Gene therapy constructs for metabolic disorders

Abstract

The present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, a human insulin-like growth factor II (IGFII) gene sequence, and a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis inserted into said IGFII gene sequence between the nucleotides encoding amino acids 28 and 42 of mature IGFII. The invention further relates to related viral particles, fusion proteins, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to the field of gene therapy, and in particular to gene delivery vehicles and methods of gene therapy for the treatment of metabolic disorders, including lysosomal storage disorders. [Background technology]

[0002] Metabolic disorders are inborn errors of metabolism that affect the metabolism of macronutrients and micronutrients, including carbohydrates, proteins, amino acids, lipids, and fatty acids. Most metabolic disorders are caused by genetic enzyme deficiencies. Lysosomal storage disorders (LSDs) are metabolic disorders characterized by lysosomal dysfunction, most of which are inherited as autosomal recessive traits. Lysosomes are membrane-enclosed organelles containing a variety of enzymes capable of breaking down all types of biological substances, namely proteins, nucleic acids, carbohydrates, and lipids. Lysosomes both break down materials ingested from outside the cell, including microorganisms, and digest the cell's own components. Many LSDs are caused by the absence or deficiency of one or more lysosomal proteins (hydrolases, transport proteins, receptor molecules, ion pumps), particularly lysosomal hydrolases. Most LSDs have a progressive degenerative disease course. Symptoms include lysosomal accumulation ("storage") of undegraded materials, cellular damage, and tissue dysfunction, resulting in morbidity and premature death.

[0003] Enzyme replacement therapy (ERT), which involves the intravenous administration of recombinant human enzymes, has been developed for several LSDs, including Pompe disease and Hunter syndrome. The treatment aims to increase the intracellular levels of enzyme activity in affected cells and tissues, thereby reducing or preventing lysosomal accumulation and ultimately the symptoms of the disease.

[0004] Normally, misclassified and therefore secreted lysosomal enzymes are transported to lysosomes through a receptor-mediated endocytosis mechanism via the CI-M6P / IGF2 receptor. This endogenous process has been exploited in several other approaches for the treatment of LSDs, indicating that hematopoietic stem cells (HSCs) are a preferred target for ex vivo gene therapy. Because HSCs divide several times throughout their lifetime, vectors capable of long-term expression of corrected genes and integrating into the host genome are preferred. Lentiviral vectors or site-specific gene editing approaches fulfill these requirements and may represent therapeutic options for the treatment of LSDs via HSC-mediated gene therapy. Once transplanted, genetically corrected HSC-derived cells can synthesize and secrete therapeutic proteins, which can be successfully incorporated into target tissues, enabling cross-correction of disease pathology.

[0005] WO2018 / 146473 describes a gene therapy strategy for mucopolysaccharidosis type II (MPSII) or Hunter syndrome, which is caused by mutations in the gene encoding iduronate-2-sulfatase (IDS gene). The construct contains repeats of (partial) IDS gene sequences and apolipoprotein E (ApoEII) gene sequences.

[0006] In WO2008 / 136670, Van Til et al. (Blood. 2010 Jul 1;115(26):5329-37) and Wagemaker et al. (Mol Ther Methods Clin Dev. 2020 May 4;17:1014-1025) described lentiviral constructs containing the acid alpha-glucosidase-encoding gene (GAA gene) defective in Pompe disease for transduction of mouse hematopoietic stem cells. Recently, Dogan et al. (https: / / doi.org / 10.1101 / 2021.12.28.474352) described several constructs for hematopoietic stem cell gene therapy, including untagged, codon-optimized GAA (GAAco) and GAA lacking the signal peptide, tagged with several IGFII and modified IGFII tags.

[0007] ERT results in variable clinical responses (due to antibody formation against the recombinant enzyme and other unknown factors), is highly invasive (4-6 hour infusion every 1-2 weeks), and does not provide a cure for LSD.

[0008] Current lentiviral gene therapy is based on lentivirus integration into genomic DNA. For safety reasons, the number of integrations per genome should be maintained at a maximum of at least about 5 copies / genome or less while achieving sufficient therapeutic levels of enzyme. Current gene therapy strategies have insufficient therapeutic levels in all target tissues, namely peripheral tissues and the central and peripheral nervous systems. In addition, the undesirable effects of current epitope tags, such as IGF2 tags, and antibody formation against therapeutic enzymes are further safety concerns of current gene therapy strategies.

[0009] Therefore, there is a need in the art for gene therapy strategies with enhanced therapeutic efficacy and safety. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2018 / 146473 [License 2] WO2008 / 136670 [License 3] US9469683B2 [License 4] Oranda Country License Application No. NL2031676 [Patent Document 5] WO2009 / 137721 [Non-licensed literature]

[0011] [Non-licensed Document 1] Van Tilら(Blood. 2010 Jul 1;115(26):5329~37 pages) [Non-licensed Document 2] Wagemakerら(Mol Ther Methods Clin Dev. 2020 May 4;17:1014~1025 pages) [Non-licensed Document 3] Doganら(https: / / doi.org / 10.1101 / 2021.12.28.474352) [Non-licensed Document 4] Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001) [Non-licensed Document 5] Wangら, 2020. Fluids and Barriers of the CNS; 17(47); doi.org / 10.1186 / s12987-020-00209-0 [Non-licensed Document 6] Hashimoto, J. Biol. Chem. 270(30):18013~8 pages (1995) [Non-licensed Document 7] Gleitz, H.F. et al., (2018, EMBO Mol Med 10. doi.10.15252 / emmm.201708730)

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Summary of the Invention

Problems to be Solved by the Invention

[0012] It is an object of the present invention to overcome one or more of the problems associated with the above-mentioned therapies, particularly gene therapies, for the treatment of metabolic disorders, particularly lysosomal storage disorders. A further object is to provide nucleic acid molecules, gene therapy vectors, and viral particles for gene therapy of such disorders. [Means for solving the problem]

[0013] In a first aspect, the present invention therefore provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - a nucleotide sequence encoding a metabolic protein or a part thereof, or a sequence having at least 90% sequence identity with said metabolic protein or a part thereof, - human insulin-like growth factor II (IGFII) gene sequence, and - a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis inserted into said IGF II gene sequence at a position between the nucleotides encoding amino acids 28 and 42 of mature IGF II. The present invention provides a nucleic acid molecule comprising:

[0014] Optional flexible linker sequences are present adjacent to the inserted sequence located between the nucleotides encoding amino acids 28 and 42 of mature IGF II in the referenced IGF II gene sequence. These linkers are designed to support proper exposure of the inserted sequence. Optional cysteine ​​residues are present around these potentially flexible linkers to support disulfide bond formation, thereby ensuring the structural integrity of IGF II.

[0015] In a further aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, and a receptor-associated protein (RAP) sequence, preferably a RAP tag, more preferably amino acids 251 to 262 of human RAP (EAKIEKHNHYQK; RAP12), or repeats thereof, such as RAP12x2 (EAKIEKHNHYQKGEAKIEKHNHYQK).

[0016] In a further aspect, the present invention provides a viral particle comprising a nucleic acid molecule according to the invention.

[0017] In a further aspect, the present invention provides a fusion protein encoded by a nucleic acid molecule according to the invention.

[0018] In a further aspect, the present invention provides a fusion protein comprising a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, fused to a human insulin-like growth factor II (IGF II) sequence and at least one peptide that promotes cellular uptake or transcytosis, wherein said metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, the human IGF II sequence, and / or said at least one peptide are separated by one or more linking sequences.

[0019] In a further aspect, the present invention provides a nucleic acid sequence encoding a fusion peptide according to the invention.

[0020] In a further aspect, the present invention provides a cell population, preferably a hematopoietic stem cell (HSC) population, provided with a nucleic acid molecule, vector or viral particle according to the invention.

[0021] In a further aspect, the present invention provides a nucleic acid molecule, a viral particle, a fusion protein, or a cell population according to the invention for use in a method for the treatment of a metabolic disorder, preferably a lysosomal storage disorder.

[0022] In a further aspect, the present invention provides a method of treatment comprising administering a nucleic acid molecule, viral particle, fusion protein or cell population according to the invention to an individual in need thereof.

[0023] In a further aspect, the present invention provides the use of a nucleic acid molecule, a viral particle, a fusion protein, or a cell population according to the invention in the preparation of a medicament for the treatment of a metabolic disorder, preferably a lysosomal storage disorder. DETAILED DESCRIPTION OF THE INVENTION

[0024] As used herein, "comprise" and its conjugations are used in their open-ended sense, meaning that the items following the phrase are included, but not that items not specifically mentioned are excluded. Additionally, the verb "consisting of" may be replaced with "consisting essentially of," which means that the compound or ancillary compound defined herein may contain additional ingredients other than those specifically identified that do not alter the unique characteristics of the invention.

[0025] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0026] The term "approximately" or "about," when used in connection with a numerical value (e.g., approximately 10, about 10), preferably means that the value can be a given value (e.g., 10), plus or minus 5% of the value (e.g., 10 plus or minus 5%), preferably plus or minus 1% of the value.

[0027] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0028] As used herein, a nucleic acid molecule of the present invention comprises a nucleotide chain of any length, preferably DNA and / or RNA. In other embodiments, a nucleic acid molecule or nucleic acid sequence of the present invention comprises other types of nucleic acid structures, such as, for example, a DNA / RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA), and / or ribozyme. Thus, the term "nucleotide sequence" also encompasses chains comprising unnatural nucleotides, modified nucleotides, and / or non-nucleotide building blocks that exhibit the same function as natural nucleotides. The term nucleic acid molecule includes recombinant and synthetic nucleic acid molecules, as well as nucleic acid molecules that have been isolated, e.g., partially, from natural sources. In preferred embodiments of the present invention, the nucleic acid molecule is DNA or RNA. The nucleic acid molecule may be single-stranded or double-stranded. Those skilled in the art are fully enabled to prepare the nucleic acid molecules and vectors of the present invention using standard molecular cloning techniques, as described, for example, in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001), incorporated herein by reference.

[0029] The term "peptide" refers to a compound containing amino acids linked via peptide bonds. In particular, "peptide" refers to small proteins containing up to 50 amino acid residues.

[0030] In the amino acid sequences defined or listed herein, amino acids are designated by their single letter code. These single-letter and three-letter symbols are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine.

[0031] As used herein in reference to an amino acid sequence of a peptide, protein, or fusion protein, the terms "N-terminus" and "C-terminus" refer to the relative positions of the amino acid sequence toward the N-terminus and C-terminus, respectively. "N-terminus" and "C-terminus" refer to the final amino- and carboxyl-terminus of a polypeptide, respectively. "Immediately N-terminus" and "immediately C-terminus" refer to the position of a first amino acid sequence relative to a second amino acid sequence when the first and second amino acid sequences are covalently linked to provide a contiguous amino acid sequence.

[0032] The percentage of identity of an amino acid or nucleic acid sequence or the term "% sequence identity" is defined herein as the percentage of residues over the entire length of an amino acid or nucleic acid sequence that are identical to residues in a reference amino acid or nucleic acid sequence, after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art, such as "Align 2." Programs for determining nucleotide sequence identity are also well known in the art, such as the BESTFIT, FASTA, and GAP programs. These programs are readily utilized with the default parameters recommended by the manufacturer.

[0033] As used herein, the term "individual" includes humans. Preferably, the subject is a human, particularly a human suffering from a metabolic disorder, particularly a lysosomal storage disorder, more preferably Hunter syndrome, Pompe disease, or CLN2. In some embodiments, the individual is a child, i.e., up to 18 years of age. In other embodiments, the individual is an adult, i.e., 18 years of age or older.

[0034] The term "therapeutically effective amount," as used herein, refers to the amount of an agent or composition being administered sufficient to relieve to some extent one or more of the symptoms of the disease or condition being treated. This may be reduction or alleviation of the symptoms of the disease or condition, reduction or alleviation of the cause of the disease or condition, or any other desired therapeutic effect.

[0035] The term "treatment" refers to inhibiting a disease or condition, i.e., arresting or reducing the onset of a disease or disorder or at least one clinical symptom, and / or alleviating the symptoms of a disease or disorder. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered even in the absence of symptoms. For example, treatment may be administered to a suspected individual prior to the onset of symptoms (e.g., in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0036] The present inventors have developed constructs encoding modular, tunable fusion proteins. The constructs contain a combination of two epitope tags. One of the epitope tags is an IGF II sequence, and the other is a separate tag based on one or more peptides that promote cellular uptake or transcytosis, such as a peptide that promotes crossing the blood-brain barrier (BBB). As demonstrated in the Examples herein, a suitable tunable construct is a lentiviral construct of a lysosomal hydrolase, an IDS in this example, and a combination of two different epitope tags. Several examples of second epitope tags are included in the Examples herein: an ApoE2-derived tag, an ApoB-derived tag, a RAP12 tag, a leptitin-30 tag, and an Angiopep2 tag. For all of these tags, uptake of the fusion protein in desired cells (see Figures 6 and 7), reduced lysosomal substrate accumulation (see, e.g., Figures 12, 14, and 17), and the ability to bind CI-M6P / IGFIIR (Figure 19A), reduced binding to the insulin receptor (Figure 19B), and additional receptors not normally bound to the IGFII tag (e.g., LRP-1) have been demonstrated.

[0037] The principles underlying the constructs of the present invention are not limited to one particular lysosomal hydrolase, but are applicable to metabolic proteins in general, particularly metabolic enzymes whose deficiency is associated with metabolic disorders. Therefore, the constructs and other products are modular, having three main components: 1) the metabolic protein sequence, 2) the human IGF II sequence, and 3) the sequence of a second epitope tag. In particular, components 1) and 3) can be varied. Alternatively, components 2) and 3) can be present in an intermediate construct, while component 1), the metabolic protein sequence, can be varied.

[0038] The modular, tunable constructs of the present invention utilize a combination of three main components: 1) a metabolic protein sequence, 2) a human IGF II sequence, and 3) a second epitope tag sequence. Thus, by selecting the relevant metabolic protein sequence, they are tunable in that they adjust the biodistribution of relevant therapeutic proteins for different diseases. Second, by utilizing a combination of two different epitope tags, two different mechanisms are utilized to increase the presence of the relevant metabolic protein at the target site. The IGF II tag targets the fusion protein to the lysosome, thereby enabling uptake of the encoded fusion protein via the cation-independent mannose 6-phosphate or IGF2 receptor (CI-M6PR / IGF2R). In the case of lysosomal hydrolases, for example, IDS and GAA already bind to CI-M6PR via M6P present in these proteins, whereas the IGF II tag increases lysosomal targeting because IGF II binds with higher affinity to the same receptor. This is particularly advantageous for the treatment of LSDs, as different disorders are characterized by differences in the organs primarily affected. For example, the organs primarily affected in Pompe disease include skeletal muscle, brain / central nervous system, and heart, while the organs primarily affected in Hunter syndrome include lung, heart, joints, connective tissue, and brain / central nervous system. In particular, the second epitope tag can be selected to target a receptor different from CI-M6PR / IGF2R. In this way, uptake of related metabolic proteins, such as lysosomal enzymes, can be specific to the relevant organs affected by a particular LSD, such as the brain and nervous system, by utilizing additional epitopes that bind to different receptors, particularly the LRP-1 receptor, and promote uptake in target cells or passage through the blood-brain barrier. Both IGFII and the second tag that promotes passage through the BBB, such as an ApoE-based tag, bind to receptors expressed by endothelial cells (ECs) that form the BBB.However, the amount of receptors expressed by ECs at the BBB, particularly those expressed by human ECs, is limited (see Wang et al., 2020. Fluids and Barriers of the CNS; 17(47); doi.org / 10.1186 / s12987-020-00209-0). This limits the amount of therapeutic protein that can cross the BBB when using only one tag, such as IGFII that binds to CI-M6PR / IGF2R. Targeting multiple receptors, i.e., using two or more different epitope tags that bind to different receptors, makes it possible to proportionally increase the amount of therapeutic protein that can cross the BBB. This is particularly true when the first receptor is saturated, so that an additional tag specific for a second and optionally different receptor increases BBB uptake. The present inventors have shown that constructs with a double epitope tag have affinity for the CI-M6PR / IGF2R receptor comparable to that of constructs with only an IGF II tag (see Figure 19A). This demonstrates that the double epitope tag does not negatively affect binding to this receptor, which is necessary for lysosomal uptake of lysosomal enzymes. Furthermore, it has been demonstrated that the affinity of the double epitope tagged construct for the insulin receptor is further reduced compared to the affinity of constructs with only an IGF II tag (see Figure 19B). This is an advantage of the double epitope tagged construct of the present invention, since binding to the insulin receptor is undesirable. This is achieved while the double tagged construct has binding affinity for receptors other than those that normally bind the IGF II tag, e.g., LRP-1 (Figures 8A and 8B).

