Transcriptional regulatory elements and their uses

Abstract

The present invention provides regulatory elements and vectors containing the regulatory elements, which can be used to stimulate the transcription of related genes in certain tissue types. The transcription regulatory elements described herein can be operably linked to a transgene, such as acid α-glucosidase (GAA), to promote the expression of the GAA transgene in cells, such as muscle cells, hepatocytes or neurons. The transcription regulatory elements described herein can be operably linked to a therapeutic transgene and can be used to treat a variety of disorders, such as lysosomal storage diseases, particularly Pompe disease.

Description

Field of the Invention

[0001] The present invention relates to the field of nucleic acid biotechnology and provides a composition and a method for promoting the expression of relevant genes. Background of the Invention

[0003] Pompe disease is a lysosomal storage disease caused by mutations in the acid α-glucosidase (GAA) gene, which encodes an enzyme responsible for processing lysosomal glycogen. Patients with Pompe disease exhibit clinical phenotypes in a variety of tissues, including glycogen accumulation in cells, defects in cardiac, respiratory and skeletal muscle function, and central nervous system symptoms. Some of these defects are significantly improved by enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA). Clinical efficacy is limited by the immunogenicity of hGAA ERT and the fact that rhGAA is not taken into some affected tissues. Gene therapy has also been studied as a potential treatment paradigm for this disease. The development of gene therapy for the treatment of Pompe disease is hindered by the difficulty of achieving the expression of a therapeutically effective amount of GAA in the affected tissues while preventing the immune system from causing an anti-GAA immune response. Still need a transcriptional regulatory element that can achieve this balance. Summary of the invention

[0004] The present invention provides compositions and methods for promoting expression of related genes (e.g., acid alpha-glucosidase (GAA)) in cells of certain tissues, including those affected by Pompe disease. The compositions and methods described herein relate to nucleic acid regulatory elements that stimulate transcription of transgenes (e.g., GAA) in muscle cells (e.g., cardiomyocytes), hepatocytes, and / or cells of the central nervous system. The nucleic acid regulatory elements described herein are operably linked to transgenes (e.g., GAA) and can be administered to patients (e.g., human patients) to treat lysosomal storage diseases, such as Pompe disease. Advantageously, the compositions and methods described herein can be used to promote expression of GAA in patients (e.g., those with Pompe disease), in muscle cells and / or neurons affected by lysosomal storage diseases, and can simultaneously stimulate expression in the liver. This provides surprising therapeutic benefits. Without being limited by mechanism, the present disclosure is based in part on the discovery that the transcriptional regulatory elements described herein can (i) promote expression of transgenes in cells affected by lysosomal storage diseases to improve the condition, and (ii) stimulate expression in the liver, which is used to promote immune tolerance. Thus, the compositions and methods described herein can be used to treat lysosomal storage diseases, such as Pompe disease, in a manner that mitigates the deleterious effects of lysosomal enzyme deficiency (e.g., GAA deficiency) in a patient while preventing or reducing the induction of an immune response to the enzyme introduced by gene therapy.

[0005] In a first aspect, the invention features a nucleic acid regulatory element comprising a first segment operably linked to a second segment, wherein the first segment comprises an apolipoprotein E liver control region (ApoE-HCR) or a functional portion thereof, and the second segment comprises a desmin promoter or a functional portion thereof. The 3' end of the first segment is operably linked to the 5' end of the second segment. In some embodiments, the 5' end of the first segment is operably linked to the 3' end of the second segment.

[0006] In some embodiments, the first segment contains an ApoE-HCR enhancer, which has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical) to the nucleic acid sequence of SEQ ID NO:3; or a functional portion of the ApoE-HCR enhancer, which has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical) to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3; or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3; or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence of SEQ ID NO: 3 or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence of SEQ ID NO: 4.

[0007] In some embodiments, the first segment contains an ApoE-HCR enhancer, which has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical) to the nucleic acid sequence of SEQ ID NO:3; or a functional portion of the ApoE-HCR enhancer, which has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical) to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3; or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3; or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence of SEQ ID NO: 3 or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence of SEQ ID NO: 1.

