV40 intron comprises at least one mutation in an ATG codon and 19S acceptor site. In some embodiments, the modified SV40 intron comprises a mutation of A to T at position 79 and a mutation of A to T at position 30, G to C at position 31, or AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10. In some embodiments, the modified SV40 intron comprises a mutation of AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10 (underlined in Table 5), which is also referred to herein as the “SV40 double mutant” (SEQ ID NO: 11). In some embodiments, the SV40 intron comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the SV40 intron comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, additional modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the SV40 intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 11.

[0320] In some embodiments, the intron is a MVM intron, including functional variants or fragments thereof. In some embodiments, the MVM intron comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the MVM intron comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the MVM intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 13.

[0321] In some embodiments, the MVM intron is a modified MVM intron. In some embodiments, the modified MVM intron comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified MVM intron comprises at least one mutation in an ATG codon. In some embodiments, the modified MVM intron comprises a mutation of A to T, A to C, or A to G at position 16 and / or a mutation of G to C, G to A, or G to T at position 40 as compared to SEQ ID NO: 13. In some , embodiments, the modified MVM intron comprises a mutation of A to T at position 16 and a mutation of G to C at position 40 as compared to SEQ ID NO: 13 (underlined in Table 5), which is also referred to herein as “MVMi” (SEQ ID NO: 14). In some embodiments, the MVM intron comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the MVM intron comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, additional modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the MVM intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 14.

[0322] In some embodiments, the intron is a RK intron, including functional variants or fragments thereof. In some embodiments, the RK intron is an RK intron described in W02009080720A1, which is incorporated by reference in its entirety. In some embodiments, the RK intron comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the RK intron comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, modifications, e.g. , substitutions, relative to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the RK intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 15.

[0323] In some embodiments, the RK intron is a modified RK intron. In some embodiments, the modified RK intron comprises at least one mutation that abolishes at least one open reading frame (ORF). In some embodiments, the modified RK intron comprises at least one mutation in an ATG codon. In some embodiments, the modified RK intron comprises a mutation of A to G, A to C, or A to T at position 78 as compared to SEQ ID NO: 15. In some embodiments, the modified RK intron comprises a mutation of A to G at position 78 as compared to SEQ ID NO: 15 (underlined in Table 5), which is also referred to herein as “RKi” (SEQ ID NO: 16). In some embodiments, the RK intron comprises or consists of a nucleic acid sequence that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the RK intron comprises or consists of a nucleic acid sequence comprising at least one, two, or three, but no more than four, additional modifications, e.g., substitutions, relative to the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the RK intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 16.

[0324] Table 5. Intron Sequences

[0325] The nucleic acid sequence of the polynucleotides of the present invention may comprise a splice donor site 5’ of the intron and a splice acceptor site 3’ of the intron. The nucleic acid sequence of the polynucleotides of the present invention may also comprise a sequence 5’ (e.g., within the splice donor site) and / or 3’ (e.g., within the splice acceptor site) of the intron sequence that matches a human consensus sequence. The nucleotides GT at positions +1 and +2 adjacent to the splice donor site and the nucleotides AG at positions -1 and -2 adjacent to the splice acceptor site are highly conserved and nearly invariant. In some embodiments, the sequence that matches the human consensus sequence at the 5 ’ end of the intron comprises GAG, CAG, AAC, or AAG. In some embodiments, the sequence that matches the human consensus sequence at the 3 ’ end of the intron comprises GTT, GTA, GCC, GAA, or GAT. In some embodiments, the splice donor site and / or the splice acceptor site is modified to match a human consensus sequence. In some embodiments, the splice donor site is modified to comprise a GAG, CAG, AAC, or AAG sequence 5 ’ of the intron. For example, an A or G mutation may be introduced into a CAG sequence 5 ’ of the intron to match a human consensus sequence. In some embodiments, the splice acceptor site is modified to comprise a GTT, GTA, GCC, GAA, or GAT sequence 3’ of the intron. For example, a CC deletion or a mutation of CC to TA may be introduced into the sequence 3 ’ of the intron to comprise the sequence GTT, GTA, GCC, GAA, or GAT to match a human consensus sequence.