[0039] Additionally, the inventors have found that epitope tagging for BBB crossing is not always successful when the tag is included in a lentiviral construct; however, this issue is successfully addressed by the so-called indel constructs of the present invention. This is illustrated for the RAP12x2 tag in Figure 8 herein. It has been demonstrated that the RAP12x2 tag coupled to an IDS sequence does not efficiently bind to its target, the LRP1 receptor, in a functional ELISA assay (see Figure 8A). When the RAP12x2 sequence is inserted into an IGFII sequence (i.e., the IDS.IGF2 indel RAP12x2 construct), it is able to exert its normal function, i.e., bind to the LRP1 receptor, thus promoting crossing of the BBB (Figure 8B). Indeed, it is generally known in the art that epitope tagging can interfere with the normal function of the protein to which it is coupled, as well as the function of the tag itself due to steric hindrance of the tagged protein. It is difficult to predict whether this will occur for a particular tag-protein combination. The present inventors have shown that the insertion of a second epitope tag between amino acids 29 and 41 of the IGF II sequence provides a strategy to circumvent non-functionality of the tag due to, for example, steric hindrance. In particular, the insertion of a second epitope tag in an IGF II sequence lacking amino acids 30-40 or 29-41 results in a preferred conformation of the second epitope tag, allowing the tag to bind to its receptor. This may be supported by the presence of flexible linker sequences flanking the inserted sequence, located between the nucleotides encoding amino acids 28 and 42 of mature IGF II in the referenced IGF II gene sequence, which results in preferred exposure of the inserted tag. Additionally, cysteine ​​residues may be added to the N- and C-termini of both flexible linkers to support the correct conformation of the IGF II tag.

[0040] Thus, by selecting an advantageous combination of a metabolic protein, e.g., a lysosomal hydrolase, and a second epitope tag, the constructs of the present invention can be tailored for specific metabolic disorders, particularly specific LSDs, as well as for tissue-specific targeting and uptake. In particular, the IGF II sequence is a fixed component of the modular construct, while the second epitope is linked to a specific metabolic protein and, consequently, to a specific disorder. The inventors have shown that successful results can be achieved by inserting the second epitope tag within the IGF II sequence, particularly at a position within IGF II required for efficient binding to the insulin receptor (see US9469683B2). In this way, undesirable side effects due to binding of the encoded fusion protein to the insulin receptor are avoided. The inventors have previously shown that efficacy in the brain can also be achieved by a vector containing an IGF II sequence with a deletion in the nucleotide sequence encoding the insulin-binding receptor or a deletion in the nucleotide sequence (unpublished data, e.g., Dutch Patent Application NL2031676). This is surprising because the insulin receptor has been reported to mediate transcytosis in vivo, but no such report has been made for CI-M6PR, and therefore the insulin receptor was thought to be important for crossing the blood-brain barrier. This can also be inferred from the lack of efficacy of intravenously applied enzyme replacement therapy (targeting CI-M6PR) in lysosomal-disordered brains.

[0041] In a first aspect, the present invention therefore provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - a nucleotide sequence encoding a metabolic protein or a part thereof, or a sequence having at least 90% sequence identity with said metabolic protein or a part thereof, - human insulin-like growth factor II (IGFII) gene sequence, and - a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis The present invention provides a nucleic acid molecule comprising:

[0042] In some embodiments, the nucleic acid molecule of the present invention comprises a human insulin-like growth factor II (IGFII or IGF2) gene sequence. Figure 1 shows the sequence of human mature IGFII, including its signal peptide. IGFII is synthesized as a 180-amino acid proprotein, which is ultimately processed into a 67-amino acid biologically active IGFII, referred to as mature IGFII. First, the 24-amino acid signal peptide is removed from the N-terminus to generate pro-IGFII (156 amino acids), which is then cleaved into a 104-amino acid protein product [IGFII(1-104)]. Subsequent endoproteolysis generates IGFII(1-87) and the 67-amino acid mature IGFII protein.

[0043] In preferred embodiments, the IGF II gene sequence comprises a sequence encoding amino acids 8-28 and 42-67 of mature human IGF II shown in Figure 1, or a sequence having at least 70% sequence identity thereto, preferably at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, more preferably at least 95% sequence identity thereto. In some preferred embodiments, the IGF II gene sequence comprises a sequence encoding amino acids 8-29 and 41-67 of mature human IGF II shown in Figure 1, or a sequence having at least 70% sequence identity thereto, preferably at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, more preferably at least 95% sequence identity thereto.

[0044] In a preferred embodiment, amino acids 2-7 are deleted, which results in a significant decrease in affinity for the human IGF-I receptor and an increase in affinity for the IGF II receptor (Hashimoto et al., J. Biol. Chem. 270(30):18013-8 (1995)). Thus, in a further preferred embodiment, the IGF II gene sequence comprises a sequence encoding amino acids 1, 8-28, and 42-67, or 1, 8-29, and 41-67, of mature human IGF II shown in Figure 1, or a sequence having at least 70% sequence identity thereto, preferably at least 80% sequence identity thereto, more preferably at least 90% sequence identity thereto, and more preferably at least 95% sequence identity thereto.

[0045] In preferred embodiments, the IGF-II sequence encodes an IGF-II amino acid sequence having a mutation that reduces the binding affinity to the insulin receptor compared to wild-type human IGF-II. In some preferred embodiments, the human IGF-II gene sequence contains a mutation, preferably a deletion, within the nucleotide region encoding amino acids 29-41 of IGF-II. This region of the IGF-II sequence contains the insulin receptor-binding domain. Thus, the IGF-II sequence is preferably mutated such that binding of the fusion protein to the insulin receptor is reduced by at least 50% or abolished. In a preferred embodiment, the IGF II gene sequence comprises at least amino acids 29-41, 30-41, 31-41, 32-41, 33-41, 34-41, 35-41, 36-41, 37-41, 29-40, 30-40, 31-40, 32-40, 33-40, 34-40, 35-40, 36-40, 37-40, 29-39, 30-39, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 29-38, 30-38 ...0-38, 31-39, 32-3 These include deletions of nucleotides encoding 31 to 38, 32 to 38, 33 to 38, 34 to 38, 35 to 38, 36 to 38, 29 to 37, 30 to 37, 31 to 37, 32 to 37, 33 to 37, 34 to 37, 35 to 37, 29 to 36, 30 to 36, 31 to 36, 32 to 36, 33 to 36, 34 to 36, 29 to 35, 30 to 35, 31 to 35, 32 to 35, 33 to 35, 29 to 34, 30 to 34, 31 to 34, 30 to 34, 29 to 33, 30 to 33, 31 to 33, 29 to 32, or 30 to 32.

[0046] In a preferred embodiment, the IGF II gene sequence comprises a deletion of nucleotides encoding amino acids 30 to 40 of mature human IGF II. Thus, in a preferred embodiment, the IGF II gene sequence comprises the nucleotide sequence encoding amino acids 1, 8 to 29, and 41 to 67 of mature IGF II shown in Figure 1.

[0047] In a preferred embodiment, the IGF II gene sequence comprises a deletion of nucleotides encoding amino acids 29 to 41 of mature human IGF II. Thus, in a preferred embodiment, the IGF II gene sequence comprises the nucleotide sequence encoding amino acids 1, 8 to 28, and 42 to 67 of mature IGF II shown in Figure 1.

[0048] In some preferred embodiments, the IGF II gene sequence consists of the nucleotide sequence encoding amino acids 1, 8-28, and 42-67 of human mature IGF II as shown in FIG.

[0049] In some preferred embodiments, the IGF II gene sequence consists of the nucleotide sequence encoding amino acids 1, 8-29, and 41-67 of human mature IGF II shown in FIG.

[0050] Further suitable IGF II mutants with reduced insulin receptor binding are described in WO2009 / 137721, which is incorporated herein by reference.

[0051] As detailed herein below, a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB, is preferably inserted into the IGFII gene sequence at a position between the nucleotides encoding amino acids 28 and 42 of mature human IGFII. If the IGFII sequence contains a mutation within the nucleotide region encoding amino acids 29-41 of IGFII as detailed herein above, then the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB, is preferably inserted into the IGFII sequence at the position of the deleted amino acids.

[0052] In other embodiments, the IGF II gene sequence comprises a nucleotide sequence encoding an IGF II signal peptide and amino acids 8-67, preferably 1 and 8-67, of IGF II shown in Figure 1, or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% sequence identity thereto. In some embodiments, the IGF II gene sequence encodes human mature IGF II (i.e., amino acids 1-67), the sequence of which is shown in Figure 1, or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% sequence identity thereto. In some embodiments, the IGF II gene sequence consists of a nucleotide sequence encoding amino acids 1 and 8-67 of human mature IGF II shown in Figure 1.

[0053] In some embodiments, the IGF II gene sequence comprises a nucleotide sequence encoding the human IGF II signal peptide shown as amino acids -24 to -1 (MGIPMGKSMLVLLTFLAFASCCIA) in Figure 1, or a sequence having at least 90% sequence identity thereto. Preferably, the IGF II gene sequence comprises a nucleotide sequence encoding the human IGF II signal peptide shown as amino acids -24 to -1 in Figure 1.

[0054] The use of nucleic acid molecules or vectors comprising such IGF II sequences with deletions in or within the nucleotide sequence encoding the insulin-binding receptor preferably leaves binding to the IGF II / mannose-6-phosphate receptor (IGFIIR / M6PR) intact. IGF II gene sequences with deletions in or within the nucleotide sequence encoding the insulin-binding receptor domain have been found to improve safety with respect to glucose metabolism, which is of clinical importance.

[0055] The nucleic acid molecules of the invention comprise a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis. As used herein, "promoting cellular uptake or transcytosis" means that a protein or polypeptide expressed from a nucleic acid molecule of the invention has increased cellular uptake or transcytosis when at least one peptide is present in the protein or polypeptide, compared to an otherwise identical protein or polypeptide lacking the at least one peptide. The peptide that promotes cellular uptake or transcytosis is not IGF II or is not derived from IGF II.

[0056] As used herein, "cellular uptake" refers to the internalization of a molecule or compound, particularly a proteinaceous compound encoded by a nucleic acid molecule of the present invention, into a cell. As used herein, "transcytosis" refers to a transcellular vesicular transport mechanism in which a cell encapsulates a molecule or compound within a vesicle formed by the cell membrane and moves the vesicle within the cell, releasing the material through the opposite cell membrane. Transcytosis is particularly transcellular vesicular transport across endothelial cells. In a preferred embodiment, transcytosis is transcellular vesicular transport across the blood-brain barrier (BBB).

[0057] In preferred embodiments, at least one peptide that promotes cellular uptake or transcytosis has a length of at most 100 amino acids. More preferably, the peptide has a length of at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids, more preferably at most 50 amino acids, more preferably at most 40 amino acids, such as at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, or at most 45 amino acids.

[0058] In a preferred embodiment, the at least one peptide that promotes cellular uptake or transcytosis is at least one peptide that promotes passage through the BBB. As used herein, "blood-brain barrier" and "BBB" refer to the functional barrier between the blood circulation and the brain and spinal cord, which tightly controls the movement of substances from the blood to neural tissue and is formed primarily by (cerebrovascular) endothelial cells. As used herein, "promoting passage through the blood-brain barrier" means that passage through the BBB of a protein or polypeptide expressed from a nucleic acid molecule of the present invention is increased when at least one peptide that promotes passage through the BBB is present in the protein or polypeptide, compared to an otherwise identical protein or polypeptide that lacks at least one peptide that promotes passage through the BBB.

[0059] Peptides that facilitate crossing the BBB are well known in the art. Preferred, but non-limiting, peptides include Angiopep-2 (TFFYGGSRGKRNNFKTEEY-OH); ApoB(3371-3409)(SSVIDALQYKLEGTTRLTRK-RGLKLATALSLSNKFVEGS); ApoE(159-167)2 ((LRKLRKRLL)2); peptide-22 (Ac-C(&)MPRLRGC(&)-NH2; & is a cyclic linker between C(&) and C(&)). peptide); THR (THRPPMWSPVWP-NH2); THR retro-enantiomer (pwvpswmpprht-NH2); CRT (C(&)RTIGPSVC(&); & indicates a cyclic peptide where C(&) and C(&) are linked); Leptin 30 (YQQILTSMPSRNVIQISND-LENLRDLLHVL); RVG29 (YTIWMPENPRPGTPCDIFT-NSRGKRASNG-OH); DCDS (GreirtGraerwsekf-OH); apamin (C(&1)NC(&2)KAPETALC(&1)-AR-RC(&2)QQH-NH2; & indicates a cyclic peptide in which C(&1) and C(&1) are bonded and C(&2) and C(&2) are bonded); MiniAp-4 ([Dap](&)KAPETALD(&); Dap = diaminopropionic acid, & indicates a cyclic peptide in which & and & are bonded); GSH (γ-L-glutamyl-CG-OH); G23 (HLNILSTLWKYRC); g 7 (GFtGFLS(O-β-Glc)-NH2), TGN (TGNYKALHPHNG), TAT(47-57)(YGRKKRRQRRR-NH2), SynB1 (RGGRLSYSRRRFSTSTGR), and PhPro ((phenylproline)4-NH2).

[0060] In a preferred embodiment, peptides that promote cellular uptake or transcytosis, particularly crossing the BBB, bind to receptors present on endothelial cells and / or involved in transcytosis. Examples of such receptors are the insulin receptor, particularly M6PR (mannose-6-phosphate receptor), the leptin receptor, the transferrin receptor, and the low-density lipoprotein receptor (LRP). For example, the peptide contains the receptor-binding domain of apolipoprotein (Apo) A, B, or E, receptor-associated protein (RAP), transferrin (Tf), lactotransferrin, melanotransferrin (p97), or leptin. Thus, in a preferred embodiment, the peptide contains the receptor-binding domain of ApoA, ApoB, ApoE, RAP, transferrin, lactotransferrin, melanotransferrin (p97), or leptin. Similarly, the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, particularly at least one peptide that promotes crossing of the BBB, encodes the receptor binding domain of ApoA, ApoB, ApoE, RAP, transferrin, lactotransferrin, melanotransferrin (p97), or leptin, or any combination and / or repeat of these domains.

[0061] In a further preferred embodiment, the peptide that enhances crossing of the BBB comprises a low-density lipoprotein receptor-related protein 1 (LRP1) binding domain. In a preferred embodiment, the peptide that enhances crossing of the BBB comprising an LRP1 binding domain has a length of at most 100 amino acids. More preferably, the peptide has a length of at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids, more preferably at most 50 amino acids, more preferably at most 40 amino acids, for example at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, or at most 45 amino acids.

[0062] In a further preferred embodiment, the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis encodes the receptor-binding domain of human apolipoprotein E2 (ApoE2), human apolipoprotein B (ApoB), human receptor-associated protein (RAP), such as RAP12 (EAKIEKHNHYQK) or RAP12x2 (EAKIEKHNHYQKGEAKIEKHNHYQK), or human leptin, or encodes the CRP peptide (CRTIGPSVC) or Angiopep-2 (TFFYGGSRGKRNNFKTEEY), or any combination and / or repeat of these domains and sequences. Preferably, the peptide has a length of at most 100 amino acids. More preferably, the peptide has a length of at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids, more preferably at most 50 amino acids, more preferably at most 40 amino acids, such as at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, or at most 45 amino acids.

[0063] In further preferred embodiments, the peptide that promotes crossing of the BBB comprises or consists of the receptor binding domain of human apolipoprotein E2 (ApoE2), human apolipoprotein B (ApoB), human receptor-associated protein (RAP), or human leptin, or comprises the CRP peptide (CRTIGPSVC) or Angiopep-2 (TFFYGGSRGKRNNFKTEEY), or any combination and / or repeat of these domains and sequences. Preferably, the peptide has a length of at most 100 amino acids. More preferably, the peptide has a length of at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids. In further preferred embodiments, the peptide has a length of at most 50 amino acids, more preferably at most 40 amino acids.

[0064] In further preferred embodiments, the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis encodes amino acids 141-149 of human ApoE2 (LRKLRKRLL), amino acids 3371-3409 of human ApoB (SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS), amino acids 251-262 of human RAP (EAKIEKHNHYQK; RAP12), amino acids 61-90 of human leptin (YQQILTSMPSRNVIQISNDLENLRDLLHVL), or any combination and / or repeats of these sequences. Preferably, the peptide has a length of at most 100 amino acids. More preferably, the peptide has a length of at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids. In further preferred embodiments, the peptide has a length of at most 50 amino acids, more preferably at most 40 amino acids.

[0065] In a further preferred embodiment, the peptide that promotes crossing of the BBB comprises or consists of amino acids 141-149 of human ApoE2 (LRKLRKRLL), amino acids 3371-3409 of human ApoB (SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS), amino acids 251-262 of human RAP (EAKIEKHNHYQK), amino acids 61-90 of human leptin (YQQILTSMPSRNVIQISNDLENLRDLLHVL), or any combination and / or repeat of these sequences. Preferably, the peptide is at most 100 amino acids in length. More preferably, the peptide is at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids in length. In a further preferred embodiment, the peptide is at most 50 amino acids, more preferably at most 40 amino acids in length.