[0008] In some embodiments, the first segment contains an ApoE-HCR enhancer, which has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical) to the nucleic acid sequence of SEQ ID NO:3; or a functional portion of the ApoE-HCR enhancer, which has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical) to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3; or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3; or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the first segment contains an ApoE-HCR enhancer having a nucleic acid sequence of SEQ ID NO: 3 or a functional portion of the ApoE-HCR enhancer having a nucleic acid sequence of SEQ ID NO: 2.

[0009] In some embodiments, the first segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the first segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the first segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the first segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4. For example, the first segment may contain the nucleic acid shown in SEQ ID NO: 4.

[0010] In some embodiments, the first segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the first segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the first segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 1. For example, the first segment may have the nucleic acid sequence of SEQ ID NO: 1.

[0011] In some embodiments, the first segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 2. The first segment may have a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the first segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2. For example, the first segment may have a nucleic acid sequence of SEQ ID NO: 2.

[0012] In some embodiments, the first segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 3. The first segment may have a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the first segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3. For example, the first segment may have a nucleic acid sequence of SEQ ID NO: 3.

[0013] In some embodiments, the second segment contains a 5' region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the 5' region has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the 5' region has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 5. For example, the 5' region may have the nucleic acid sequence of SEQ ID NO: 5.

[0014] In some embodiments, the second segment may contain a 3' region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the 3' region has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the 3' region has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 6. For example, the 3' region may have the nucleic acid sequence of SEQ ID NO: 6.

[0015] In some embodiments, the second segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the second segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the second segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 7. For example, the 3' region can have the nucleic acid sequence of SEQ ID NO: 7.

[0016] In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 10. For example, the nucleic acid regulatory element may have the nucleic acid sequence of SEQ ID NO: 10.

[0017] In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 12. For example, the nucleic acid regulatory element may have the nucleic acid sequence of SEQ ID NO: 12.

[0018] In some embodiments, the nucleic acid regulatory element further comprises a third segment located 5' or 3' relative to the first segment and the second segment. The third segment is operably linked to the first segment and the second segment.

[0019] In some embodiments, the third segment contains a promoter that stimulates expression of a transgene operably linked to the promoter in a central nervous system cell (e.g., in particular a neuron, a glial cell, or an astrocyte). In some embodiments, the third segment contains a promoter selected from the group consisting of a synapsin promoter, a glial fibrillary acidic protein (GFAP) promoter, a calcium / calmodulin-dependent protein kinase III promoter, a tubulin αI promoter, a microtubulin-associated protein IB (MAP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a calcium / calmodulin-dependent protein kinase III promoter, a microtubulin αI ... IB) promoter, neuron-specific enolase promoter, platelet-derived growth factor β chain promoter, neurofilament light chain promoter, neuron-specific VGF gene promoter, neuronal nuclear (NeuN) promoter, adenomatous polyposis coli (APC) promoter, ionized calcium binding adapter molecule 1 (Iba-1) promoter and homeobox protein 9 (HB9) promoter, or a variant of the promoter (e.g., a variant having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater sequence identity) to the nucleic acid sequence of the wild-type promoter locus and capable of stimulating transcription of a transgene operably linked to it after introduction into central nervous system cells) or a functional portion thereof.

[0020] In some embodiments, the third segment contains a promoter that stimulates expression of a transgene operably linked to the promoter in neurons. In some embodiments, the third segment contains the synapsin promoter or a functional portion thereof.

[0021] In some embodiments, the third segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the third segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the third segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the third segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 8.

[0022] In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the nucleic acid regulatory element has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 11. For example, the nucleic acid regulatory element may have the nucleic acid sequence of SEQ ID NO: 11.

[0023] In another aspect, the invention features a nucleic acid regulatory element comprising a first segment operably linked to a second segment, wherein the first segment comprises a desmin promoter or a functional portion thereof, and the second segment comprises a promoter or a functional portion thereof that stimulates expression of a transgene operably linked to the promoter or a functional portion thereof in cells of the central nervous system (e.g., in particular neurons, glial cells, or astrocytes). In some embodiments, the second segment contains a promoter selected from the group consisting of a synapsin promoter, a GFAP promoter, a calcium / calmodulin-dependent protein kinase III promoter, a microtubule protein αI promoter, a MAP IB promoter, a neuron-specific enolase promoter, a platelet-derived growth factor β chain promoter, a neurofilament light chain promoter, a neuron-specific VGF gene promoter, a NeuN promoter, an APC promoter, an Iba-1 promoter, and an HB9 promoter, or a variant of said promoter (e.g., a variant having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater sequence identity) to the nucleic acid sequence of the wild-type promoter locus and capable of stimulating transcription of a transgene operably linked to it upon introduction into a central nervous system cell) or a functional portion thereof.