[0326] The nucleic acid sequence of the polynucleotides of the present invention may comprise a promoter sequence and a LIPA transgene sequence, or functional variants or fragments thereof. The nucleic acid sequence of the polynucleotides of the present invention may comprise a promoter sequence, an intron sequence, and a LIPA transgene sequence, or functional variants or fragments thereof. The nucleic acid sequence of the polynucleotides of the present invention may comprise any of the promoters, introns, or LIPA transgene sequences as disclosed herein, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises a promoter sequence selected from an alpha- 1 antitrypsin (AAT) promoter sequence (e.g., hAAT) or a chicken P- actin (CBA) promoter sequence (e.g., CB7); an intron sequence selected from a simian virus 40 (SV40) intron sequence, a minute virus of mice (MVM) intron sequence, or a RK intron sequence; and a lipase A (LIPA) transgene sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises the following nucleic acid sequences from 5 ’ to 3 ’ : a promoter sequence selected from an AAT promoter sequence (e.g, hAAT) or a CBA promoter sequence (e.g., CB7); an intron sequence, wherein the intron sequence is selected from a SV40 intron sequence (or a functional variant (e.g. , SV40 double mutant or SV40 ATG mutant) or fragment thereof), a MVM intron sequence (or a functional variant (e.g., MVMi) or fragment thereof), or a RK intron sequence (or a functional variant (e.g., RKi) or fragment thereof); and a LIPA transgene sequence, or functional variants or fragments thereof. In some embodiments, the promoter sequence and the intron sequence are selected from any one of the following: i) a CBA promoter sequence (e.g. , CB7) and a SV40 intron sequence (or a functional variant (e.g. , SV40 double mutant or SV40 ATG mutant) or fragment thereof); ii) an AAT promoter sequence (e.g. , hAAT) and a SV40 intron sequence; iii) an AAT promoter sequence (e.g. , hAAT) and a MVM intron sequence (or a functional variant (e.g., MVMi) or fragment thereof); or iv) an AAT promoter sequence (e.g., hAAT) and an RK intron sequence (or a functional variant (e.g., RKi) or fragment thereof).

[0327] In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence, a SV40 intron nucleic acid sequence, and a LIPA transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence, a simian virus 40 (SV40) intron nucleic acid sequence (or a functional variant (e.g. , SV40 double mutant or SV40 ATG mutant) or fragment thereof), and a lipase A (LIPA) codon optimized transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence, a SV40 intron nucleic acid sequence, and a LIPA transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence, a SV40 intron nucleic acid sequence (or a functional variant (e.g., SV40 double mutant or SV40 ATG mutant) or fragment thereof), and a. LIP A codon optimized transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence, a SV40 intron nucleic acid sequence (or a functional variant (e.g. , SV40 double mutant or SV40 ATG mutant) or fragment thereof), and a. LIPA transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence and a LIPA transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence, a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon and 19S acceptor site (e.g. , a mutation of A to T at position 79 and a mutation of A to T at position 30, G to C at position 31, or AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10), and a LIPA transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence, a MVM intron nucleic acid sequence (or a functional variant (e.g., MVMi) or fragment thereof), and a LIPA transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence, a RK intron nucleic acid sequence (or a functional variant (e.g., RKi) or fragment thereof), and a LIPA transgene nucleic acid sequence, or functional variants or fragments thereof. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence, a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon (e.g., a mutation of A to T at position 79 as compared to SEQ ID NO: 10), and a LIPA transgene nucleic acid sequence, or functional variants or fragments thereof.

[0328] In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein does not comprise an intron. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, and a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, and a. LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, and a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, and a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.

[0329] In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10, 11, or 12 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, 11, or 12, aLIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, a SV40 intron nucleic acid sequence comprising SEQ ID NO: 10, 11, or 12, or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, 11, or 12, and a. LIPA codon optimized transgene nucleic acid sequence comprising SEQ ID NO: 7 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, a SV40 intron nucleic acid sequence comprising SEQ ID NO: 10 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, and a LIPA codon optimized transgene nucleic acid sequence comprising SEQ ID NO: 7 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.

[0330] In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10, 11, or 12, or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, 11, or 12, and a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10, 11, or 12, or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, 11, or 12, and a LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.