[0066] In a further preferred embodiment, the peptide that facilitates crossing of the BBB comprises or consists of amino acids 141-149 of human ApoE2 (LRKLRKRLL). Preferably, the peptide is at most 100 amino acids in length. More preferably, the peptide is at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids in length. In a further preferred embodiment, the peptide is at most 50 amino acids, more preferably at most 40 amino acids in length. In a further preferred embodiment, the peptide comprising amino acids 141-149 of human ApoE2 (LRKLRKRLL) is at most 30, more preferably at most 25, more preferably at most 20, more preferably at most 15, more preferably at most 12 amino acids in length.

[0067] In a further preferred embodiment, the peptide that enhances crossing of the BBB comprises or consists of a repeat of amino acids 141-149 of human ApoE2 ((LRKLRKRLL)2). Preferably, the peptide is at most 100 amino acids in length. More preferably, the peptide is at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids in length. In a further preferred embodiment, the peptide is at most 50 amino acids, more preferably at most 40 amino acids in length.

[0068] In a further preferred embodiment, the peptide that promotes crossing of the BBB comprises or consists of amino acids 3371 to 3409 of human ApoB (SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS).

[0069] In a further preferred embodiment, the peptide that promotes crossing of the BBB comprises or consists of amino acids 61-90 of human leptin (YQQILTSMPSRNVIQISNDLENLRDLLHVL). Preferably, the peptide is at most 100 amino acids in length. More preferably, the peptide is at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids in length. In a further preferred embodiment, the peptide is at most 50 amino acids, more preferably at most 40 amino acids in length. In a further preferred embodiment, the peptide comprising amino acids 61-90 of human leptin (YQQILTSMPSRNVIQISNDLENLRDLLHVL) is at most 35 amino acids in length.

[0070] In a further preferred embodiment, the peptide that promotes crossing of the BBB comprises or consists of a repeat of amino acids 251-262 of human RAP (EAKIEKHNHYQKGEAKIEKHNHYQK; RAP12x2). Preferably, the peptide is at most 100 amino acids in length. More preferably, the peptide is at most 90 amino acids, more preferably at most 80 amino acids, more preferably at most 70 amino acids, more preferably at most 60 amino acids in length. In a further preferred embodiment, the peptide is at most 50 amino acids, more preferably at most 40 amino acids in length. In a further preferred embodiment, the peptide comprising a repeat of amino acids 251-262 of human RAP (EAKIEKHNHYQKGEAKIEKHNHYQK; RAP12x2) is at most 35, more preferably at most 30 amino acids in length.

[0071] In some aspects, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, and a receptor-associated protein (RAP) sequence, preferably a RAP tag, more preferably amino acids 251 to 262 of human RAP (EAKIEKHNHYQK; RAP12), or repeats thereof, such as RAP12x2 (EAKIEKHNHYQKGEAKIEKHNHYQK).

[0072] The nucleic acid molecules of the present invention comprise a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to the metabolic protein or a portion thereof. In particular, the metabolic protein is a protein associated with a metabolic disorder. For example, the metabolic protein is a protein that is absent, mutated, reduced, or otherwise impaired, thereby causing or contributing to a metabolic disorder. The nucleic acid molecules of the present invention aim to restore the expression, level, and / or function of the metabolic protein.

[0073] The nucleic acid molecules of the present invention comprise nucleotide sequences encoding metabolic proteins that may be suitable for use in restoring the expression, level, and / or function of any metabolic protein. In a preferred embodiment, the metabolic protein is a metabolic enzyme. In another preferred embodiment, the metabolic protein is a lysosomal protein. As used herein, "lysosomal protein" refers to a protein that is present and functionally active in lysosomes. In a further preferred embodiment, the metabolic protein is a lysosomal enzyme. As used herein, "lysosomal enzyme" refers to an enzyme that is present and functionally active in lysosomes, particularly in the degradation of extracellular material or cellular waste products. There are over 50 known genetic disorders in which linkage has been established between the disorder and mutations in genes encoding lysosomal proteins, particularly lysosomal enzymes. Such mutations result in deficiencies or dysfunction of lysosomal proteins. These disorders are called lysosomal storage disorders (LSDs) and are characterized by the accumulation of a metabolite or metabolites that cannot be degraded or are insufficiently degraded due to deficiencies or dysfunction of lysosomal proteins.

[0074] In a preferred embodiment, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding a metabolic protein, preferably a lysosomal protein, more preferably a lysosomal enzyme, more preferably a lysosomal hydrolase, or a portion thereof, or a sequence having at least 95% sequence identity to said metabolic protein or portion thereof, more preferably a sequence having at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to a metabolic protein, preferably a lysosomal protein, more preferably a lysosomal enzyme, more preferably a lysosomal hydrolase, or a portion thereof.

[0075] As used herein, a "portion" of a metabolic protein, lysosomal protein, or lysosomal enzyme refers to an active portion thereof, particularly a portion having the same activity, though not necessarily at the same level, as the metabolic protein, lysosomal protein, or lysosomal enzyme from which it is derived. In particular, a portion of a lysosomal enzyme is an enzymatically active portion thereof. As another example, a portion of a lysosomal hydrolase is an enzymatically active portion thereof.

[0076] Examples of lysosomal proteins include lysosomal structural proteins, lysosomal membrane proteins, and soluble lysosomal proteins, the latter including lysosomal enzymes. In a preferred embodiment, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding a lysosomal enzyme or a portion thereof, or a sequence having at least 90% sequence identity with the lysosomal enzyme or a portion thereof. In a more preferred embodiment, the lysosomal enzyme is a lysosomal hydrolase. In a preferred embodiment, the lysosomal enzyme or lysosomal hydrolase is human GLA (α-galactosidase A; Fabry disease), ASAH1 (acid ceramidase; Farber lipogranulomatosis), GBA (glucocerebrosidase; Gaucher disease), GLB1 (β-galactosidase; GM1 gangliosidosis), HEXA (β-hexosaminidase A; GM2 gangliosidosis or Tay-Sachs disease), GM2A (GM2 activator; GM2 gangliosidosis), or GM2B (GM2 activator; GM2 gangliosidosis). or GM2 activator deficiency), GALC (galactocerebrosidase; Krabbe disease), ARSA (arylsulfatase A; metachromatic leukodystrophy), PSAP (saposin B; metachromatic leukodystrophy), SMPD1 (sphingomyelin phosphodiesterase 1; Niemann-Pick A / B), LIPA (lysosomal acid lipase; acid lipase deficiency: Wolman disease and cholesterol ester storage disease), IDUA (alpha-L-iduronidase; PMS I or Hurler syndrome), IDS (iduronate-2-sulfatase; PMS II or Hunter syndrome), SGSH (N-sulfoglucosamine sulfohydrolase; PMS IIIA), NAGLU (α-N-acetylglucosaminidase; PMS IIIB), HGSNAT (heparan-alpha-glucosaminide N-acetyltransferase; PMS IIIC), GNS (N-acetylglucosamine-6-sulfatase; PMS IIID), GALNS (N-acetylgalactosamine-6-sulfatase; PMS IVVA), GLB1 (beta-galactosidase-1; PMS IVB), ARSB (arylsulfatase B; PMS VI), GUSB (β-glucuronidase; PMS VII), HYAL1 (hyaluronoglucosaminidase-1;PMS IX), SUMF1 (sulfatase modifying factor-1; multiple sulfatase deficiency), GNPTAB (N-acetylglucosamine-1-phosphotransferase; mucolipidosis II α / β, I-cell disease, and pseudo-Hurler polydystrophy), GNPTG (N-acetylglucosamine-1-phosphotransferase gamma subunit; mucolipidosis III γ, variant pseudo-Hurler polydystrophy), MAN2B1 (alpha-mannosidase; α-mannosidosis ), MANBA (beta-mannosidase; beta-mannosidosis), FUCA1 (alpha-L-fucosidase; fucosidosis), AGA (aspartylglucosaminidase; aspartylglucosaminuria), NAGA (alpha-N-acetylgalactosaminidase; Schindler disease), NEU1 (neuraminidase-1; sialidosis types I and II), CTSA (protective protein / cathepsin A; galactosialidosis), HPS1 (Hermansky-Pudlak syndrome 1 protein protein; Hermansky-Pudlak disease type 1), AP3BA (beta 3A subunit of the heterotetrameric AP3 complex; HPS2 or Hermansky-Pudlak disease type 2), HPS3 (Hermansky-Pudlak syndrome 3 protein; Hermansky-Pudlak disease type 3), HPS4 (Hermansky-Pudlak syndrome 4 protein; Hermansky-Pudlak disease type 4), HPS5 (Hermansky-Pudlak syndrome 5 protein; Hermansky-Pudlak disease type 5), HPS6 (Hermansky-Pudlak syndrome 6 protein; Hermansky-Pudlak disease type 6), HPS7 (Hermansky-Pudlak syndrome 7 protein; Hermansky-Pudlak disease type 7), BLOC1S3 (synthesis of lysosomal organelle complex 1 subunit 3; HPS8 or Hermansky-Pudlak disease type 8), HPS9 (Hermansky-Pudlak syndrome 9 protein; Hermansky-Pudlak disease type 9), MYO5A (myosin Va; Griscelli syndrome 1), RAB27A (Rab GTPase family 27A; Griscelli syndrome 2), LYST (lysosomal trafficking regulator; Chediak-Higashi disease), GAA (acid alpha-glucosidase; Pompe disease), PPT1 (palmitoyl protein thioesterase-1;Neuronal ceroid lipofuscinosis (CLN) 1), TPP1 (tripeptidyl peptidase 1; CLN2), CLN3 (neuronal ceroid lipofuscinosis protein 3; CLN3), DNAJC5 (DnaJ homolog subfamily C member 5; CLN4), CLN5 (neuronal ceroid lipofuscinosis protein 5; CLN5), CLN6 (neuronal ceroid lipofuscinosis protein 6; CLN6), MFSD8 (neuronal ceroid The LSD associated with the lysosomal enzyme deficiency is selected from the group consisting of lipofuscinosis protein 7 (CLN7), CLN8 (neuronal ceroid lipofuscinosis protein 8 (CLN8), CTSD (cathepsin D (CLN10)), GRN (progranulin (CLN11)), ATP13A2 (ATPase cation transporter 13A2 (CLN12)), CTSF (cathepsin F (CLN13)), and KCTD7 (potassium channel tetramerization domain containing 7 (CLN14)). The LSD associated with the relevant lysosomal enzyme deficiency is indicated in parentheses.

[0077] In preferred embodiments, the lysosomal enzyme or lysosomal hydrolase is selected from the group consisting of human iduronate-2-sulfatase (IDS), acid alpha glucosidase (GAA), and tripeptidyl peptidase 1 (TPP1).

[0078] In some preferred embodiments, the nucleic acid molecule of the invention comprises a nucleotide sequence encoding amino acids 1-550 or 26-550 of human IDS, or an enzymatically active portion thereof, or a sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity to amino acids 1-550 or 26-550 of human IDS, or an enzymatically active portion thereof. In some preferred embodiments, the nucleic acid molecule of the invention comprises a nucleotide sequence encoding amino acids 1-550 or 26-550 of human IDS, or an enzymatically active portion thereof.

[0079] In some preferred embodiments, nucleic acid molecules of the invention comprise a nucleotide sequence encoding amino acids 70-952, 28-952, or 1-952 of human GAA, or an enzymatically active portion thereof, or a sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity to amino acids 70-952, 28-952, or 1-952 of GAA, or an enzymatically active portion thereof. In some preferred embodiments, nucleic acid molecules of the invention comprise a nucleotide sequence encoding human amino acids 70-952, 28-952, or 1-952 of GAA, or an enzymatically active portion thereof.

[0080] In some preferred embodiments, the nucleic acid molecules of the invention comprise a nucleotide sequence encoding human amino acids 1-563 or 20-563 of TPP1, or an enzymatically active portion thereof, or a sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity to amino acids 1-563 or 20-563 of TPP1, or an enzymatically active portion thereof. In some preferred embodiments, the nucleic acid molecules of the invention comprise a nucleotide sequence encoding human amino acids 1-563 or 20-563 of TPP1, or an enzymatically active portion thereof.

[0081] Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is a lysosomal storage disorder caused by mutations in the IDS gene. These mutations result in deficiency of the enzyme iduronate-2-sulfatase. IDS belongs to the sulfatase family of enzymes and catalyzes the hydrolysis of the C2-sulfate ester bond at the non-reducing end of 2-O-sulfo-α-1-iduronic acid residues in dermatan sulfate and heparan sulfate. Deficiency of IDS results in lysosomal accumulation of dermatan sulfate (DS) and heparan sulfate (HS). Hunter syndrome symptoms are not present at birth but typically begin around age 2–4 years. Clinical manifestations of Hunter syndrome range from mild to severe and are mostly present in the lungs, heart, joints, connective tissue, and brain and nervous system. Current treatment approaches for Hunter syndrome are tailored to specific patients and include enzyme replacement therapy (ERT) and symptom management.

[0082] The human IDS amino acid sequence encoded by the human IDS gene is shown in Figure 2A. This sequence contains a signal peptide (amino acids 1-25). The human IDS cDNA sequence, including the sequence encoding the signal peptide, is shown in Figure 2B.

[0083] In some preferred embodiments, the metabolic protein is iduronate-2-sulfatase (IDS). In preferred embodiments, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising a sequence having at least 90% sequence identity with amino acids 26 to 550 of human IDS. In even more preferred embodiments, the nucleotide sequence encodes amino acids 26 to 550 of human IDS. In even more preferred embodiments, a nucleotide sequence encoding an IDS signal peptide is present. That is, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising a sequence having at least 90% sequence identity with amino acids 1 to 550 of human IDS. Alternatively, another signal peptide is present, such as the IGF II signal peptide shown in FIG. 1.

[0084] In some preferred embodiments, the nucleic acid molecules of the invention comprise amino acids 26-550 of human IDS, or an amino acid sequence having at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 26-550 of human IDS, or a nucleotide sequence encoding an amino acid sequence comprising or consisting of amino acids 26-550 of human IDS.

[0085] In some preferred embodiments, the nucleic acid molecules of the invention comprise amino acids 1-550 of human IDS, or an amino acid sequence having at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 1-550 of human IDS, or a nucleotide sequence encoding an amino acid sequence comprising or consisting of amino acids 1-550 of human IDS.

[0086] In some preferred embodiments, the amino acid sequence diversity exists only in amino acids 26 to 550. That is, in some preferred embodiments, the nucleic acid molecule encodes an amino acid sequence having at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 26 to 550 of human IDS, and encodes amino acids 1 to 25 of human IDS.

[0087] In one preferred embodiment, the nucleotide sequence encoding amino acids 26-550 of IDS comprises or consists of nucleotides 245-1822 of the human IDS cDNA shown in Figure 2B. In another preferred embodiment, the nucleotide sequence encoding amino acids 1-550 of IDS comprises or consists of nucleotides 170-1822 of the human IDS cDNA shown in Figure 2B. However, the IDS nucleotide sequence can also be a codon-optimized version of the wild-type IDS nucleotide sequence. Thus, in another preferred embodiment, the nucleotide sequence encoding amino acids 26-550 or amino acids 1-550 of IDS comprises or consists of the codon-optimized version of nucleotides 245-1822 or nucleotides 170-1822 of the human IDS cDNA shown in Figure 2B, respectively. Codon optimization of protein-encoding nucleotide sequences is a well-known and commonly used technique in the art, and those skilled in the art are well-versed in the ability to prepare and select suitable codon-optimized sequences. A suitable codon-optimized IDS nucleotide sequence is used in Gleitz, HF et al. (2018, EMBO Mol Med 10. doi.10.15252 / emmm.201708730).