[0024] In some embodiments, the second segment contains a promoter that stimulates expression of a transgene operably linked to the promoter in neurons. In some embodiments, the second segment contains the synapsin promoter or a functional portion thereof.

[0025] In some embodiments, the 3' end of the first segment is operably linked to the 5' end of the second segment. In some embodiments, the 5' end of the first segment is operably linked to the 3' end of the second segment.

[0026] In some embodiments, the first segment contains a 5' region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the 5' region has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the 5' region has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 5. For example, the 5' region may have the nucleic acid sequence of SEQ ID NO: 5.

[0027] In some embodiments, the first segment contains a 3' region having a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the 3' region has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the 3' region has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 6. For example, the 3' region may have the nucleic acid sequence of SEQ ID NO: 6.

[0028] In some embodiments, the first segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the first segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the first segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 7. For example, the first segment may have the nucleic acid sequence of SEQ ID NO: 7.

[0029] In some embodiments, the second segment has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the second segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the second segment has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the second segment has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 8.

[0030] On the other hand, the present invention is characterized in that the vector of the nucleic acid regulatory element of any one of the embodiments of the aforementioned aspects or aspects is contained. The nucleic acid regulatory element can be operably connected to the transgene, and the expression of the transgene can be induced after the vector is introduced into the cell (e.g., mammalian cell, e.g., human cell). The cell can be, for example, a myocyte (e.g., cardiomyocyte or skeletal muscle cell), a neuron, or a hepatocyte. In some embodiments, the transgene encodes a lysosomal enzyme, such as GAA. In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, or a vaccinia virus. In some embodiments, the viral vector is AAV, such as AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, or AAVrh74 serotype. The viral vector can be a pseudotyped AAV, such as a recombinant AAV (rAAV) 2 / 8 or rAAV2 / 9.

[0031] In another aspect, the invention features a composition containing the nucleic acid regulatory element of any of the above aspects or embodiments of the aspects. The composition can be, for example, a liposome, a vesicle, a synthetic vesicle, an extracellular body, a synthetic extracellular body, a dendrimer, or a nanoparticle. In the composition, the nucleic acid regulatory element can be operably linked to a transgene (e.g., a transgene encoding a lysosomal enzyme (e.g., GAA)).

[0032] In another aspect, the invention features a method of expressing a transgene in a cell by contacting the cell with the vector or composition of any of the preceding aspects or embodiments of the aspects for a time sufficient to mimic transcription of the transgene in the cell.

[0033] In another aspect, the invention features a method of treating a lysosomal storage disease (e.g., Pompe disease) in a patient (e.g., a mammalian patient, e.g., a human patient) in need thereof by administering to the patient a therapeutically effective amount of a vector or composition described herein.

[0034] In another aspect, the invention features a kit containing a vector or composition described herein. The kit can contain a package insert, for example, that instructs the user of the kit to contact the vector or composition with a cell (e.g., a mammalian cell, such as a human cell), thereby expressing a transgene operably linked to a regulatory element.

[0035] definition

[0036] As used herein, the term "about" refers to a value that is within 10% above or below the stated value.

[0037] The term "ApoE-HCR enhancer" as used herein refers to the human apolipoprotein E liver control region, the nucleic acid sequence of which is shown in SEQ ID NO: 3, as well as nucleic acids that have at least 85% identity (e.g., 85%, 86%, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% or greater identity) to the nucleic acid sequence of SEQ ID NO: 3 and that promote expression of a transgene in a cell (e.g., a eukaryotic cell, such as a mammalian cell, a human cell or a human hepatocyte) when the transgene is operably linked to the enhancer.

[0038] As used herein, the term "conservative mutation", "conservative substitution" or "conservative amino acid substitution" refers to one or more amino acids being substituted with one or more different amino acids exhibiting similar physicochemical properties (e.g., polarity, electrostatic charge, and steric bulk). These properties for each of the 20 natural amino acids are summarized in Table 1 below.