[0331] In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5’ to 3’, a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, and a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6 or a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence of a polynucleotide as provided herein comprises, optionally from 5 ’ to 3’, a hAAT promoter nucleic acid sequence ...

Claims

CLAIMSWhat is claimed is:

1. A recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ inverted terminal repeat (ITR) b. a promoter, or a variant or fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter), wherein the promoter or fragment thereof is selected from an alpha- 1 antitrypsin (AAT) promoter or a chicken -actin (CBA) promoter; c. an intron, wherein the intron is selected from a simian virus 40 (SV40) intron, a minute virus of mice (MVM) intron, or a RK intron, or a functional variant or fragment thereof; d. a lipase A (LIPA) transgene, or a biologically active fragment thereof; and e. a 3’ ITR.

2. The recombinant expression vector of claim 1, wherein the promoter and the intron are selected from any one of the following: a. a CBA promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and a SV40 intron, or a functional variant or fragment thereof; b. an AAT promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and a SV40 intron, or a functional variant or fragment thereof; c. an AAT promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and a MVM intron, or a functional variant or fragment thereof; or d. an AAT promoter, or a variant or fragment thereof which retains regulatory control or promoter activity, and an RK intron, or a functional variant or fragment thereof.

3. The recombinant expression vector of claim 1 or 2, wherein the promoter is an AAT promoter, optionally a human AAT (hAAT) promoter, or a variant fragment thereof which retains regulatory control or promoter activity (e.g. , retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter), optionally wherein the AAT promoter sequence comprises or consists of SEQ ID NO: 9 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9.

4. The recombinant expression vector of claim 1 or 2, wherein the promoter is a CBA promoter, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter), optionally wherein the CBA promoter sequence comprises or consists of SEQ ID NO: 8 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

5. The recombinant expression vector of any one of claims 1 to 4, wherein the intron is a SV40 intron or a modified SV40 intron sequence.

6. The recombinant expression vector of any one of claims 1 to 5, wherein the SV40 intron sequence comprises or consists of SEQ ID NO: 10, or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10.

7. The recombinant expression vector of claim 5, wherein the modified SV40 intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF), optionally wherein the modified SV40 intron sequence comprises at least one mutation in an ATG codon and / or the 19S acceptor site.

8. The recombinant expression vector of claim 7, wherein the modified SV40 intron sequence comprises one or more mutations selected from the following as compared to SEQ ID NO: 10: a) A to T at position 79; b) A to T at position 30; c) a G to C at position 31 ; and / or d) AG to TC at positions 30 and 319. The recombinant expression vector of claim 7 or 8, wherein the SV40 intron sequence comprises or consists of SEQ ID NO: 11 or SEQ ID NO: 12, or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11 or SEQ ID NO: 12.

10. The recombinant expression vector of any one of claims 1 to 4, wherein the intron is a MVM intron, a modified MVM intro sequence, or a functional variant or fragment thereof.

11. The recombinant expression vector of claim 10, wherein the MVM intron sequence comprises or consists of SEQ ID NO: 13 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13.

12. The recombinant expression vector of claim 10, wherein the modified MVM intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF), optionally wherein the modified intro sequence comprises at least one mutation in an ATG codon.

13. The recombinant expression vector of claim 12, wherein the modified MVM intron sequence comprises at least one mutation selected from the following as compared to SEQ ID NO: 13: a) a mutation of A to T , A to C, or A to G at position 16; and / or b) a mutation of G to C, G to A, or G to T at position 40.

14. The recombinant expression vector of claim 12 or 13, wherein the modified MVM intron sequence comprises or consists of SEQ ID NO: 14 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14.

15. The recombinant expression vector of any one of claims 1 to 4, wherein the intron is a RK intron, a modified RK intro sequence, or a functional variant or fragment thereof.

16. The recombinant expression vector of claim 15, wherein the RK intron sequence comprises or consists of SEQ ID NO: 15, or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15.

17. The recombinant expression vector of claim 15, wherein the modified RK intron sequence comprises at least one mutation that abolishes at least one open reading frame (ORF), optionally wherein the modified RK intron sequence comprises at least one mutation in an ATG codon.