[0088] Pompe disease, also known as acid maltase deficiency or glycogen storage disease type II, is an autosomal recessive metabolic disorder caused by the accumulation of glycogen in lysosomes. In Pompe disease, the lysosomal hydrolase protein α-D-glucoside glucohydrolase, or acid α-glucosidase (GAA, EC 3.2.1.20, also known as acid maltase), is deficient. This protein normally cleaves the α-1,4 and α-1,6 linkages in glycogen, maltose, and isomaltose, an enzyme required for the breakdown of 1-3% of cellular glycogen. Deficiency of this enzyme results in the accumulation of structurally normal glycogen in the lysosomes and cytoplasm of affected individuals. Excessive glycogen accumulation in lysosomes can disrupt the normal function of other organelles and lead to cellular damage. A lack of acid alpha-glucosidase protein, or reduced amount or activity of acid alpha-glucosidase protein, is the result of mutations (or variants) in the acid alpha-glucosidase (GAA) gene. The GAA gene is located on the long arm of chromosome 17 at 17q25.2-q25.3 (base pairs 80,101,556 to 80,119,879 in GRCh38.p10). Severe mutations that completely suppress GAA enzyme activity cause a classic infantile disease course with hypertrophic cardiomyopathy, generalized skeletal muscle weakness, and respiratory failure, resulting in death within the first year of life. Milder mutations retain partial GAA enzyme activity, resulting in a milder phenotype with onset ranging from childhood to adulthood. Overall, high residual enzyme activity in primary fibroblasts is associated with later onset of Pompe disease. Some GAA mutations in Pompe disease patients can result in alternative splicing, resulting in the absence or reduced amount or activity of α-glucosidase protein. One of the most common mutations in Pompe disease is the IVS1 mutation, c.-32-13T>G, a transversion (T to G) mutation, which occurs in infants, children, adolescents, and adults with this disorder (Huie ML et al., 1994. Hum Mol Genet. 3(12):2231-6).In childhood and adult Pompe disease, 90% of patients in the Caucasian population are affected by the common c.32-13T>G(IVS1) variant, which results in aberrant splicing of exon 2, resulting in partial or complete skipping of exon 2. The absence of exon 2 in the mRNA results in the absence of the normal AUG translation start site for the protein, leading to mRNA degradation and inability to produce the GAA protein.

[0089] The human GAA amino acid sequence encoded by the human GAA gene is shown in Figure 3A. This sequence contains a signal peptide (amino acids 1-27) and a 110 kD preproprotein (amino acids 57-952), which is further proteolytically processed to generate multiple intermediate forms and the mature, active 76 kD and 70 kD forms of the GAA enzyme. The human GAA cDNA sequence, including the sequence encoding the signal peptide and the sequence encoding the preproprotein, is shown in Figure 3B.

[0090] In some embodiments, the metabolic protein is acid alpha-glucosidase (GAA). In a preferred embodiment, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising a sequence having at least 90% sequence identity to amino acids 70-952 of human GAA. In a further preferred embodiment, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising amino acids 28-952 of human GAA, or an amino acid sequence having at least 90% sequence identity to amino acids 28-952 of human GAA. In a further preferred embodiment, the nucleotide sequence encodes an amino acid sequence having at least 90% sequence identity to amino acids 28-69 of human GAA, and encodes amino acids 70-952 of human GAA. In a further preferred embodiment, a nucleotide sequence encoding a GAA signal peptide is present. That is, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising amino acids 1-985 or a sequence having at least 90% sequence identity to amino acids 1-27 and 70-952 of human GAA. Alternatively, another signal peptide is present, such as an IGFII signal peptide. See Figure 1.

[0091] In some preferred embodiments, the nucleic acid molecules of the invention comprise a nucleotide sequence encoding amino acids 28-952 of human GAA, or an amino acid sequence having at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 28-952 of human GAA, or an amino acid sequence having at least 90% sequence identity to amino acids 28-952 of human GAA.

[0092] In some preferred embodiments, the amino acid sequence diversity exists only in amino acids 28-69. That is, in preferred embodiments, the nucleic acid molecule encodes an amino acid sequence having at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 28-69 of human GAA, and encodes amino acids 70-952 of human GAA.

[0093] In one preferred embodiment, the nucleotide sequence encoding amino acids 70-952 of GAA comprises or consists of nucleotides 365-3013 of the human GAA cDNA shown in Figure 3B. In another preferred embodiment, the nucleotide sequence encoding amino acids 28-952 of GAA comprises or consists of nucleotides 239-3013 of the human GAA cDNA shown in Figure 3B. However, the GAA nucleotide sequence can also be a codon-optimized version of the wild-type GAA amino acid sequence. Thus, in another preferred embodiment, the nucleotide sequence encoding amino acids 70-952 or amino acids 28-952 of GAA comprises or consists of the codon-optimized version of nucleotides 365-3013 or nucleotides 239-3013 of the human GAA cDNA shown in Figure 3B, respectively. Codon optimization of protein-encoding nucleotide sequences is a well-known and commonly used technique in the art, and those skilled in the art are well-versed in the ability to prepare and select suitable codon-optimized sequences. A suitable codon-optimized GAA nucleotide sequence is described in Stok et al., 2020 (Molecular Therapy - Methods & Clinical Development 17: 1014-1025).

[0094] Neuronal ceroid lipofuscinosis (NCL) is a heterogeneous group of LSDs. CLN2 disease is caused by mutations in the tripeptidyl peptidase 1 (TPP1) / CLN2 gene, resulting in TPP1 enzyme deficiency. CLN2 most commonly presents with seizures and / or ataxia in late childhood (ages 2-4 years), and symptoms include language delay, progressive childhood dementia, motor and visual deterioration, and early death. Atypical phenotypes of CLN2 can occur, characterized by later disease onset. Some atypical phenotypes are further characterized by longer life expectancies. While many mutations in TPP1 have been identified, two variants, c.509-1 G>C and c.622 C>T (p.(Arg208*)), occur in 60% of affected individuals and account for 50% of disease-associated alleles. The only current treatment available for CLN2 is cerliponase alfa (Brineura®).

[0095] The human TPP1 amino acid sequence encoded by the human TPP1 / CLN2 gene is shown in Figure 4. This sequence contains a signal peptide (amino acids 1-19). The human TPP1 cDNA sequence, including the sequence encoding the signal peptide, is shown in Figure 4B.

[0096] In some preferred embodiments, the metabolic protein is TPP1. In preferred embodiments, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising a sequence having at least 90% sequence identity with amino acids 20 to 563 of human TPP1. In even more preferred embodiments, the nucleotide sequence encodes amino acids 20 to 563 of human TPP1. In even more preferred embodiments, a nucleotide sequence encoding a TPP1 signal peptide is present. That is, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising a sequence having at least 90% sequence identity with amino acids 1 to 563 of human TPP1. Alternatively, another signal peptide is present, such as the IGFII signal peptide shown in FIG. 1.

[0097] In some preferred embodiments, the nucleic acid molecules of the invention comprise amino acids 20 to 563 of human TPP1, or an amino acid sequence having at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 20 to 563 of human TPP1, or a nucleotide sequence encoding an amino acid sequence comprising or consisting of amino acids 20 to 563 of human TPP1.

[0098] In some preferred embodiments, the nucleic acid molecules of the invention comprise amino acids 1 to 563 of human TPP1, or an amino acid sequence having at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 1 to 563 of human TPP1, or a nucleotide sequence encoding an amino acid sequence comprising or consisting of amino acids 1 to 563 of human TPP1.

[0099] In some preferred embodiments, the amino acid sequence diversity exists only in amino acids 20 to 563. That is, in some preferred embodiments, the nucleic acid molecule encodes an amino acid sequence having at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to amino acids 20 to 563 of human TPP1, and encodes amino acids 1 to 19 of human TPP1.

[0100] In one preferred embodiment, the nucleotide sequence encoding amino acids 20-563 of TPP1 comprises or consists of nucleotides 80-1711 of the human TPP1 cDNA shown in Figure 4B. In another preferred embodiment, the nucleotide sequence encoding amino acids 1-563 of TPP1 comprises or consists of nucleotides 23-1711 of the human TPP1 cDNA shown in Figure 4B. However, the TPP1 nucleotide sequence may also be a codon-optimized version of the wild-type TPP1 amino acid sequence. Thus, in another preferred embodiment, the nucleotide sequence encoding amino acids 20-563 or amino acids 1-563 of TPP1 comprises or consists of the codon-optimized sequence of nucleotides 80-1711 or nucleotides 23-1711 of the human TPP1 cDNA shown in Figure 4B, respectively. Codon optimization of protein-encoding nucleotide sequences is a well-known and commonly used technique in the art, and those skilled in the art are well-versed in the ability to prepare and select suitable codon-optimized sequences.

[0101] The nucleotide sequence encoding a metabolic protein or a portion thereof or a sequence having at least 90% sequence identity to said metabolic protein or portion thereof, the human IGF II gene sequence and the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB, are linked, which means that expression of the nucleotide sequence encoding a metabolic protein or a portion thereof or a sequence having at least 90% sequence identity to said metabolic protein or portion thereof, the human IGF II gene sequence and the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB, results in a proteinaceous compound, in particular a fusion protein, comprising the amino acid sequence encoded by the nucleotide sequence encoding a metabolic protein or a portion thereof or a sequence having at least 90% sequence identity to said metabolic protein or portion thereof, the human IGF II gene sequence and the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB.

[0102] In a preferred embodiment, the nucleotide sequence encoding the human IGF II gene sequence and at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB, is located 3' to a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof. For example, this is preferably the case when the metabolic protein is an IDS sequence, or a portion or variant thereof, or a TPP1 sequence, or a portion or variant thereof.

[0103] In another preferred embodiment, the nucleotide sequence encoding the human IGF II gene sequence and at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB, is located 5' to a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof. For example, this is preferably the case when the metabolic protein is GAA, or a portion or variant thereof.

[0104] A nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB, is inserted into the IGFII gene sequence at a position between the nucleotides encoding amino acids 28 and 42 of mature human IGFII. This nucleotide sequence is hypothesized to form a loop within the IGFII sequence such that it is exposed to the outside of the IGFII sequence. Insertion at this specific position is hypothesized to provide excellent accessibility for the at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB. This accessibility allows binding of the at least one peptide to its receptor, particularly to receptors expressed on ECs that are part of the BBB. Furthermore, the at least one peptide is positioned in a specific, fixed conformation relative to the IGFII peptide, compared to positioning the at least one peptide at the N- or C-terminus of the IGFII peptide. This is hypothesized to be useful for receptor binding and / or dimerization and to avoid steric hindrance between different epitope tags. Finally, by inserting at least one peptide at this position, insulin binding affinity is reduced without compromising the brain targeting of the IGF II peptide and without optimizing C- or N-terminal tagging with double or multiple tags.

[0105] It is preferred to combine the insertion of a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis with an IGF II sequence having a mutation, preferably a deletion of one or more amino acids, within the nucleotide region encoding amino acids 29 to 41 of mature human IGF II. For example, a nucleotide sequence can be inserted at the site of the deleted amino acids. In a preferred embodiment, the nucleotide sequence comprises at least amino acids 29-41, 30-41, 31-41, 32-41, 33-41, 34-41, 35-41, 36-41, 37-41, 29-40, 30-40, 31-40, 32-40, 33-40, 34-40, 35-40, 36-40, 37-40, 29-39, 30-39, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 29-38, 30-38, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 29-38, 30-38, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 29-38, 30-38, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 29-40, 30-40, 31-40, 32-40, 33-40, 34-40 ... and inserted at the deletion site of nucleotides encoding 8, 32 to 38, 33 to 38, 34 to 38, 35 to 38, 36 to 38, 29 to 37, 30 to 37, 31 to 37, 32 to 37, 33 to 37, 34 to 37, 35 to 37, 29 to 36, 30 to 36, 31 to 36, 32 to 36, 33 to 36, 34 to 36, 29 to 35, 30 to 35, 31 to 35, 32 to 35, 33 to 35, 29 to 34, 30 to 34, 31 to 34, 30 to 34, 29 to 33, 30 to 33, 31 to 33, 29 to 32, or 30 to 32. In a particularly preferred embodiment, the nucleotides encoding amino acids 29-41 of mature human IGF II are deleted and a nucleotide sequence is inserted between the nucleotides encoding amino acids 28 and 42 of mature human IGF II. More preferably, such an IGF II sequence comprises or consists of the nucleotides encoding amino acids 1, 8-28, and 42-67 of mature human IGF II as shown in Figure 1. Optionally, nucleotides encoding one or more amino acids may be present on either or both sides of the insertion between the IGF II sequence and the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis. That is, nucleotides encoding one or more amino acids are located at both ends of the inserted nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis.In a preferred embodiment, nucleotides encoding at least one cysteine ​​are present on both sides of the insertion. This would place the amino acids of the IGF II sequence closest to the inserted nucleotide sequence, e.g., in the case of a deletion of amino acids 29-41, amino acids 28 and 42 of mature human IGF II, in close proximity, allowing disulfide bond formation between the cysteine ​​residues at both ends of the insertion in the encoded fusion protein. In addition to the nucleotides encoding the cysteine ​​residues, additional linking sequences may be present on one or both sides of the insertion. In the Examples herein, constructs 5-14 are exemplary constructs in which a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis is inserted between the nucleotides encoding amino acids 28 and 42 of the IGF II gene sequence. Constructs 6, 8, and 9 contain two linking sequences located at both ends of the inserted nucleotide sequence. Constructs 5, 7, and 10-14 contain two linking sequences and cysteine-encoding nucleotides located at both ends of the inserted nucleotide sequence.

[0106] Thus, in a preferred embodiment, in the nucleic acid molecule of the present invention, the IGF II gene sequence comprises or consists of nucleotides encoding amino acids 1, 8-28, and 42-67 of mature human IGF II, a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB, is inserted between the nucleotides encoding amino acids 28 and 42 of the IGF II gene sequence, and a cysteine-encoding nucleotide sequence and / or linking sequence is present between the nucleotide encoding amino acid 28 of the mature human IGF II sequence and the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB, and between the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB, and the nucleotide encoding amino acid 42 of mature human IGF II. When both the cysteine-encoding nucleotide and the linking sequence are present, the linking sequence is preferably flanked on both sides by the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB.

[0107] As indicated hereinabove, one or more linking sequences may be present in the nucleic acid molecules of the present invention. In a preferred embodiment, the nucleic acid molecule comprises linking sequences flanking a nucleotide sequence encoding at least one peptide which, when inserted between the nucleotides encoding amino acids 28 and 42 of human mature IGF II, promotes cellular uptake or transcytosis.

[0108] In a preferred embodiment, the linking sequence is a nucleotide sequence encoding an amino acid sequence of 2 to 30 amino acids, preferably 2 to 25 amino acids, more preferably 2 to 22 amino acids, for example, 4, 6, 10, 14, 15, 20, or 21 amino acids. In a preferred embodiment, the linking sequence encodes an amino acid sequence having a high percentage of G and S residues, for example, at least 50%, or at least 60%, or at least 70%. Suitable, but non-limiting, linking sequences are nucleotide sequences encoding the amino acid sequences LGGGGSGGGGSGGGGSGGGGS, SGGGG, SGGGGSG, GAPLGGGGSGGGGS, SGGSGGGGSGGGGSG, SGGS, GGSGGSGGSG.

[0109] The linking sequences flanking the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis and the linking sequence inserted between amino acids 28 and 42 of the IGF II gene sequence may be the same or different.

[0110] As detailed herein above, when linking sequences are present at both ends of a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, nucleotides encoding cysteine ​​residues are preferably also present adjacent to amino acids 28 and 42 of the IGF II sequence and attached to both linking sequences.

[0111] The nucleic acid molecule of the present invention preferably contains a signal peptide. The signal peptide can be any signal peptide suitable for secretion of the fusion protein encoded by the nucleic acid molecule of the present invention or the fusion protein of the present invention. Suitable signal peptides are known in the art. In a preferred embodiment, the signal peptide is a signal peptide of a metabolic protein, preferably a lysosomal enzyme, preferably a lysosomal hydrolase. In a more preferred embodiment, the signal peptide is a signal peptide of a metabolic protein, preferably a lysosomal enzyme, preferably a lysosomal hydrolase present in the nucleic acid molecule of the present invention or the fusion protein of the present invention. For example, if the metabolic protein is an IDS, the signal peptide is preferably an IDS signal peptide. The signal peptide is preferably located 5' to a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to the metabolic protein or a portion thereof, a human insulin-like growth factor II (IGFII) gene sequence, and a nucleotide sequence encoding at least one peptide promoting cellular uptake or transcytosis. The signal peptide is preferably located at the N-terminus of the fusion protein encoded by the nucleic acid molecule of the present invention.

[0112] Nucleic acid molecules of the invention can further comprise one or more regulatory sequences that direct the expression of one or more of the nucleotide sequences present in the nucleic acid molecule, including promoters, enhancers, transcription termination signals, and polyadenylation sequences.

[0113] In a preferred embodiment, the nucleic acid molecule of the present invention comprises a promoter operably linked to a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to the metabolic protein or a portion thereof, a human IGF II gene sequence, and a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB. As used herein, "operably linked" means that a nucleotide sequence is functionally linked to one or more other nucleotide sequences. For example, a promoter nucleotide sequence is operably linked to a nucleotide sequence encoding a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to the metabolic protein or a portion thereof, a human IGF II gene sequence, and a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, such that the promoter sequence affects or directs the expression of the other nucleotide sequences. Various promoters, including inducible promoters, may be used to direct the expression of nucleotide sequences contained in the nucleic acid molecule of the present invention, including viral promoters. Both cell-type-specific and ubiquitous promoters can be used. Suitable promoters include the SV40 promoter, the Rous sarcoma virus (RSV) promoter, the cytomegalovirus (CMV) promoter, the CD11b promoter, and the MND promoter, or any derivatives of these promoters.