[0039] Table 1. Representative physicochemical properties of natural amino acids

[0040]

[0041]

[0042] Based on A 3 The volume of the meter: 50-100 is small, 100-150 is medium,

[0043] 150-200 is large, and >200 is huge

[0044] It will be appreciated from this table that conservative amino acid families include, for example, (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S, and T; (iv) H, K, and R; (v) N and Q; and (vi) F, Y, and W. Thus, a conservative mutation or substitution is one in which an amino acid is replaced by a member of the same amino acid family (e.g., Ser for Thr or Lys for Arg).

[0045] The term "desmin promoter" as used herein refers to the nucleic acid set forth in SEQ ID NO:7, as well as nucleic acids having at least 85% identity (e.g., 85%, 86%, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% or greater identity) to the nucleic acid sequence of SEQ ID NO:7 and promoting expression of a transgene in a cell (e.g., a eukaryotic cell, such as a mammalian cell, a human cell or a human muscle cell) when the transgene is operably linked to an enhancer.

[0046] As used herein in the context of transcriptional regulatory elements, the term "functional part" refers to the part of the transcriptional ability of the retention stimulation related gene in the target cell of a larger nucleic acid. For example, apolipoprotein E liver control region (ApoE-HCR) is a 774 nucleotide enhancer, and the sequence of the 774 nucleotide enhancers is shown in SEQ ID NO:3. The part (the nucleotide sequence of the part is shown in SEQ ID NO:1) containing 193 nucleotides of this locus can retain the transcriptional activation properties of a larger locus. Therefore, the part containing 193 nucleotides shown in SEQ ID NO:1 is the "functional part" of the ApoE-HCR locus. As another example, another part of the transcriptional activation properties that can retain full-length enhancers of the ApoE-HCR locus is the section containing 320 nucleotides shown in SEQ ID NO:2. Therefore, the part containing 320 nucleotides shown in SEQ ID NO:2 is the "functional part" of the ApoE-HCR locus. As another example, another portion of the ApoE-HCR locus that is capable of retaining the transcriptional activation properties of the full-length enhancer is the 50-nucleotide segment shown in SEQ ID NO: 4. Therefore, the 50-nucleotide portion shown in SEQ ID NO: 4 is also a "functional portion" of the ApoE-HCR locus.

[0047] As used herein, the term "operably connected" refers to a first molecule being linked to a second molecule, wherein the arrangement of the molecules causes the first molecule to affect the function of the second molecule. Two molecules may or may not be a part of a single adjacent molecule and may or may not be adjacent. For example, if a promoter regulates the transcription of a related transcribable polynucleotide molecule in a cell, then the promoter is operably connected to a transcribable polynucleotide molecule. In addition, if the two parts of the transcriptional regulatory element are connected so that the transcriptional activation function of a part is not adversely affected by the presence of another part, then the two parts of the transcriptional regulatory element are operably connected to each other. Two transcriptional regulatory elements can be operably connected to each other by means of a joint nucleic acid (e.g., an intervening non-coding nucleic acid) or can be operably connected to each other in the absence of an intervening nucleotide.

[0048] "Percentage (%) of sequence identity" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are consistent with the nucleic acids or amino acids in a reference polynucleotide or polypeptide sequence after the sequences are aligned and spaces are introduced (if necessary) to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining nucleic acid or amino acid sequence identity percentage can be achieved in a variety of ways within the capabilities of those skilled in the art, such as using publicly available computer software (such as BLAST, BLAST-2 or Megalign software). Those skilled in the art can determine the parameters applicable to the alignment sequence, including any algorithm required for achieving the maximum alignment within the full length of the compared sequence. For example, a sequence identity percentage value can be generated using a sequence comparison computer program BLAST. As an illustration, the percentage of sequence identity of a given nucleic acid or amino acid sequence A to, with or for a given nucleic acid or amino acid sequence B (alternatively expressed as a given nucleic acid or amino acid sequence A with a certain percentage of sequence identity to, with or for a given nucleic acid or amino acid sequence B) is calculated as follows:

[0049] 100 times (fraction X / Y)

[0050] wherein X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in the program's alignment of A and B, and wherein Y is the total number of nucleic acids in B. It will be appreciated that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0051] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic compounds to be administered to a subject (eg, a mammal, such as a human) to prevent, treat, or control a particular disease or disorder that affects or may affect the subject.