18. The recombinant expression vector of claim 17, wherein the modified RK intron sequence comprises an A to G, A to C, or A to T mutation at position 78 as compared to SEQ ID NO: 15.

19. The recombinant expression vector of claim 17 or 18, wherein the modified RK intron sequence comprises or consists SEQ ID NO: 16 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16.

20. The recombinant expression vector of any one of claims 1 to 19, further comprising a splice donor site 5 ’ of the intron and a splice acceptor site 3 ’ of the intron, optionally wherein the splice donor site and / or splice acceptor site is modified.

21. The recombinant expression vector of any one of claims 1 to 20, wherein the polynucleotide comprises a sequence 5’ (e.g., within the splice donor site) and / or 3’ (e.g., within the splice acceptor site) of the intron sequence that matches a human consensus sequence, optionally wherein the sequence that matches the human consensus sequence at the 5 ’ end of the intron comprises GAG, CAG, AAC, or AAG, and / or the sequence that matches the human consensus sequence at the 3 ’ end of the intron comprises GTT, GT A, GCC, GAA, or GAT.

22. The recombinant expression vector of any one of claims 1 to 21, wherein the lipase A (LIPA) transgene is a human LIPA transgene, optionally wherein the LIPA transgene comprises or consists of SEQ ID NO: 6 or SEQ ID NO: 7 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6 or SEQ ID NO: 7.

23. The recombinant expression vector of any one of claims 1 to 22, further comprising a post-transcriptional regulatory element (PRE) (e.g., WPRE or HPRE), a Kozak sequence, or a functional variant or fragment thereof.

24. The recombinant expression vector of any one of claims 1 to 23, further comprising a polyadenylation (PolyA) signal, optionally wherein the PolyA signal is a bovine growth hormone polyadenylation (bgh-PolyA) signal, synthetic polyA signal, or an SV40 polyA signal.

25. The recombinant expression vector of claim 24, wherein the PolyA signal comprises or consists of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a functional fragment thereof.

26. The recombinant expression vector of any one of claims 1 to 25, further comprising an enhancer, optionally wherein the enhancer is selected from a CMV enhancer and an ApoE HCR enhancer.

27. The recombinant expression vector of claim 26, wherein the enhancer is an ApoE HCR enhancer, optionally wherein the ApoE HCR enhancer comprises or consists of the polynucleotide sequence of SEQ ID NO: 22 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 22.

28. The recombinant expression vector of claim 26, wherein the enhancer is a CMV enhancer, optionally wherein the CMV enhancer comprises or consists of the polynucleotide sequence of SEQ ID NO: 21 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21.

29. The recombinant expression vector of any one of claims 1 to 28, further comprising an untranslated region (UTR), e.g., a LIPA 3’UTR, optionally wherein the UTR comprises or consists of the polynucleotide sequence of SEQ ID NO: 29 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29.

30. The recombinant expression vector of any one of claims 1 to 29, further comprising a stuffer, optionally wherein the staffer comprises or consists of the polynucleotide sequence of SEQ ID NO: 34 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34..

31. The recombinant expression vector of any one of claims 1 to 30, wherein the 5’ ITR is a 5 ’ AAV2 ITR (e.g. , 5 ’ ITR AAV2 R short) and / or 3 ’ ITR is an AAV2 ITR (e.g. , 3 ’ ITR AAV2 R short), or a variant or fragment thereof, optionally wherein the 5’ ITR comprises or consists of the polynucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17or SEQ ID NO: 18, and / or the 3’ ITR comprises or consists of the polynucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 80 or comprises or consists of a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 80.

32. The recombinant expression vector of any one of claims 1 to 31, further comprising at least one antibiotic resistant gene, e.g, selected from kanamycin and / or ampicillin.

33. The recombinant expression vector of any one of claims 1 to 32, further comprising at least one origin of replication polynucleotide sequence, e.g., selected from an Ori, M13 Ori, and / or Ori pUC sequence.

34. The recombinant expression vector of any one of claims 20 to 33, further comprising at least one mutation in one or more adjacent nucleotides to the donor site and / or 16S acceptor site of the intron, optionally comprising an AAC, CAG, or GAG sequence adjacent to the donor site of the intron and / or a GCC or GTA sequence adjacent to the 16S acceptor site of the intron.