[0114] In the case of LSD, accumulation and symptoms in several organs (including lung, heart, skeletal and smooth muscle, joints, connective tissue, peripheral nervous system, and brain) and the peripheral nervous system (see below) need to be corrected. Therefore, in a preferred embodiment, the promoter is a ubiquitous promoter rather than a cell-type-specific promoter. In a preferred embodiment, the promoter is the MND promoter or a derivative thereof, such as the MND promoter with a 174-bp deletion at the 5' end. The MND promoter is described in Robbins et al. (Proc. Natl. Acad. Sci. USA 1994, Vol. 95, pp. 10182-10187) and Astrakhan et al. (Blood. 2012;119(19):4395-4407), both of which are incorporated herein by reference. In a preferred embodiment, the metabolic protein is IDS, and the nucleic acid molecule of the present invention comprises a ubiquitous promoter.

[0115] Another strategy is to use a myeloid promoter, i.e., a promoter specific for cells from the myeloid lineage, such as the lysM, csflr, CDllc, CD68, macrophage SRA, and CDllb promoters, since the secreted enzyme is preferably expressed from HSCs in the myeloid lineage. Thus, in another preferred embodiment, the promoter is a myeloid promoter, such as the lysM, csflr, CDllc, CD68, macrophage SRA, and CDllb promoters, preferably the CDllb promoter.

[0116] Expression of the nucleic acid molecule present in the nucleic acid molecule of the present invention results in a fusion protein comprising a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, fused to a human insulin-like growth factor II (IGF II) sequence and at least one peptide that promotes cellular uptake or transcytosis.

[0117] Thus, also provided is a fusion protein encoded by a nucleic acid molecule according to the invention.

[0118] Further provided is a fusion protein comprising a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, fused to a human insulin-like growth factor II (IGF II) sequence and at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB.

[0119] In a preferred embodiment, the human IGF II sequence and at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes passage through the BBB, are located on the same side of the fusion protein of the invention as a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof.

[0120] In a preferred embodiment of the fusion protein of the present invention, the metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity with the metabolic protein or a portion thereof, the human IGF II sequence, and / or the at least one peptide promoting cellular uptake or transcytosis, preferably at least one peptide promoting crossing of the BBB, are separated by one or more linking sequences and / or cysteine ​​residues. Such linking sequences, cysteine ​​residues, and the locations of the nucleotide sequences encoding them in the nucleic acid molecule of the present invention have been discussed herein above. The same disclosures and embodiments are applicable to the fusion protein of the present invention.

[0121] In a further preferred embodiment, the human IGF II amino acid sequence in the fusion protein of the present invention is the amino acid sequence encoded by the IGF II gene sequence defined herein above.

[0122] In a preferred embodiment, the IGF II amino acid sequence comprises amino acids 1 and 8-67 of human IGF II shown in FIG.

[0123] In a further embodiment, the IGFII amino acid sequence comprises the IGFII signal peptide and amino acids 1-67 of IGFII shown in FIG.

[0124] In a further preferred embodiment, the IGF II amino acid sequence comprises a mutation within amino acids 29-41 of the IGF II sequence, preferably a deletion within amino acids 29-41 or 30-40 of IGF II.

[0125] In a further preferred embodiment, the IGF II amino acid sequence comprises the IGF II signal peptide and amino acids 29-41, 30-41, 31-41, 32-41, 33-41, 34-41, 35-41, 36-41, 37-41, 29-40, 30-40, 31-40, 32-40, 33-40, 34-40, 35-40, 36-40, 37-40, 29-39, 30-39, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 29-38, 39-40, 39-41 ... The present invention also includes human mature IGF II containing deletions of 30 to 38, 31 to 38, 32 to 38, 33 to 38, 34 to 38, 35 to 38, 36 to 38, 29 to 37, 30 to 37, 31 to 37, 32 to 37, 33 to 37, 34 to 37, 35 to 37, 29 to 36, 30 to 36, 31 to 36, 32 to 36, 33 to 36, 34 to 36, 29 to 35, 30 to 35, 31 to 35, 32 to 35, 33 to 35, 29 to 34, 30 to 34, 31 to 34, 30 to 34, 29 to 33, 30 to 33, 31 to 33, 29 to 32, or 30 to 32.

[0126] In particularly preferred embodiments, the IGF II amino acid sequence comprises a deletion of amino acids 29 to 41 or 30 to 40 of IGF II. Thus, in preferred embodiments, the IGF II amino acid sequence comprises amino acids 1, 8 to 28 and 42 to 67, or amino acids 1, 8 to 29, and 41 to 67 of mature IGF II as shown in Figure 1. Further suitable IGF II variants with reduced insulin receptor binding are described in WO2009 / 137721, which is incorporated herein by reference.

[0127] In a preferred embodiment, at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB, is inserted between amino acids 28 and 42 of the human IGF II sequence in the fusion protein of the invention. In particular, at least one peptide is inserted at the position of the mutation within amino acids 29 to 41 or 30 to 40 of the IGF II sequence. In particular, at least one peptide is located at the position of the deleted amino acid in the IGF II sequence. That is, the IGF II amino acid sequence comprises a deletion of amino acids 29 to 41 or 30 to 40 of IGF II, and at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing of the BBB, is located at the site of the mutated amino acid. In a preferred embodiment, the IGF II amino acid sequence comprises amino acids 1, 8-28, and 42-67, or amino acids 1, 8-29, and 41-67, of mature IGF II as shown in Figure 1, with at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB, located between amino acids 28 and 42, or 29 and 41, respectively, of the IGF II sequence. In a preferred embodiment, a cysteine ​​is present on each side of the insert, i.e., on each side of the at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB. In addition to the cysteine ​​residue, an additional linking sequence may be present on one or both sides of the insert. When both a cysteine ​​and a linking sequence are present, the linking sequence is preferably flanked on both sides by at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB. That is, the cysteine ​​is preferably located adjacent to a portion of the IGF II sequence; for example, if amino acids 29 to 41 are deleted, the cysteine ​​is adjacent to amino acids 28 and 41, and if amino acids 30 to 40 are deleted, the cysteine ​​is adjacent to amino acids 29 and 41.

[0128] The fusion protein preferably contains a linking sequence, as defined herein, between the IGFII amino acid sequence and the GAA amino acid sequence. The linking sequence preferably consists of 2 to 10 amino acids, more preferably 2 to 5 amino acids, for example, 2, 3, or 4 amino acids. In a preferred embodiment, the linking sequence is an amino acid sequence of 3 amino acids. A preferred, but non-limiting, linking sequence is the amino acid sequence GAP.

[0129] Similarly, nucleic acid sequences encoding the fusion peptides according to the invention are provided.

[0130] After transplantation of HSCs transfected or transduced with a nucleic acid molecule of the invention, the cells express a fusion protein in vivo that contains an IGFII-tagged GAA preproprotein, as opposed to the known IGFII-tagged GAA fusion protein that is expressed after transfection, transduction, and transplantation of HSCs.

[0131] In a preferred embodiment, the nucleic acid molecule of the present invention is a vector or part of a vector. In a preferred embodiment, the nucleic acid molecule of the present invention is a gene therapy vector. Thus, a vector, preferably a gene therapy vector, comprising the nucleic acid molecule of the present invention is also provided by the present invention.

[0132] Gene therapy vectors are preferably vectors that are suitable for ex vivo transfection or transduction of hematopoietic stem cells.

[0133] Vectors can be viral or non-viral vectors. Non-limiting examples of suitable expression vectors include retroviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors, non-viral vectors, and engineered vectors.

[0134] The vector, particularly the gene therapy vector, is preferably a viral vector. The viral vector is preferably a recombinant adeno-associated viral vector, a herpes simplex virus-based vector, or a lentivirus-based vector, such as a human immunodeficiency virus-based vector. The viral vector is more preferably a retrovirus-based vector, such as a lentivirus-based vector, such as a human immunodeficiency virus-based vector, or a gamma-retrovirus-based vector. The retrovirus can be packaged in a suitable complementation cell that provides the group antigen polyprotein (Gag)-polymerase (Pol) and / or envelope (Env) proteins. Suitable packaging cells are human embryonic kidney-derived 293T or 293 cells, Phoenix cells (Swift et al., 2001. Curr Protoc Immunol, Chapter 10: Unit 10 17C), PG13 cells (Loew et al., 2010. Gene Therapy 17: 272-280), and Flp293A cells (Schucht et al., 2006. Mol Ther 14: 285-92). Methods for generating such non-viral expression vectors are well known in the art.

[0135] Non-viral expression vectors include naked DNA and nucleic acids packaged in synthetic or engineered compositions, such as liposomes, polymers, nanoparticles, and molecular conjugates. Methods for producing such non-viral expression vectors are well known in the art.

[0136] Alternatively, the nucleic acid molecules used in accordance with the present invention can be provided to a subject by gene editing techniques, including CRISPR / Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), to insert a receptor transgene into a specific genomic locus with or without an exogenous promoter. Preferred genomic loci include the AAVS1 locus and the PD-1 locus, as known to those skilled in the art.

[0137] In a particularly preferred embodiment, the nucleic acid molecule or vector of the present invention is a gene delivery vehicle of lentiviral origin. In a preferred embodiment, the vector, particularly a gene therapy vector, is a lentiviral vector. Typically, the terms "gene delivery vehicle of lentiviral origin" and "lentiviral vector" refer to any gene delivery vehicle of lentiviral origin. Gene delivery vehicles of lentiviral origin are all vehicles containing genetic and / or proteinaceous material derived from lentiviruses. Typically, the most important feature of such vehicles is their ability to integrate the genetic material into the genome of target cells and transduce stem cells. These elements appear to be functionally essential, meaning that the sequence does not need to be identical to the lentiviral sequence as long as the essential functions are present. However, the method of the present invention is particularly suitable for recombinant lentiviral particles that possess most, if not all, of the replication and reproduction characteristics of lentiviruses. Typically, the lentiviral particles that constitute the gene delivery vehicle are themselves replication-deficient. In a preferred embodiment, the gene delivery vehicle of lentiviral origin used is a gene delivery vehicle of HIV lentiviral origin, and even more preferably an HIV-1-derived self-inactivating lentiviral vector.

[0138] The present invention also provides a viral particle, preferably a lentiviral particle, comprising a nucleic acid molecule according to the invention.

[0139] Methods and means (e.g., producer cells) for producing desired lentiviral particles are well known in the art. An example of a preferred producer cell is 293T cells co-transfected with VSV-G (vesicular stomatitis virus-G protein envelope). Another pseudotype used in this context is RD114 (feline immunodeficiency virus).

[0140] The present invention also provides uses of the nucleic acid molecules, vectors, fusion proteins, compositions and cell populations, particularly HSC populations, particularly in the treatment of metabolic disorders.

[0141] In one aspect, the present invention therefore also provides a method for the treatment of a metabolic disorder, comprising administering a nucleic acid molecule, a fusion protein, or a cell population, in particular HSCs, according to the invention to an individual in need thereof.

[0142] Also provided is a nucleic acid molecule, a fusion protein or a cell population, in particular HSCs, according to the invention for use in a method for the treatment of a metabolic disorder.

[0143] Also provided is the use of a nucleic acid molecule, a fusion protein or a cell population, particularly HSCs, according to the invention in the preparation of a medicament for the treatment of a metabolic disorder.

[0144] As used herein, "metabolic disorder," also referred to as metabolic disease, refers to a disorder or disease in which normal cellular metabolism is disrupted. A metabolic disorder is a disorder or disease in which the expression and / or functional activity of, inter alia, metabolic enzymes is impaired or absent. Metabolic enzymes are enzymes involved in cellular metabolism. In a preferred embodiment, the metabolic disorder is a lysosomal disorder, more preferably a lysosomal storage disorder. As used herein, "lysosomal disorder" or "lysosomal disease" refers to a metabolic disorder resulting from defective lysosomal function. "Lysosomal storage disorder" (LSD) refers to a metabolic disorder characterized by lysosomal dysfunction and the accumulation of undegraded substrates in lysosomes. Non-limiting examples of LSDs include Fabry disease, Farber lipogranulomatosis, Gaucher disease, GM1 gangliosidosis, Tay-Sachs disease, GM2 gangliosidosis, Krabbe disease, metachromatic leukodystrophy, Niemann-Pick A / B, Wolman disease, cholesterol ester storage disease, PMS I or Hurler syndrome, PMS II or Hunter syndrome, PMS IIIA, PMS IIIB, PMS IIIC, PMS IIID, PMS IVA, PMS IVB, PMS VI, PMS VII, PMS IX, multiple sulfatase deficiency, mucolipidosis II alpha / beta, I-cell disease, pseudo-Hurler polydystrophy, mucolipidosis III gamma, alpha-mannosidosis, beta-mannosidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, sialidosis type I or type II, galactosialidosis, Hermansky-Pudlak disease types 1, 2, 3, 4, 5, 6, 7, 8, or 9, Griscelli syndrome 1 or 2, Chediak-Higashi disease, Pompe disease, and neuronal ceroid lipofuscinosis (CLN) 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, or 14. In one preferred embodiment, the LSD treated in accordance with the present invention is Hunter syndrome or PMSII, and the metabolic protein is IDS or a portion thereof, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to IDS or a portion thereof.In another preferred embodiment, the LSD treated according to the present invention is Pompe disease, and the metabolic protein is GAA or a portion thereof, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to GAA or a portion thereof. In another preferred embodiment, the LSD treated according to the present invention is CLN2, and the metabolic protein is TPPI or a portion thereof, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to TPP1 or a portion thereof.

[0145] In a preferred embodiment of the invention, the nucleic acid molecule or vector of the invention is for use in transfecting or transducing cells, in particular hematopoietic stem cells (HSCs), preferably for ex vivo transfection or transduction of cells, in particular HSCs.

[0146] In one aspect, the present invention therefore provides a method of transfecting or transducing HSCs with a nucleic acid molecule or vector, preferably a gene delivery vehicle of lentiviral origin according to the invention, comprising contacting HSCs with said nucleic acid molecule or vector, preferably a gene delivery vehicle. In a preferred embodiment, the HSCs are human, more preferably isolated from an individual suffering from an LSD, in particular Hunter syndrome, Pompe disease or CLN2. As one skilled in the art will appreciate, the particular LSD will depend on the particular metabolic protein, or portion thereof, encoded by the nucleotide sequence present in the nucleic acid molecule or vector.

[0147] As used herein, the term "transfection" refers to the transfer of genetic material from a non-viral particle into a hematopoietic stem cell. As used herein, the term "transduction" refers to the transfer of genetic material from a viral particle into a hematopoietic stem cell.

[0148] HSCs are stem cells and early progenitor cells that give rise to all blood cell types, including both myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, and some dendritic cells) and lymphoid (T cells, B cells, NK cells, and some dendritic cells) lineages. Hematopoietic tissues possess cells with long-term and short-term regenerative potential, as well as committed multipotent, micropotent, and unipotent progenitors. HSCs can be obtained from different sources, for example, found in human bone marrow, including the femur, hip, rib, sternum, and other bones. Cells can be obtained directly from the hip joint using a needle and syringe or by withdrawing them from the blood after pretreatment with cytokines, such as G-CSF (granulocyte colony-stimulating factor), which induces the release of cells from the bone marrow compartment into the blood (mobilized peripheral blood).

[0149] In a preferred embodiment, autologous HSCs, i.e., HSCs obtained from a patient, are transfected or transduced with a nucleic acid molecule or vector of the present invention. In such an embodiment, HSCs are preferably isolated from an individual suffering from the LSD to be treated, particularly Hunter syndrome, Pompe disease, or CLN2, and transfected or transduced ex vivo with a nucleic acid molecule or vector of the present invention. After transfection or transduction, the transfected or transduced HSCs are preferably returned to the individual. Those skilled in the art will understand that the specific LSD will depend on the specific metabolic protein or portion thereof encoded by the nucleotide sequence present in the nucleic acid molecule or vector.

[0150] The present invention also provides compositions obtainable by the methods of the present invention, thus providing compositions comprising HSCs transfected or transduced with a nucleic acid molecule or vector, preferably a gene delivery vehicle of lentiviral origin, of the present invention.

[0151] The present invention further provides a composition comprising a viral particle, preferably a lentiviral particle, provided with a nucleic acid molecule or vector of the invention. Preferably, said lentiviral particle is a gene delivery vehicle and is capable of transducing HSCs and / or progenitor cells.

[0152] The present invention further provides a cell population, preferably a hematopoietic stem cell (HSC) population, provided with a nucleic acid molecule, vector, or viral particle according to the invention. In a preferred embodiment, the cell population or HSC is transfected or transduced with a nucleic acid molecule, vector, or viral particle according to the invention. The cell population or HSC is further preferably capable of expressing a fusion protein according to the invention, in particular a fusion protein comprising a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, fused to a human insulin-like growth factor II (IGF II) sequence and at least one peptide that promotes cellular uptake or transcytosis, preferably at least one peptide that promotes crossing the BBB. In a preferred embodiment, the cell population, preferably an HSC population, expresses a fusion protein comprising a metabolic protein or a portion thereof, or a sequence having at least 90% sequence identity to said metabolic protein or a portion thereof, fused to a human insulin-like growth factor II (IGF II) sequence and at least one peptide that promotes cellular uptake or transcytosis of the invention.