[0052] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of subjects (e.g., mammals, such as humans) without excessive toxicity, irritation, allergic response and other problematic complications and are commensurate with a reasonable benefit / risk ratio.

[0053] As used herein, the term "sample" refers to a specimen (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic juice, chorionic villus sample, or cells) isolated from a subject. The subject can be, for example, a patient suffering from a disease described herein (e.g., a lysosomal storage disease, such as Pompe disease).

[0054] As used herein, the phrases "specific binding" and "binding" refer to a binding reaction that determines the presence of a specific molecule (e.g., a polypeptide) in a heterogeneous population of polypeptides and other biomolecules, which is specifically recognized by, for example, a ligand (e.g., an antibody or an antigen-binding fragment thereof). A ligand (e.g., a complementary polynucleotide) that specifically binds to a protein can, for example, bind with a K of less than 100 nM. D For example, a ligand that specifically binds to a protein may have a K of up to 100 nM (e.g., between 1 pM and 100 nM). D A ligand that does not exhibit specific binding to another molecule or a domain of the other molecule may exhibit a K for that particular molecule or domain of the particular molecule greater than 100 nM (e.g., greater than 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 100 μM, 500 μM, or 1 mM). D A variety of assay formats can be used to determine the affinity of a ligand for a particular protein. For example, solid phase ELISA assays are often used to identify ligands that specifically bind to a target protein. For descriptions of assay formats and conditions that can be used to determine binding of a specific protein, see, for example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999).

[0055] As used herein, the terms "subject" and "patient" refer to an organism that is treated for a particular disease or disorder as described herein (e.g., a lysosomal storage disorder, such as Pompe disease). Examples of subjects and patients include mammals, such as humans, that are treated for a disease or disorder as described herein.

[0056] The term "synapsin promoter" as used herein refers to the nucleic acid set forth in SEQ ID NO:8, and variants of the nucleic acid set forth in SEQ ID NO:8 that have at least 85% identity (e.g., 85%, 86%, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% or greater identity) to the nucleic acid sequence of SEQ ID NO:8 and that promote expression of a transgene in a cell (e.g., a eukaryotic cell, such as a mammalian cell, a human cell, or a human neuron) when the transgene is operably linked to an enhancer.

[0057] As used herein, the term "transcriptional regulatory element" refers to a nucleic acid that at least partially controls the transcription of a related gene. A transcriptional regulatory element may include a promoter, enhancer, and other nucleic acids (e.g., a polyadenylation signal) that control or help control gene transcription. Examples of transcriptional regulatory elements are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).

[0058] As used herein, the term "treat" or "treatment" refers to therapeutic treatment, wherein the object is to prevent or slow (lessen) an undesirable physiological change or condition, such as, inter alia, the progression of a lysosomal storage disorder (e.g., Pompe disease). Beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, reduction in extent of disease, a stable (i.e., non-worsening) disease state, a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In the context of a lysosomal storage disease (e.g., Pompe disease), treatment of a patient can be manifested in one or more detectable changes, such as an increase in the concentration of acid alpha-glucosidase (GAA) protein or a nucleic acid (e.g., DNA or RNA, such as mRNA) encoding GAA, or an increase in GAA activity (e.g., an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 200%, , 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 500 times, 1,000 times or more. The concentration of GAA protein can be determined using protein detection assays known in the art (including ELISA assays described herein). The concentration of nucleic acids encoding GAA can be determined using nucleic acid detection assays described herein (e.g., RNA Seq assays). Exemplary protocols for detecting GAA proteins and nucleic acids are provided in Example 1 below. In addition, treatment of patients with lysosomal storage diseases (e.g., Pompe disease) can be manifested in improvements in the patient's muscle function (e.g., cardiac or skeletal muscle function) and improvements in muscle coordination. Exemplary procedures for measuring muscle function are described in Example 1 below.

[0059] The term "vector" as used herein refers to a nucleic acid, such as DNA or RNA, that can be used as a medium for delivering a gene of interest to a cell (e.g., a mammalian cell, such as a human cell), for example, for replication and / or expression purposes. Exemplary vectors that can be used in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of the expression vectors are disclosed in, for example, WO 1994 / 11026, the disclosure of which is incorporated herein by reference. The expression vectors described herein contain polynucleotide sequences and other sequence elements, such as for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express transgenics described herein include plasmids containing regulatory sequences (e.g., promoter regions and enhancer regions) that direct gene transcription. Other vectors that can be used to express transgenics contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA derived from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of the genes carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is a diagram showing the arrangement of elements in each vector. GAAco, human codon-optimized acid α-glucosidase gene (GAA); ITR, inverted terminal repeat; SD, splice donor; SA, splice acceptor.