35. A recombinant expression vector selected from:1) a recombinant expression vector comprising, optionally from 5’ to 3’: a. a 5 ’ ITR nucleic acid sequence; b. a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a simian virus 40 (SV40) intron nucleic acid sequence, or a functional variant or fragment thereof; d. a lipase A (LIPA jtransgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR nucleic acid sequence;2) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d. a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR nucleic acid sequence;3) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR nucleic acid sequence;4) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d. a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR nucleic acid sequence;5) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR AAV2 R short nucleic acid sequence; b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; d. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR AAV2 R short nucleic acid sequence;6) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and d. a 3 ’ ITR nucleic acid sequence; wherein the recombinant expression vector does not contain an intron nucleic acid sequence;7) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter) c. a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon and 19S acceptor site (e.g., a mutation of A to T at position 79 and a mutation of A to T at position 30, G to C at position 31, or AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10); d. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR nucleic acid sequence;8) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence;b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a MVM intron nucleic acid sequence, or a functional variant or fragment thereof; d. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR nucleic acid sequence;9) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a RK intron nucleic acid sequence, or a functional variant or fragment thereof; d. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e. a 3 ’ ITR nucleic acid sequence; and10) a recombinant expression vector comprising, optionally from 5’ to 3’: a. a 5 ’ ITR nucleic acid sequence; b. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); c. a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon (e.g. , a mutation of A to T at position 79 as compared to SEQ ID NO: 10); d. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; and e . a 3 ’ ITR nucleic acid sequence .

36. A recombinant expression vector selected from:1) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; c. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80;2) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; c. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d. a LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; and e. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80;3) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80;4) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17;b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; and d. a LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; e. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80;5) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 18; b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e. a 3’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 20;6) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and d. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; wherein the recombinant expression vector does not contain an intron nucleic acid sequence;7) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9;c. a SV40i intron nucleic acid sequence comprising or consisting of SEQ ID NO: 11; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80;8) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c. a MVM intron nucleic acid sequence comprising or consisting of SEQ ID NO: 14; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80;9) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c. a RK intron nucleic acid sequence comprising or consisting of SEQ ID NO: 16; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; and e. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; and10) a recombinant expression vector comprising, optionally from 5’ to 3’: a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; c. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 12; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; ande. a 3’ 777? nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80.

37. The recombinant expression vector of claim 35 or 36, further comprising a post- transcriptional regulatory element (PRE) (e.g., WPRE or HPRE) nucleic acid sequence, a polyadenylation (Poly A) signal (e.g., bovine growth hormone polyadenylation ( / >g / ?-PolyA) signal) nucleic acid sequence, and / or an enhancer (e.g., CMV enhancer or ApoE HCR enhancer) nucleic acid sequence, or a functional variants or fragments thereof.