[0153] The compositions, cell populations, or HSCs according to or used in accordance with the present invention may be administered to an individual by a variety of routes, preferably parenteral administration. Parenteral administration may include, for example, intra-articular, intramuscular, intravenous, intraventricular, intra-arterial, or intrathecal administration. In a preferred embodiment, the compositions or HSCs of the present invention may be administered to an individual via infusion or injection, particularly in a hospital setting by a medical professional.

[0154] The compositions of the present invention preferably contain at least one pharmaceutically acceptable carrier, diluent, and / or excipient. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and preferably not harmful, e.g., toxic, to the recipient. Generally, any pharmaceutically suitable additive that does not interfere with the function of the active compound can be used. The compositions according to the present invention containing at least one pharmaceutically acceptable carrier, diluent, and / or excipient are preferably suitable for human use.

[0155] Compositions, cell populations, or HSCs for intravenous administration may be, for example, a solution of the HSCs of the invention in sterile aqueous or nonaqueous solution, for example, in sterile isotonic aqueous buffer. If necessary, intravenous compositions may also contain, for example, one or more buffers, solubilizing agents, stabilizers, and / or a local anesthetic to ease pain at the site of the injection.

[0156] Typically, the use of compositions comprising lentiviral particles involves the transduction of HSCs, such as bone marrow cells, umbilical cord blood cells, or mobilized peripheral blood stem cells. Such transduced cells are preferably prepared ex vivo and are also part of the present invention. Thus, the present invention provides compositions for the treatment of metabolic disorders, preferably LSDs, more preferably Hunter syndrome, Pompe disease, or CNL2, comprising a plurality of HSCs transduced with a lentiviral vector or particle composition according to the present invention.

[0157] In yet another embodiment, the present invention provides the use of the above composition in the preparation of a medicament for the treatment of a metabolic disorder, preferably an LSD, more preferably Hunter Syndrome, Pompe Disease, or CNL2.

[0158] In one aspect, the present invention provides a method for treating a metabolic disorder, preferably an LSD, more preferably Hunter Syndrome, Pompe Disease, or CNL2, comprising administering a cell population according to the present invention, particularly a cell population comprising HSCs, to an individual in need thereof.

[0159] Similarly, there is provided a method for treating a metabolic disorder, preferably an LSD, more preferably Hunter syndrome, Pompe disease, or CNL2, comprising administering to an individual in need thereof a cell population, particularly HSCs, transfected or transduced ex vivo with a nucleic acid molecule, vector, or viral particle, preferably a lentiviral vector or lentiviral particle, according to the invention. Similarly, there is provided a cell population, preferably HSCs, transfected or transduced with a nucleic acid molecule or vector according to the invention for use in a method for treating a metabolic disorder, preferably an LSD, more preferably Hunter syndrome, Pompe disease, or CNL2, in an individual. In a preferred embodiment, the cells, particularly HSCs, are isolated from the individual and transfected or transduced ex vivo with a nucleic acid molecule or vector of the invention. That is, in a preferred embodiment, the individual is treated with autologous transfected or transduced HSCs. Thus, in a preferred embodiment, the treatment method of the present invention comprises harvesting HSCs from an individual, providing the HSCs with a nucleic acid molecule, vector, or viral particle according to the present invention, preferably a lentiviral vector or lentiviral particle, and administering the HSCs provided with the nucleic acid molecule, vector, or viral particle, preferably a lentiviral vector or lentiviral particle, to the individual. In a preferred embodiment, the method or use comprises transfecting or transducing the HSCs with a nucleic acid molecule, vector, or viral particle according to the present invention, preferably a lentiviral vector or lentiviral particle, and administering the HSCs transfected or transduced with the nucleic acid molecule, vector, or viral particle, preferably a lentiviral vector or lentiviral particle, to the individual. However, allogeneic HSCs can also be used in the methods and uses of the present invention.

[0160] In a preferred embodiment, the method or use of the present invention comprises administering myeloablative treatment to an individual prior to administering a treatment according to the present invention, particularly transfected or transduced HSCs. Myeloablative treatment is also referred to in the art as myeloablative treatment or myeloablative conditioning. The term refers to a treatment, such as chemotherapy or radiation, that removes hematopoietic cells in the individual being treated. Preferably, most (e.g., more than 80%) of the hematopoietic cells are removed. In addition, a reduced conditioning regimen may be applied. The myeloablative treatment is preferably carried out by chemotherapy. Myeloablative treatment of human individuals is well known in the art, and suitable chemotherapeutic agents are well known and can be selected by those skilled in the art. Such agents are used, for example, for allogeneic stem cell transplantation. Suitable, but non-limiting, examples of such myeloablative agents are busulfan, melphalan, treosulfan, fludarabine, cyclophosphamide, and combinations thereof. In a preferred embodiment, the myeloablative treatment is treatment with an agent selected from the group consisting of busulfan, treosulfan, fludarabine, and combinations thereof.

[0161] In a further preferred embodiment, treatment according to the present invention, particularly HSC transplantation, is combined with enzyme replacement therapy (ERT). The enzyme of ERT is preferably the same as the metabolic protein or a portion thereof present in the fusion protein or encoded by the nucleotide sequence present in the nucleic acid molecule of the present invention. For example, if the fusion protein includes an IDS or a portion thereof, or the nucleic acid molecule includes a nucleotide sequence encoding an IDS or a portion thereof, the enzyme used in ERT is IDS or a portion or variant thereof, optionally combined with or fused to a chaperone, agent, or peptide that facilitates passage through the blood-brain barrier. If the fusion protein includes GAA or a portion thereof, or the nucleic acid molecule includes a nucleotide sequence encoding GAA or a portion thereof, the enzyme used in ERT is GAA or a portion or variant thereof, optionally combined with or fused to a chaperone, agent, or peptide that facilitates passage through the blood-brain barrier. A "portion" or "variant" is a portion or variant of a related enzyme having the same type of enzymatic activity.

[0162] In some embodiments, an individual is treated with ERT before the treatment according to the present invention, particularly before HSC transplantation. In such examples, ERT serves to improve the patient's condition, for example, before the treatment according to the present invention, particularly before HSC transplantation. In some preferred embodiments, an individual is treated with an immunosuppressant before ERT or simultaneously with ERT. Immunosuppressants are well known in the art, and suitable immunosuppressants are well known and can be selected by those skilled in the art. As used herein, an immunosuppressant refers to an agent capable of suppressing an immune response in an individual. In a preferred embodiment, B cells are suppressed. In a preferred embodiment, the immunosuppressant is selected from the group consisting of B cell depleting agents, such as anti-CD20 antibodies, T cell depleting agents, such as anti-CD3 antibodies (e.g., OKT3, muronomab) or anti-T cell receptor antibodies (e.g., muromonab-CD3), anti-IL-2 receptor antibodies (e.g., basiliximab and daclizumab), azathioprine, calcineurin inhibitors, corticosteroids, cyclosporine, methotrexate, IVIG, mercaptopurine, mycophenolate mofetil, and combinations thereof. In a further preferred embodiment, the immunosuppressant is a B cell depleting agent, such as an anti-CD20 antibody, particularly rituximab. As used herein, a B cell depleting agent is an agent that reduces the amount of B cells in an individual treated with the agent. Treatment with an immunosuppressant may reduce or prevent the formation of antibodies specific for enzymes involved in ERT. Thus, such treatment optimizes the combination of ERT and treatment according to the present invention, particularly HSC transplantation.

[0163] In some embodiments, the individual is treated with a treatment according to the invention, e.g., ERT during and / or after HSC transplantation. In some embodiments, the individual is treated with a treatment according to the invention, e.g., ERT both before and during and / or after HSC transplantation. In some embodiments, the individual is not treated with ERT, but is treated with a treatment according to the invention, particularly HSC transplantation alone.

[0164] Features may be described herein as part of the same or separate aspects or embodiments of the invention for purposes of clarity and conciseness of description. It will be recognized by those skilled in the art that the scope of the invention may include embodiments having all or partial combinations of the features described herein as part of the same or separate embodiments.

[0165] The invention will now be described in more detail in the following non-limiting examples. [Brief explanation of the drawings]

[0166] [Figure 1] FIG. 1 shows the human IGF II amino acid sequence (P01344; based on UniProt). [Figure 2] Figure 2A shows the IDS sequence: the human IDS amino acid sequence (NP_000193.1). Figure 2B shows the IDS sequence: the human IDS mRNA sequence (nucleotides 170-1822 of NM_000202.8). [Figure 3-1] Figure 3A shows the GAA sequence. The human GAA amino acid sequence (NP_000143.2; GenPept). Figure 3B shows the GAA sequence. The human GAA mRNA sequence (NM_001079803.3; GenBank). [Figure 3-2] This is a continuation of Figure 3B. [Figure 4-1] Figure 4A shows the TPP1 sequence. The human TPP1 amino acid sequence (NP_000382.3). Figure 4B shows the TPP1 sequence. The human TPP1 mRNA sequence (nucleotides 23-1711 of NM_000391.4). [Figure 4-2] This is a continuation of Figure 4B. [Figure 5]1 shows lentiviral vectors encoding IDS and tagged variants of IDS. IDSco: codon-optimized nucleotide sequence encoding IDS amino acids 26-550. IDSSP: codon-optimized nucleotide sequence encoding IDS amino acids 1-25, corresponding to the IDS signal peptide. IGF2: codon-optimized nucleotide sequence encoding human mature IGF2 amino acids 1, 8-67. Amino acid 1 is intended to be the first amino acid of human mature IGF2 after deletion of signal peptide amino acids 2-7. ApoE2x2: codon-optimized nucleotide sequence encoding amino acids (LRKLRKRLL)x2. The sequence LRKLRKRLL is amino acids 141-149 of human apolipoprotein E. ApoE2: codon-optimized nucleotide sequence encoding amino acids LRKLRKRLL, which correspond to amino acids 141-149 of human apolipoprotein E. RAP12x2: a codon-optimized nucleotide sequence encoding amino acids EAKIEKHNHYQKGEAKIEKHNHYQK, where the sequence EAKIEKHNHYQK is amino acids 251-262 of domain 3 of human receptor-associated protein (RAP, also known as alpha2-macroglobulin receptor-associated protein). RAP12x1: a codon-optimized nucleotide sequence encoding amino acids EAKIEKHNHYQK, where amino acids 251-262 of domain 3 of human receptor-associated protein (RAP, also known as alpha2-macroglobulin receptor-associated protein). ApoB: a codon-optimized nucleotide sequence encoding amino acids SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS, which correspond to amino acids 3371-3409 of human apolipoprotein B. IGF2a: the sequence ALCGGELVDTLQFVCGDRGFYF, which corresponds to amino acids 1, 8-28 of mature human IGF2. Amino acid 1 is intended to be the first amino acid of mature human IGF2 after the signal peptide. IGF2b: sequence SG corresponding to amino acids 29 and 41 of mature human IGF2. Amino acid 1 is intended to be the first amino acid of mature human IGF2 after the signal peptide.IGF2c: the sequence IVEECCFRSCDLALLETYCATPAKSE, corresponding to amino acids 42-67 of mature human IGF2. Amino acid 1 is intended to be the first amino acid of mature human IGF2 after the signal peptide. Angiopep2: the codon-optimized nucleotide sequence encoding the amino acids TFFYGGSRGKRNNFKTEEY. Leptin-30: the codon-optimized nucleotide sequence encoding the amino acids YQQILTSMPSRNVIQISNDLENLRDLLHVL. SP: signal peptide. co: optimized codons. L: linker sequence of amino acids LGGGGSGGGGSGGGGSGGGGS. L1: linker sequence SGGGG. L2: linker sequence SGGGGSG. L3: linker sequence GAPLGGGGSGGGGS. L4: linker sequence SGGSGGGGSGGGGSG. L5: linker sequence SGGS. L6: linker sequence GGSGGSGGSG. C: cysteine ​​residue. [Figure 6] Figure 1 shows the selection of the best indel strategy: uptake of IDS.IGF2 indel variants into MPS II fibroblasts. Data represent mean ± SEM and are analyzed by one-way ANOVA with Bonferroni multiple test correction. Significance is expressed relative to IDS. ***P≦0.001, ****P≦0.0001. [Figure 7] Figure 1 shows the application of an optimal indel strategy containing cysteine ​​residues flanking the insert: uptake of IDS.IGF2 indel variants into MPS II fibroblasts. For clarity, all indel types modeled after the IDS.IGF2 indel APoE2-C contain double cysteine ​​residues flanking the insert, although this is not indicated in the name. See legend to Figure 1. Data represent mean ± SEM and are analyzed by one-way ANOVA with Bonferroni multiple testing correction. Significance is expressed relative to IDS. ns, not significant; ***P ≤ 0.001, ****P ≤ 0.0001. [Figure 8]Figure 1 shows LRP-1 functional ELISA. A. LRP-1 functional ELISA with IDS, IDS.ApoE2, and IDS.RAP12x2. B. LRP-1 functional ELISA with IDS.ApoE2, IDS.IGF2 indel RAP12x2, IDS.IGF2 indel RAP12, IDS.IGF2 indel ApoE, and IDS.IGF2 indel ApoB. Nonlinear fit curves are shown. Km and Vmax values ​​are indicated. [Figure 9] Hematopoietic stem cell (HSC)-mediated lentiviral gene therapy in MPS II mice results in supraphysiological levels of IDS activity in the bone marrow. Two- to five-month-old CD45.1 ids- / - mice were sacrificed, and lineage cells were purified from the bone marrow as previously described. Lineage cells were cultured ex vivo for a short period and transduced with lentiviral vectors encoding constructs 1 to 9 or GFP as a mock control at the indicated multiplicity of infection (MOI). Two- to three-month-old CD45.2 ids- / - mice were pretreated with a dose of 9 Gy of total body irradiation. 24 hours after pretreatment, mice were transplanted with 10^6 cells. Six months after gene therapy, mice were sacrificed, and tissues were harvested for histology and molecular analysis. (A) Mean integrated vector copy number (VCN) in the bone marrow. VCN was determined by qPCR as previously described. (B) IDS enzyme activity in the bone marrow. Data in (A) and (B) represent the mean ± SEM, and data were analyzed by one-way ANOVA with Bonferroni multiple testing correction. *P≦0.05. [Figure 10] Figure 1 shows that gene therapy results in increased levels of IDS activity in the brain. (A) IDS enzyme activity in the brain 6 months after gene therapy. Data in (A) represent mean ± SEM. [Figure 11-1] Figure 1 shows that gene therapy results in supraphysiological IDS activity levels in plasma. (A, B) IDS enzyme activity in plasma 6 months after gene therapy. (B, C) VCN in the brain is plotted against activity in plasma. Results of linear regression are shown in (B, C). Data in (A) represent mean ± SEM. [Figure 11-2]Figure 1 shows that gene therapy results in supraphysiological IDS activity levels in plasma. (A, B) IDS enzyme activity in plasma 6 months after gene therapy. (B, C) VCN in the brain is plotted against activity in plasma. Results of linear regression are shown in (B, C). Data in (A) represent mean ± SEM. [Figure 12-1] Figure 12A shows that gene therapy results in a reduction of brain heparan sulfate. (A) HPLC-MS quantification of brain heparan sulfate (HS) as previously described. Multiple comparison results are shown in (B). Data were analyzed by one-way ANOVA with Bonferroni multiple test correction and are presented as mean ± SEM. *P≦0.05, **P≦0.01, ****P≦0.0001. [Figure 12-2] Figure 12B shows that gene therapy results in a reduction of brain heparan sulfate. (A) HPLC-MS quantification of brain heparan sulfate (HS) as previously described. Multiple comparison results are shown in (B). Data were analyzed by one-way ANOVA with Bonferroni multiple test correction and are presented as mean ± SEM. *P≦0.05, **P≦0.01, ****P≦0.0001. [Figure 13] Relationship between VCN in the bone marrow and HS levels in the brain. (A, B) VCN in the brain is plotted against brain HS. Nonlinear regression results are shown. [Figure 14-1] Figure 1 shows that gene therapy results in normalization of Alcian blue staining in the spleen, liver, kidneys, heart valves, and aortic wall. (A-F) Scoring of Alcian blue staining in the spleen, liver, kidneys, heart valves, and aortic wall. Scoring was performed by two independent operators blinded to the experimental conditions. The scoring system is shown in Table 1. n = 3. AB = Alcian blue. Data represent mean ± SEM. Data were analyzed by one-way ANOVA with Bonferroni multiple testing correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. [Figure 14-2]Figure 1 shows that gene therapy results in normalization of Alcian blue staining in the spleen, liver, kidneys, heart valves, and aortic wall. (A-F) Scoring of Alcian blue staining in the spleen, liver, kidneys, heart valves, and aortic wall. Scoring was performed by two independent operators blinded to the experimental conditions. The scoring system is shown in Table 1. n = 3. AB = Alcian blue. Data represent mean ± SEM. Data were analyzed by one-way ANOVA with Bonferroni multiple testing correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. [Figure 14-3] Figure 1 shows that gene therapy results in normalization of Alcian blue staining in the spleen, liver, kidneys, heart valves, and aortic wall. (A-F) Scoring of Alcian blue staining in the spleen, liver, kidneys, heart valves, and aortic wall. Scoring was performed by two independent operators blinded to the experimental conditions. The scoring system is shown in Table 1. n = 3. AB = Alcian blue. Data represent mean ± SEM. Data were analyzed by one-way ANOVA with Bonferroni multiple testing correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. [Figure 14-4] This is a continuation of Figure 14F. [Figure 14-5] This is a continuation of Figure 14F. [Figure 15] Figure 1 shows that gene therapy leads to a strong reduction in lysosomal pathology in the brain. Quantification of lamp1 deposition in the brain 6 months after gene therapy. Lamp1 DAB staining was performed on sagittal brain sections, and quantification was performed by counting the number of lamp1-positive cells in the indicated regions. n=3; data are means ± SEM. Data represent means ± SEM, and data were analyzed by two-way ANOVA with Bonferroni multiple test correction using brain region and treatment as categorical variables. *P≦0.05, ***P≦0.001, ****P≦0.0001. [Figure 16-1]Figure 1 shows that gene therapy results in a reduction of neuroinflammation in the brain. Quantification of lamp1 deposition in the brain 6 months after gene therapy. GFAP (a marker for astrocytes) and CD68 (a marker for activated microglia) DAB staining was performed on sagittal brain sections, and quantification was performed by counting the number of GFAP- or CD68-positive cells in the indicated regions. n=3; data represent mean ± SEM. Data were analyzed by two-way ANOVA with Bonferroni multiple test correction using brain region and treatment as categorical variables. *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. [Figure 16-2] Figure 1 shows that gene therapy results in a reduction of neuroinflammation in the brain. Quantification of lamp1 deposition in the brain 6 months after gene therapy. GFAP (a marker for astrocytes) and CD68 (a marker for activated microglia) DAB staining was performed on sagittal brain sections, and quantification was performed by counting the number of GFAP- or CD68-positive cells in the indicated regions. n=3; data represent mean ± SEM. Data were analyzed by two-way ANOVA with Bonferroni multiple test correction using brain region and treatment as categorical variables. *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. [Figure 17] 1 shows that gene therapy results in normalization of Alcian blue staining in the brain. Quantification of Alcian blue staining in the brain. Alcian blue staining was performed on sagittal sections of the brain, and quantification was performed by counting the number of Alcian blue-positive cells in the indicated areas. AB=Alcian blue. Data are mean±SEM. n=3. [Figure 18] (B) Vector copy number in mice used for brain histology. VCN was determined by 3qPCR as previously described. (C) IDS enzyme activity in bone marrow. Data are means ± SEM. n=3. [Figure 19]Figure 1 shows competitive IGF2R (A) and IR-A (B) ELISAs with the indicated concentrations of the indicated ligands. Data represent mean ± SD. n=2 technical replicates per condition. [Example]