[0061] Figure 2 It is displayed for 1×10 13 Graph of GAA activity assessed one month post-dosing in quadriceps muscle tissue of male (black dots) and female (grey triangles) mice treated with control and vehicle treated mice vg / kg.

[0062] Figure 3 It is displayed for 1×10 13Figure 3. Plot of hGAA transcripts measured by RNA-seq analysis in liver tissue of male (dots) and female (triangles) mice treated with control and vehicle at 400 μg / kg. The horizontal line indicates the median expression level of endogenous murine GAA.

[0063] Figure 4 It is displayed for 1×10 13 Graph of GAA activity assessed one month after dosing in liver tissues of male (black dots) and female (grey triangles) mice treated with control and vehicle treated mice at 400 vg / kg.

[0064] Figure 5A and Figure 5B It is displayed for 1×10 13 vg / kg( Figure 5A ) or 3×10 13 vg / kg( Figure 5B ) was administered to control-treated mice and vehicle-treated mice, with anti-GAA antibody levels assessed one month after administration in the sera of male mice (dots) and female mice (triangles).

[0065] Fig. 6A and Figure 6B It is displayed for 1×10 13 vg / kg( Fig. 6A ) or 3×10 13 vg / kg( Figure 6B ) is a graph of GAA activity assessed one month after dosing in the serum of male mice (dots) and female mice (triangles) treated with control and vehicle-treated mice.

[0066] Fig. 7A and Figure 7B It is displayed for 1×10 13 vg / kg( Fig. 7A ) or 3×10 13 vg / kg( Figure 7B Figure 5 shows the pathological scores (reported in Table 5) of sectioned quadriceps muscle tissues from male (dots) and female (triangles) mice, control-treated mice and vehicle-treated mice, evaluated one month after dosing.

[0067] Figure 8 Images of H&E and PAS-stained sections of representative mice are shown and corresponding measurements are shown, indicating elevated GAA activity, decreased glycogen levels, and repair of muscle pathology in mice dosed with vectors containing the hGAA transgene directed by a hybrid promoter and a liver-directed promoter. 13Serum GAA activity and quadriceps GAA activity and glycogen accumulation in quadriceps muscle of representative control treated mice and vehicle treated mice dosed with vg / kg. Glycogen accumulation scores were evaluated in tissue sections of isopentane frozen quadriceps muscle sectioned according to standard procedures and stained with H&E and PAS.

[0068] Fig. 9 It is shown that for 3×10 13 Figure 3. Graph of GAA activity assessed one month after dosing in the spinal cords of male (black dots) and female (grey triangles) mice dosed with vg / kg control-treated and vehicle-treated mice.

[0069] Fig.10 It is displayed for 1×10 13 Figure quantification of hGAA transcripts measured by RNA-seq analysis in spinal cord tissue of male (dots) and female (triangles) mice treated with control and vehicle at 50 vg / kg doses. The horizontal line indicates the median expression level of endogenous murine GAA.

[0070] Fig.11 is a graph showing the ratio of RNA expression levels in the liver and spinal cord one month after administration determined by RNA-Seq analysis divided by the average vector copy number (VCN) in liver or brain tissue to estimate the expression level per vector in liver or CNS tissue, respectively. DETAILED DESCRIPTION

[0071] Described herein are transcriptional regulatory elements that stimulate transcription of relevant genes, such as genes encoding lysosomal enzymes, such as acid alpha-glucosidase (GAA), in cells of certain tissues. Specifically, the transcriptional regulatory elements described herein can promote expression of target lysosomal enzyme genes in tissues affected by lysosomal storage diseases, such as Pompe disease. The tissues include muscle tissues, such as cardiac muscle tissue and skeletal muscle tissue, and central nervous system tissues. The nucleic acid regulatory elements described herein can be operably linked to a transgene, such as GAA, and incorporated into a vehicle for administration to a patient, such as a human patient, to treat a lysosomal storage disease, such as Pompe disease. The delivery vehicle can be a vector, such as a viral vector described herein, or other agents that introduce nucleic acids into relevant cells, such as liposomes, vesicles, exosomes, dendrimers, or nanoparticles described herein.