38. A recombinant expression vector selected from:1) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a CMV enhancer nucleic acid sequence; c. a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence;2) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. a CMV enhancer nucleic acid sequence; c. a CB7 promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter);d. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e. a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence;3) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence;4) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. an ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter);d. a SV40 intron nucleic acid sequence, or a functional variant or fragment thereof; e. a LIPA codon optimized transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence;5) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR AAV2 R short nucleic acid sequence; b. an ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d. a SV40 intron nucleic acid sequence; e. a. LIP A 3’ UTR nucleic acid sequence; f. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; g. a bGH poly (A) signal nucleic acid sequence; h. a staffer nucleic acid sequence; i. a 3 ’ ITR AAV2 R short nucleic acid sequence; j . a P syn E. coli nucleic acid sequence; k. a M KanR* nucleic acid sequence; and l. an ori pUC nucleic acid sequence;6) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. an ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or tall length promoter);d. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; e. a bGH poly (A) signal nucleic acid sequence; f. a 3 ’ ITR nucleic acid sequence; g. an AmpR promoter nucleic acid sequence; h. a KanR nucleic acid sequence; i. an ori nucleic acid sequence; and j . a M 13 ori nucleic acid sequence; wherein the recombinant expression vector does not contain an intron nucleic acid sequence;7) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. an ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d. a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon and 19S acceptor site (e.g., a mutation of A to T at position 79 and a mutation of A to T at position 30, G to C at position 31, or AG to TC at positions 30 and 31 as compared to SEQ ID NO: 10); e. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence;8) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. an ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d. a MVM intron nucleic acid sequence, or a functional variant or fragment thereof; e. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence;9) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5 ’ ITR nucleic acid sequence; b. an ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d. a RK intron nucleic acid sequence, or a functional variant or fragment thereof; e. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence; and10) a recombinant expression vector comprising, optionally from 5’ to 3’: a. a 5 ’ ITR nucleic acid sequence; b. an ApoE HCR1 enhancer nucleic acid sequence; c. a hAAT promoter nucleic acid sequence, or a variant fragment thereof which retains regulatory control or promoter activity (e.g., retains at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the wild-type or full length promoter); d. a SV40 intron nucleic acid sequence comprising one or more mutations in an ATG codon (e.g. , a mutation of A to T at position 79 as compared to SEQ ID NO: 10); e. a LIPA transgene nucleic acid sequence, or a biologically active fragment thereof; f. a bGH poly (A) signal nucleic acid sequence; g. a 3 ’ ITR nucleic acid sequence; h. an AmpR promoter nucleic acid sequence; i. a KanR nucleic acid sequence; j . an ori nucleic acid sequence; and k. a M13 ori nucleic acid sequence.

39. A recombinant expression vector selected from:1) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a CMV enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 21; c. a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; d. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e. a. LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f. a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; andk. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27;2) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. a CMV enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 21; c. a CB7 promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 8; d. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e. a lipase A (LIPA) codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; f. a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27;3) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e. a. LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6;f. a bGH poly (A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27;4) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e. a LIPA codon optimized transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 7; f. a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27;5) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ :a. a 5’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 18; b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 10; e. a LIPA 3’ UTR nucleic acid sequence comprising or consisting of SEQ ID NO: 29; f. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; g. a bGH poly (A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; h. a staffer nucleic acid sequence comprising or consisting of SEQ ID NO: 34; i. a 3’ ITR AAV2 R short nucleic acid sequence comprising or consisting of SEQ ID NO: 20; j . a P syn E. coli nucleic acid sequence comprising or consisting of SEQ ID NO: 31; k. a M KanR* nucleic acid sequence comprising or consisting of SEQ ID NO: 33; and l. an ori pUC nucleic acid sequence comprising or consisting of SEQ ID NO: 28;6) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a LIPA transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; e. a bGH poly (A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23;f. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; g. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; h. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; i. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and j. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27; wherein the recombinant expression vector does not contain an intron nucleic acid sequence;7) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 11; e. a. LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f. a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27;8) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17;b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a MVM intron nucleic acid sequence comprising or consisting of SEQ ID NO: 14; e. a. LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f. a bGH poly (A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27;9) a recombinant expression vector comprising, optionally from 5 ’ to 3 ’ : a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a RK intron nucleic acid sequence comprising or consisting of SEQ ID NO: 16; e. a LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f. a bGH poly (A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80;h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27; and10) a recombinant expression vector comprising, optionally from 5’ to 3’: a. a 5’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 17; b. an ApoE HCR1 enhancer nucleic acid sequence comprising or consisting of SEQ ID NO: 22; c. a hAAT promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 9; d. a SV40 intron nucleic acid sequence comprising or consisting of SEQ ID NO: 12; e. a. LIP A transgene nucleic acid sequence comprising or consisting of SEQ ID NO: 6; f. a bGH poly(A) signal nucleic acid sequence comprising or consisting of SEQ ID NO: 23; g. a 3’ ITR nucleic acid sequence comprising or consisting of SEQ ID NO: 19 or SEQ ID NO: 80; h. an AmpR promoter nucleic acid sequence comprising or consisting of SEQ ID NO: 30; i. a KanR nucleic acid sequence comprising or consisting of SEQ ID NO: 32; j. an ori nucleic acid sequence comprising or consisting of SEQ ID NO: 26; and k. a M13 ori nucleic acid sequence comprising or consisting of SEQ ID NO: 27.