[0167] Materials and Methods MPS II uptake into fibroblasts Dosage based on enzyme activity HEK 293T cells, as previously shown 3 The cells were transfected with a self-inactivating lentiviral pCCL transfer vector encoding constructs 1-15. 48 hours after transfection, the culture supernatant containing the secreted IDS variants was collected, and the IDS concentration was determined using a 4MU assay to determine IDS activity. 100 nmol / ml IDS for 4 h was incubated with MPS II fibroblasts for 18 h. After washing with PBS, the captured IDS was measured in the cell lysates using an IDS sandwich ELISA. The results are shown in Figure 6.

[0168] In other experiments, HEK 293T cells were transfected with 100 ng / ml of 10 ... 3 The cells were transfected with a self-inactivating lentiviral pCCL transfer vector encoding constructs 1 to 15. 48 hours after transfection, the culture supernatant containing the secreted IDS variants was collected, and the IDS concentration was determined using a sandwich IDS ELISA. 4 100 ng of IDS was incubated with MPS II fibroblasts for 18 hours. After washing with PBS, captured IDS was measured in cell lysates using an IDS sandwich ELISA. The results are shown in Figure 7.

[0169] LRP-1 functional ELISA HEK 293T cells, as previously shown 3Forty-eight hours after transfection, the culture supernatant containing the secreted IDS variants was collected, and the IDS concentration was determined using a sandwich IDS ELISA (DY2449, R&D). Human recombinant LRP-1 receptor (5395-L4; R&D) was immobilized with a human anti-IgG antibody (62-8400; Invitrogen) in a Nunc-immuno microwell plate M9410. Culture medium supernatant containing (A) 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875, or 0.0234375 μg / ml IDS or (B) 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015635, or 0.0078125 μg / ml IDS was added to LRP-1-coated wells. LRP-1-bound IDS protein was detected using a biotinylated anti-IDS capture antibody and HRP-streptavidin (DY2449, R&D). Optical density was determined using a microplate reader (Varioskan, Thermo Electron Corporation) set at 450 nm and corrected for optical imperfections measured at 570 nm.

[0170] Construction and production of lentiviral vectors The codon-optimized human IDS was cloned into the third-generation self-inactivating (SIN) lentiviral vector pCLL.PPT.MND.RAG1.WPRE.SIN (pCCL.MND.coRAG1, a gift from Dr. Frank Staal; Garcia-Perez et al., Mol Ther Methods Clin Dev. 2020 Mar 31;17:666-682. doi: 10.1016 / j.omtm.2020.03.016) by replacing the RAG1 gene using BamHI and SalI restriction sites to generate pCLL.PPT.MND.IDSco.WPRE.SIN (IDSco). Codon-optimized cassettes encoding the tags described in Figure 5, constructs 2-15, were cloned into the IDSco backbone after double digestion with SbfI and SalI. Lentiviral vectors were produced in HEK 293T cells by calcium phosphate transfection of the third-generation lentiviral vector packaging plasmids pMDL-g / pRRE, pMD2-VSVg, and pRSV-Rev (Dull et al., 1998 J. Virol. 72, 8463; Zufferey et al., 1998 J. Virol. 72, 9873-9880). Viral concentration was performed by ultracentrifugation (Beckman, SW32Ti rotor) at 20,000 rpm for 2 hours at 4°C, and titration was performed in HeLa cells by quantitative polymerase chain reaction (qPCR) with primers targeting the U3 and Psi sequences of HIV. A standard curve was generated using HeLa cells transduced with an average of one copy of integrated lentiviral vector per genome. Viral titer concentrations were determined as the mean VCN multiplied by the cell number and dilution factor.

[0171] Hematopoietic stem cell (HSC)-mediated lentiviral gene therapy in MPS II mice 2-5 month old CD45.1 ids - / - Mice were sacrificed and lineage cells were isolated as previously described. 2,5Purified from bone marrow. Lineage cells were briefly cultured ex vivo and transduced with lentiviral vectors encoding constructs 1 to 9 or GFP as a mock control at the indicated multiplicity of infection (MOI). 2- to 3-month-old CD45.2 ids - / - Mice were pretreated with a dose of 9 Gy of total body irradiation. 24 hours after pretreatment, mice were transplanted with 10^6 cells. Six months after gene therapy, mice were sacrificed and tissues were collected for histological and molecular analysis.

[0172] immunohistochemistry Spleen, liver, kidney, heart, and brain were fixed in metacharn solution (30% chloroform, 60% methanol, 10% acetic acid) for 24 hours. Samples were embedded in paraffin in a Leica tissue processor TP1020 Histokinette and sectioned at 8 or 10 μm thickness. Alcian blue staining was performed according to the method of Alliegro and coworkers. 6 and scored as shown in Table 1. Dab staining was performed as described by Doyle and coworkers. 7 The results were as shown by

[0173] [Table 1]

[0174] molecular analysis VCN in bone marrow was cultured as previously described. 2 HPLC-MS quantification of brain heparan sulfate was performed as previously described. 1 Quantification of IDS enzyme activity was performed as previously described. 8 carried out.

[0175] result Construction of third-generation lentiviral vectors encoding IDS and tagged variants of IDS The constructs are shown in Figure 5. All constructs were cloned into the pCCL third generation lentiviral vector. Construct 1. / IDS encodes the IDS without a tag. Construct 2. / IDS.IGF2 encodes an IDS tagged at the C-terminus with amino acids 1, 8-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). Construct 3. / IDS.ApoE2 encodes an IDS tagged at the C-terminus with two amino acids (141-149) of human apolipoprotein E (amino acid 1 is the first amino acid after the signal peptide). Construct 4. / IDS.RAP12x2 encodes an IDS tagged at the C-terminus with amino acids (251–262) of human receptor-associated protein (RAP) in tandem repeats separated by glycine residues (amino acid 1 is the first amino acid after the signal peptide). Construct 5. / IDS.IGF2 indel ApoE-C encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence LRKLRKRLL, corresponding to amino acids 141-149 of human apolipoprotein E and flanked by the sequences CSGGGG (N-terminus) and SGGGGSGC (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 6. / IDS.IGF2indelApoE2 encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence LRKLRKRLL, corresponding to amino acids 141-149 of human apolipoprotein E and flanked by the sequences SGGGG (N-terminus) and SGGGGSG (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 7. / IDS.IGF2 indel ApoE2x2-C encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence LRKLRKRLLLRKLRKRLL, corresponding to amino acids 141-149 of human apolipoprotein E and flanked by the sequences CSGGGG (N-terminus) and SGGGGSGC (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 8. / IDS.IGF2 indel ApoE2x2-a encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence LRKLRKRLLLRKLRKRLL, corresponding to amino acids 141-149 of human apolipoprotein E and flanked by the sequences SGGGG (N-terminus) and SGGSGGGGSGGGGSG (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 9. / IDS.IGF2 indel ApoE2x2-b encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence LRKLRKRLLLRKLRKRLL, corresponding to amino acids 141-149 of human apolipoprotein E and flanked by the sequences SGGS (N-terminus) and GGSGGSGGSG (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 10. / IDS.IGF2 indel RAP12x2 encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence EAKIEKHNHYQK, which is amino acids 251-262 of human receptor-associated protein (RAP), was inserted between amino acids 28 and 42 of human IGF2, flanked by the sequences CSGGGG (N-terminus) and SGGGGSGC (C-terminus). Construct 11. / IDS.IGF2 indel RAP12 encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). It consists of amino acids 251-262 of human receptor-associated protein (RAP), with the sequence EAKIEKHNHYQK, flanked by the sequences CSGGGG (N-terminus) and SGGGGSGC (C-terminus), inserted between amino acids 28 and 42 of human IGF2. Construct 12. / IDS.IGF2indelApoB encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS, corresponding to amino acids 3371-3409 of human apolipoprotein B and flanked by the sequences CSGGGG (N-terminus) and SGGGGSGC (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 13. / IDS.IGF2indelleptin-30 encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence YQQILTSMPSRNVIQISNDLENLRDLLHVL, flanked by the sequences CSGGGG (N-terminus) and SGGGGSGC (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 14. / IDS.IGF2 indel Angiopep2 encodes an IDS tagged at the C-terminus with amino acids 1, 8-28, and 42-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide). The sequence TFFYGGSRGKRNNFKTEEY, flanked by the sequences CSGGGG (N-terminus) and SGGGGSGC (C-terminus), was inserted between amino acids 28 and 42 of human IGF2. Construct 15. / IDS.IGF2delta30-40 encodes an IDS tagged at the C-terminus with amino acids 1, 8-29, and 41-67 of mature human IGF2 (amino acid 1 is the first amino acid after the signal peptide).

[0176] MPS II uptake into fibroblasts The IGF2 indel design was optimized by tagging the IDS at the C-terminus with one of four IGF2 indel variants containing an insertion of ApoE2, a peptide capable of binding to members of the LDL receptor family (LDLrf) (Figure 6). The four IGF2 indel variants differed in the length and composition of linkers flanking the ApoE2 insertion (different combinations used in the four constructs; see brief description of the figures on page 39) and in the presence or absence of a cysteine ​​residue flanking the insertion. a. IDS.IGF2 indel ApoE2-C (construct 5 in Figure 5): one ApoE2 repeat; cysteines on either side of the insert; linkers L1 and L2; b. IDS.IGF2 indel ApoE2 (construct 6 in Figure 5): one ApoE2 repeat; no cysteines; linkers L1 and L2; c. IDS.IGF2 indel ApoE2x2-a (construct 8 in Figure 5): two tandem ApoE2 repeats; no cysteines; linkers L1 and L4; d. IDS.IGF2 indel ApoE2x2-b (construct 9 in Figure 5): two tandem ApoE2 repeats; no cysteines; linkers L5 and L6.

[0177] The IGF2 indel design was evaluated by comparing its uptake into patient-derived MPS II fibroblasts with those of an IGF2-tagged IDS (IDS.IGF2), an IGF2-tagged IDS with a deletion of amino acids 30-40 (IDS.IGF2delta30-40), and an ApoE2-tagged IDS (in tandem repeats, IDS.ApoE2).

[0178] As shown in Figure 6, IDS.IGF2 showed slightly higher uptake values ​​compared to IDS.IGF2 delta 30-40 and approximately threefold higher uptake levels compared to IDS.ApoE2. IGF2 indels with tandem repeats of ApoE2 (IDS.IGF2 indel ApoE2x2-a and IDS.IGF2 indel ApoE2x2-b) resulted in uptake levels comparable to IDS.ApoE2 and approximately one-third lower than IDS.IGF2. In contrast, insertion of a single repeat of ApoE2 resulted in slightly lower (IDS.IGF2 indel ApoE2) or equivalent (IDS.IGF2 indel ApoE2-C) uptake levels compared to IDS.IGF2 delta 30-40.

[0179] In particular, the inclusion of cysteine ​​residues on both sides of the insert appeared to be beneficial for uptake into MPS II fibroblasts (Figure 6). For this reason, we chose the IDS.IGF2 indel ApoE2-C design, characterized by a "SGGG" linker at the N-terminus of the insert, a "SGGGGSG" linker at the C-terminus of the insert, and cysteine ​​residues at the N- and C-termini of the two linkers, to further test IGF2 indel variants with additional inserted sequences (Figure 7).

[0180] To evaluate the applicability of the selected IGF2 indel designs to epitope modular switching, we replaced the ApoE2 sequence in IDS.IGF2 indel ApoE2-C with alternative sequences (Figure 7). Furthermore, to examine the loading capacity of the selected IGF2 indel designs, we specifically selected epitopes of various lengths, ranging from as short as 9 amino acids in ApoE2 to as long as 39 amino acids in ApoB. This resulted in six IGF2 indel versions containing six different insertions: - ApoE2 (IDS.IGF2 indel ApoE2-C), - ApoE2x2 (ApoE2 tandem repeat, IDS.IGF2 indel ApoE2x2-C), - RAP12 (IDS.IGF2 indel RAP12), - RAP12x2 (tandem repeats of RAP12 separated by glycine residues, IDS.IGF2 indel RAP12x2), - ApoB (IDS.IGF2 indel ApoB), and - Leptin-30 (IDS.IGF2 indel leptin-30). All of these constructs contain cysteine ​​residues at the N- and C-termini of the two linkers flanking the insert.

[0181] Next, we tested these versions in MPS II fibroblast uptake. As shown in Figure 7, all tested constructs provided improved uptake compared to untagged IDS. While IDS.IGF2 indel RAP12x2 demonstrated comparable uptake values ​​to IDS.IGF2, IDS.IGF2 indel ApoE2-C, IDS.IGF2 indel RAP12, and IDS.IGF2 indel ApoB yielded slightly reduced values ​​compared to IDS.IGF2 and comparable values ​​to IDS.IGF2 delta 30-40. We selected IDS.IGF2 indel ApoE2, IDS.IGF2 indel RAP12x2, and IDS.IGF2 indel ApoB for further in vitro testing (Figure 19).

[0182] To examine binding to CI-M6P / IGF2R, we performed a competitive binding assay using domain 11 of CI-M6P / IGF2R, which is known to specifically bind IGF2. In this assay, we used a fixed concentration of biotinylated IGF2 (8 nM; referred to as the hot ligand) to bind the following concentration range (1-500 nM; referred to as the cold ligand): - IDS.IGF2, - IDS.IGF2 indel ApoE2-C, - IDS.IGF2 indel RAP12x2, - IDS.IGF2 indel ApoB, or - IGF2 peptide with mutations in amino acids 1 to 6 (Δ1-6.IGF2 peptide) was made to compete with.

[0183] Binding of IGF2 to CI-M6P / IGF2R, when used as a peptide or tagged with IDS, was increased approximately 12.3-fold in IC compared to the untagged Δ1-6.IGF2 peptide, unlike IDS.IGF2. 50 The value is shown (IC 50 IDS.IGF2: 61.69nM; IC 50 Δ1-6.IGF2 peptide: 5.008 nM) (FIG. 19A), suggesting that binding of IGF2 to CI-M6P / IGF2R is partially inhibited when tagged with IDS.

[0184] The IDS.IGF2 indel versions examined showed slightly higher affinity for CI-M6P / IGF2R compared to IDS.IGF2, with IC values ​​of IDS.IGF2 indel ApoE2, IDS.IGF2 indel RAP12x2, and IDS.IGF2 indel ApoB being significantly higher. 50 are the IC of IDS and IGF2, respectively. 50 were approximately 1.69, 1.44, and 1.21 times lower than those of the IC 50 IDS.IGF2 indel ApoE2: 36.45nM; IC 50 IDS.IGF2 indel RAP12x2: 42.92 nM; IC 50 IDS.IGF2 indel ApoB: 50.92 nM) (Figure 19A).

[0185] Binding to the insulin receptor (IR) was also investigated. Similar to the competitive CI-M6P / IGF2R ELISA assay, we used a fixed concentration of biotinylated insulin (0.8 nM; referred to as the hot ligand) and competed it with a range of concentrations of IDS.IGF2 indel versions and IDS.IGF2 and Δ1-6.IGF2 peptides (1–500 nM; referred to as the cold ligands) for binding to IR isoform A (IR-A).

[0186] Similar to the observations made during the CI-M6P / IGF2R ELISA assay, we noted an effect of IDS tagging on IGF2 binding to IR-A (Figure 19B). Specifically, IDS.IGF2 had an affinity (IC) that was approximately 150-fold lower than that of the Δ1-6.IGF2 peptide. 50 IDS.IGF2: 250.7nM;IC 50 Δ1-6.IGF2 peptide: 1.607 nM) to IR-A, suggesting partial interference with IGF2 binding to IR-A when tagged with IDS.

[0187] In contrast, none of the IDS.IGF2 indel versions tested showed any appreciable binding to IR-A, as evidenced by the complete lack of competition for biotinylated insulin.

[0188] LRP-1 functional ELISA Binding of IDS.RAP12x2 to the LRP-1 receptor was less efficient than that of IDS.IGF2 indel RAP12x1 and IDS.IGF2 indel RAP12x2 to the same receptor, which may be caused by the inaccessibility of the RAP12 tag for LRP-1 binding when present in IDS.RAP12 (Figure 8A). IDS.IGF2 indel versions containing either the ApoE2, RAP12, or ApoB tag can bind to the LRP-1 receptor with affinity comparable to IDS.ApoE2. IDS.IGF2 does not bind efficiently to the LRP-1 receptor (Figure 8B).

[0189] The insertion of RAP12 as an indel version within the accessible loop structure of the IGF2 tag likely provides accessibility of the RAP12 tag, enabling binding to the LRP-1 receptor. IDS.IGF2 indel ApoE2-C and IDS.IGF2 indel ApoB have superior affinity for the LRP-1 receptor compared to IDS.IGF2 indel RAP12 and IDS.IGF2 indel RAP12x2.

[0190] Hematopoietic stem cell (HSC)-mediated lentiviral gene therapy in MPS II mice As demonstrated in Figure 9, HSC-mediated lentiviral gene therapy in MPS II mice results in supraphysiological levels of IDS activity in the bone marrow. IDS activity in the brain is comparable among the different lentiviral vectors tested (Figure 10).

[0191] IDS activity in plasma is lower after gene therapy with IDS.IGF2 or IDS.IGF2 indel versions compared to IDS, IDS.ApoE2, IDS.RAP12x2, and IDS.IGF2 delta30-40 (Figure 11). This lower IDS activity in plasma is likely due to higher uptake of IDS.IGF2 and IDS.IGF2 indel versions compared to IDS and IDS.ApoE2 constructs, which may reduce the plasma half-life of these constructs, as shown in Figures 6 and 7.

[0192] In the brain, IDS.IGF2 and IDS.ApoE2 are comparable in reducing heparan sulfate (HS), a compound that accumulates in Hunter disease. IDS.IGF2, IDS.ApoE2, IDS.IGF2 indel ApoE2-C, IDS.IGF2 indel RAP12x2, and IDS.IGF2 delta 30-40 are the most effective LV treatments for reducing HS in the brain (see Figure 12). IDS.IGF2 indel ApoE2-C reaches a plateau in brain HS levels compared to both IDS.IGF2 and IDS.IGF2 delta 30-40 (Figure 13, lower panel; plateau levels are 0.34, 0.46, and 0.52, respectively). IDS.IGF2 indel RAP12x2 showed lower efficacy in treating brain HS compared to IDS.IGF2 indel ApoE2-C, consistent with the lower affinity of the IDS.IGF2 indel RAP12x2 construct for LRP-1 compared to IDS.IGF2 indel ApoE2-C (and shown in Figure 8).

[0193] Alcian blue specifically binds to acidic mucins, such as heparan sulfate and dermatan sulfate glycosaminoglycans. Alcian blue staining was performed to visualize glycosaminoglycan accumulation in the spleen, liver, kidney, heart valve, and aortic wall. All indel vectors and other vectors can normalize Alcian blue-positive staining in the spleen, liver, kidney, and heart valve (see Figure 14). As demonstrated in Figure 17, the IDS.IGF2 indel vector, as well as the IDS.IGF2 and IDS.ApoE2 constructs, were more effective than untagged IDS in normalizing AB staining in the brain.

[0194] Lysosome-associated membrane protein 1 (Lamp1) is a marker of lysosomes. Widespread increases in Lamp1 deposition are observed in the brains of MPS II mice (Figure 15). Lentiviral gene therapy resulted in small (IDS and IDS.RAP12x2 treatment) to strong (IDS.IGF2, IDS.IGF2 indel version, IDS.IGF2 delta 30-40, and IDS.ApoE2) reductions in Lamp1 deposition in the brains of MPS II mice. IDS.IGF2 indel version and IDS.IGF2 showed comparable efficacy. In the cortex, the IGF2 indel version showed better correction of Lamp1 pathology than IDS.IGF2 delta 30-40, whereas in the thalamus, IDS.IGF2 and IDS.IGF2 indel ApoE2-C showed better results than IDS.IGF2 delta 30-40.

[0195] GFAP is a marker of activated astrocytes, a hallmark of neuroinflammation. GFAP deposition is increased in the cortex, thalamus, hypothalamus, midbrain, brainstem, and cerebellum of MPS II mice compared with wild-type mice, but is reduced in the corpus callosum and hippocampus (Figure 16). IDS.IGF2, IDS.IGF2 indel version, and IDS.IGF2 delta 30-40 produced comparable therapeutic outcomes in all regions, but IDS.IGF2 and IDS.IGF2 indel version outperformed IDS.IGF2 delta 30-40 in the cortex and corpus callosum.

[0196] Neuroinflammation is also characterized by activated microglial cells that upregulate CD68. Consequently, MPS II mice showed widespread increases in CD68 deposition in the brain (Figure 16). After lentiviral gene therapy, correction of altered CD68 deposition in the brain of MPS II mice followed the same trend as reported for Lamp1. While IDS.IGF2, IDS.IGF2 indel version, and IDS.IGF2 delta 30-40 resulted in comparable therapeutic outcomes, IDS.IGF2 indel version treatment appeared to result in better correction of CD68 deposition in the cortex, hippocampus, hypothalamus, and midbrain compared with IDS.IGF2 delta 30-40. The VCN of the mice used for brain Alcian blue, Lamp1, GFAP, and CD68 staining is shown in Figure 18.

[0197] (References) 1. Langereis, EJ, Wagemans, T., Kulik, W., Lefeber, DJ, van Lenthe, H., Oussoren, E., van der Ploeg, AT, Ruijter, GJ, Wevers, RA, Wijburg, FA, et al. (2015). A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses. PLoS One 10. 10.1371 / JOURNAL.PONE.0138622. 2. Pike-Overzet, K, Baum, C, Bredius, RGM, Cavazzana, M, Driessen, GJ, Fibbe, WE, Gaspar, HB, Hoeben, RC, Lagresle-Peyrou, C, Lankester, A, et al. (2014). Successful RAG1-SCID gene therapy depends on the level of RAG1 expression. Journal of Allergy and Clinical Immunology 134, 242–243. doi:10.1016 / j.jaci.2014.04.033. 3. Bergsma, AJ, Stijn, ·, in 't Groen, LM, Catalano, F., Yamanaka, M., Takahashi, S., Okumiya, T., Ans, ·, van der Ploeg, T., and Pim Pijnappel, · WWM (2021). A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease. European Journal of Human Genetics 29, 422–433. 10.1038 / s41431-020-00751-3 4. Human Iduronate 2-Sulphatase / IDS DuoSet ELISA DY2449-05: R&D Systems https: / / www.rndsystems.com / products / human-iduronate-2-sulfatase-ids-duoset-elisa_dy2449-05. 5. Gleitz, H.F., Liao, A.Y., Cook, J.R., Rowlston, S.F., Forte, G.M., D’Souza, Z., O’Leary, C., Holley, R.J., and Bigger, B.W. (2018). Brain‐targeted stem cell gene therapy corrects mucopolysaccharidosis type II via multiple mechanisms. EMBO Mol Med 10. 10.15252 / emmm.201708730. 6. Alliegro, M., Ferla, R., Nusco, E., de Leonibus, C., Settembre, C., and Auricchio, A. (2016). Low-dose Gene Therapy Reduces the Frequency of Enzyme Replacement Therapy in a Mouse Model of Lysosomal Storage Disease. Mol Ther 24, 2054-2063. 10.1038 / MT.2016.181. 7. Doyle, B.M., Turner, S.M.F., Sunshine, M.D., Doerfler, P.A., Poirier, A.E., Vaught, L.A., Jorgensen, M.L., Falk, D.J., Byrne, B.J., and Fuller, D.D. (2019). AAV Gene Therapy Utilizing Glycosylation-Independent Lysosomal Targeting Tagged GAA in the Hypoglossal Motor System of Pompe Mice. Mol Ther Methods Clin Dev 15, 194-203. 10.1016 / J.OMTM.2019.08.009. 8. Voznyi, Y. v., Keulemans, J.L.M., and van Diggelen, O.P. (2001). A fluorimetric enzyme assay for the diagnosis of MPS II (hunter disease). J Inherit Metab Dis 24, 675-680. 10.1023 / A:1012763026526.

Claims

1. - a nucleotide sequence encoding a lysosomal hydrolase or an enzymatically active portion thereof, or a sequence having at least 90% sequence identity with said lysosomal hydrolase or a portion thereof, - a human IGF II gene sequence comprising a deletion of nucleotides encoding amino acids 30-40, 30-41, 29-30, or 29-41 of human mature insulin-like growth factor II (IGFII); and - a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis inserted into said IGFII gene sequence at the position of the deletion in said IGFII gene sequence, at a position between the nucleotides encoding amino acids 28 and 42 of mature IGFII. A nucleic acid molecule comprising:

2. The nucleic acid molecule of claim 1, wherein the lysosomal hydrolase is selected from the group consisting of human iduronate-2-sulfatase (IDS), acid alpha-glucosidase (GAA), and tripeptidyl peptidase 1 (TPP1).

3. The lysosomal hydrolase - an IDS, wherein the nucleotide sequence encodes an amino acid sequence comprising a sequence having at least 90% identity with amino acids 26 to 550 of human IDS; - GAA, wherein the nucleotide sequence encodes an amino acid sequence comprising a sequence having at least 90% sequence identity with amino acids 70 to 952 of human GAA, or - TPP1, wherein the nucleotide sequence encodes an amino acid sequence comprising a sequence having at least 90% sequence identity with amino acids 20 to 563 of human TPP1.

3. The nucleic acid molecule of claim 1 or 2.

4. 4. The nucleic acid molecule according to claim 1, wherein the peptide that promotes cellular uptake or transcytosis is a peptide that promotes crossing the blood-brain barrier (BBB).

5. 5. The nucleic acid molecule of claim 4, wherein the peptide that promotes crossing of the BBB comprises a low-density lipoprotein receptor-related protein 1 (LRP1) binding domain and / or encodes the receptor binding domain of human apolipoprotein E2 (ApoE2), human apolipoprotein B (ApoB), human receptor-associated protein (RAP), or human leptin, or encodes the CRP peptide (CRTIGPSVC) or Angiopep-2 (TFFYGGSRGKRNNFKTEEY), or any combination and / or repeat of these domains and sequences.

6. 6. The nucleic acid molecule of claim 4 or 5, wherein the nucleotide sequence encodes amino acids 141 to 149 of human ApoE2 (LRKLRKRLL), amino acids 3371 to 3409 of human ApoB (SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS), amino acids 251 to 262 of human RAP (EAKIEKHNHYQK), amino acids 61 to 90 of human leptin (YQQILTSMPSRNVIQISNDLENLRDLLHVL), or combinations and / or repeats of any of these sequences.

7. 7. The nucleic acid molecule of any one of claims 1 to 6, wherein the human IGF II gene sequence comprises a nucleotide sequence encoding amino acids 8 to 28 and 42 to 67, or amino acids 8 to 29 and 41 to 67, of mature human IGF II, preferably amino acids 1, 8 to 28 and 42 to 67, or amino acids 1, 8 to 29 and 41 to 67, of mature human IGF II, or a sequence having at least 90% sequence identity thereto.

8. 8. The nucleic acid molecule of claim 1, comprising a nucleotide encoding at least one cysteine ​​on each side of a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis.

9. 9. The nucleic acid molecule of claim 1, comprising a linking sequence on one or both sides of a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis.

10. 10. The nucleic acid molecule of any one of claims 1 to 9, comprising a linking sequence and a nucleotide encoding a cysteine ​​on either side of a nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis, whereby the linking sequence flanks the nucleotide sequence encoding at least one peptide that promotes cellular uptake or transcytosis.

11. 11. The nucleic acid molecule of any one of claims 1 to 10, which is a gene therapy vector, preferably a lentiviral vector.

12. A viral particle comprising a nucleic acid molecule according to any one of claims 1 to 11.

13. 12. A fusion protein encoded by a nucleic acid molecule according to any one of claims 1 to 11, preferably comprising a human IGF II sequence comprising a deletion of nucleotides encoding amino acids 30-40 or 29-41 of human mature insulin-like growth factor II (IGFII), and a lysosomal hydrolase or an enzymatically active part thereof, or a sequence with at least 90% sequence identity to said lysosomal hydrolase or a part thereof, fused to at least one peptide which promotes cellular uptake or transcytosis, wherein said lysosomal hydrolase or a part thereof, or a sequence with at least 90% sequence identity to said lysosomal hydrolase or a part thereof, the human IGF II sequence, and / or said at least one peptide are separated by one or more linking sequences, more preferably - the lysosomal hydrolase or a part thereof, or a sequence having at least 90% sequence identity with the lysosomal hydrolase or a part thereof, is as defined in claim 2 or 3, - the human IGF II amino acid sequence is as defined in claim 7, and - the at least one peptide that promotes cellular uptake or transcytosis is as defined in any one of claims 4 to 6, Fusion proteins.

14. 13. A cell population, preferably a hematopoietic stem cell (HSC) population, provided with a nucleic acid molecule, vector or viral particle according to any one of claims 1 to 12, wherein preferably the cells have been transfected or transduced with said nucleic acid molecule, vector or viral particle, and more preferably the cells express a fusion protein comprising a lysosomal hydrolase or an enzymatically active part thereof, or a sequence having at least 90% sequence identity to said lysosomal hydrolase or a part thereof, fused to a human insulin-like growth factor II (IGF II) sequence and at least one peptide which promotes cellular uptake or transcytosis, preferably the fusion protein according to claim 13.

15. A nucleic acid molecule according to any one of claims 1 to 11, a viral particle according to claim 12, a fusion protein according to claim 13, or a cell population according to claim 14, for use in a method for the treatment of a lysosomal storage disorder.

16. 15. A method for treating a lysosomal storage disorder, comprising administering to an individual in need thereof a nucleic acid molecule described in any one of claims 1 to 11, a viral particle described in claim 12, a fusion protein described in claim 13, or a cell population described in claim 14.

17. 17. A nucleic acid molecule, fusion protein or cell population for the use according to claim 15 or the method according to claim 16, comprising obtaining cells, preferably HSCs, from an individual, providing said cells, preferably HSCs, with a nucleic acid molecule or viral particle according to any one of claims 1 to 12, preferably transfecting or transducing said cells, preferably HSCs, with a viral particle according to claim 12, and administering said cells, preferably HSCs, provided with said nucleic acid molecule or viral particle to said individual.

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