40. The recombinant expression vector of any one of claims 1 to 39, wherein the recombinant expression vector comprises or consists any one of SEQ ID NOs: 38-47, or comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-47.

41. The recombinant expression vector of any one of claims 1 to 40, wherein the recombinant expression vector is a recombinant viral vector, preferably an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex vector, or a retroviral vector, more preferably an adeno-associated viral (AAV) vector (e.g., an AAV vector that has liver tropism).

42. The recombinant expression vector of claim 41, wherein the AAV vector is a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh, AAVrhlO, AAVrh74, AAV-DJ, AAV-DJ / 8, Anc80, and AAV7m8, or a derivative, hybrid or chimeric serotype thereof.

43. The recombinant expression vector of claim 41 to 42, wherein the AAV vector is an AAV9 serotype, optionally wherein the AVV vector is a single stranded AAV9 (ssAAV9) vector or a self-complementary AAV9 (scAAV9) vector.

44. The recombinant expression vector of any one of claims 41 to 43, wherein the AAV vector is a recombinant adeno-associated viral (rAAV) vector.

45. A viral particle comprising the recombinant expression vector of any one of claims 1 to 44.

46. A host cell comprising a recombinant expression vector of any one of claims 1 to 44, or a viral particle of claim 45, optionally wherein the host cell is a mammalian or an insect cell.

47. The host cell of claim 46, further comprising a helper construct, optionally wherein the helper construct is a pHelp plasmid encoding at least one adenovirus regulatory element (e.g., E2A, E4, and / or VAI / II RNA genes).

48. The host cell of any claim 46 or 47, further comprising a Rep2-Cap9 plasmid.

49. A pharmaceutical composition comprising a recombinant expression vector of any one of claims 1 to 44, or a viral particle of claim 45, and a pharmaceutically acceptable excipient, optionally wherein the composition is formulated for intravenous (IV) delivery, portal vein delivery, or intramuscular delivery.

50. A method of treating a subject having or suspected of having a Lysosomal Acid Lipase Deficiency (LAL-D) or ameliorating one or more symptoms thereof comprising administering to the subject a therapeutically effective amount of a recombinant expression vector of any one of claims 1 to 44, a viral particle of claim 45, or a pharmaceutical composition of claim 49.

51. A method of treating hepatomegaly or hepatosplenomegaly or ameliorating one or more symptoms thereof in a subject comprising administering a therapeutically effective amount of a recombinant expression vector of any one of claims 1 to 44, a viral particle of claim 45, or a pharmaceutical composition of claim 49.

52. The method of claim 50, wherein the LAL-D is Wolman disease or cholesterol ester storage disease (CESD).

53. The method of any one of claims 50 to 52, wherein the subject is a human subject.

54. The method of any one of claims 50 to 53, wherein said administering is via portal vein delivery, intramuscular delivery, or intravenous (IV) injection.

55. The method of any one of claims 50 to 54, wherein said therapeutically effective amount is at a dose of at least about lei 3 vg / kg, at least about lel4 vg / kg, at least about lei 5 vg / kg, or at least about le 16 vg / kg.

56. The method of any one of claims 50 to 55, wherein at least one additional therapeutic agent or therapy is administered to the subject, optionally wherein the at least one additional therapeutic agent or therapy is selected from corticosteroids, enzyme replacement therapy (ERT), rapamycin, immunoadsorption, and rituximab.

57. A method of making a viral particle, said method comprising incubating the host cell of any one of claims 46 to 48 under conditions suitable to enclose a recombinant expression vector in a viral capsid, e.g., an AAV capsid, thereby making the viral particle, e.g., AAV particle.

58. A method of making a pharmaceutical composition, said method comprising culturing the host cell of any of one of claims 46 to 48, collecting the supernatant of the cultured host cells, concentrating and purifying recombinant viral vectors from the collected supernatant, and adding pharmaceutically acceptable excipients to the purified recombinant viral vectors.

59. A kit comprising a recombinant expression vector of any one of claims 1 to 44, a viral particle of claim 45, or a pharmaceutical composition of claim 49, optionally further comprising instructions and / or means for administering the polynucleotide, the recombinant expression vector, the viral particle, or the pharmaceutical composition to a subject in need thereof.

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