[0072] The present invention is based in part on the discovery that nucleic acid regulatory elements can be used to (i) promote expression of transgenes in cells affected by lysosomal storage diseases to improve pathology and (ii) stimulate expression in the liver, which is used to promote immune tolerance. Therefore, the compositions and methods described herein can be used to treat lysosomal storage diseases, such as Pompe disease, to treat the deleterious effects on muscle tissue of lysosomal enzyme deficiency (e.g., GAA deficiency), while preventing or reducing the immune response to the introduced enzyme (e.g., GAA).

[0073] The following section provides a description of transcriptional regulatory elements that exhibit the aforementioned advantageous properties. The following section also describes various transgenes, viral vectors and transfection agents that can be used in conjunction with the transcriptional regulatory elements described herein, as well as methods of treating various disorders using the compositions described herein.

[0074] Transcriptional regulatory elements

[0075] The transcriptional regulatory element that can be used in combination with the compositions and methods described herein can contain multiple parts that are operably connected to each other. For example, the transcriptional regulatory element described herein can contain apolipoprotein E liver control region (ApoE-HCR) or its functional part as shown in SEQ ID NO:3. The exemplary functional part of ApoE-HCR is Dang et al., J.Biol.Chem.270:22577-22585 (1995) described in SEQ ID NO:2 shown in the part containing 320 nucleotides, and the document relates to the disclosure of ApoE-HCR locus and its functional part and is incorporated herein by reference. Another example of ApoE-HCR nucleic acid that can be used in combination with the compositions and methods described herein is ApoE-HCR containing 193 nucleotides of the section, and the nucleic acid sequence of the section containing 193 nucleotides is shown in SEQ ID NO:1. Another example of ApoE-HCR nucleic acid that can be used in combination with the compositions and methods described herein is SEQ ID NO:4 shown in the section containing 50 nucleotides. Other nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater sequence identity) relative to the above nucleic acid sequences.

[0076] Additionally or alternatively, the transcriptional regulatory elements described herein may contain a desmin promoter or a functional portion thereof. For example, a regulatory element may contain a desmin promoter containing nucleic acid -984 to -644 of the desmin transcription start site of a human desmin locus. The nucleic acid sequence of this construct is shown in SEQ ID NO:5. A regulatory element may contain a desmin promoter containing nucleic acid -269 to +76 of the desmin transcription start site of a human desmin locus. The nucleic acid sequence of this construct is shown in SEQ ID NO:6. A regulatory element may contain the nucleic acid of SEQ ID NO:5 fused to the nucleic acid of SEQ ID NO:6 without intervening nucleic acid to form a desmin promoter containing nucleotides -984 to nucleotide -644 and nucleotides -269 to nucleotide +76 of the desmin transcription start site of a human desmin locus. The nucleic acid sequence of this regulatory element is shown in SEQ ID NO:7. Other nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater sequence identity) relative to the above nucleic acid sequences.

[0077] Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include promoters that stimulate expression of a transgene operably linked to the promoter in cells of the central nervous system, such as neurons, glial cells, or astrocytes. Examples of such promoters are the synapsin promoter, the glial fibrillary acidic protein (GFAP) promoter, the calcium / calmodulin-dependent protein kinase III promoter, the tubulin αI promoter, the tubulin-associated protein IB (MAP IB) promoter, neuron-specific enolase promoter, platelet-derived growth factor β chain promoter, neurofilament light chain promoter, neuron-specific VGF gene promoter, neuronal nuclear (NeuN) promoter, adenomatous polyposis coli (APC) promoter, ionized calcium binding adapter molecule 1 (Iba-1) and homeobox protein 9 (HB9) promoter, or a variant of the promoter (e.g., a variant having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater sequence identity) to the nucleic acid sequence of the wild-type promoter locus and capable of stimulating transcription of a transgene operably linked to it upon introduction into central nervous system cells) or a functional portion thereof.

[0078] For example, the transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein can contain the synapsin promoter or a functional portion thereof. An exemplary regulatory element containing the synapsin promoter region is shown in SEQ ID NO: 8. This construct contains nucleotides -465 to -90 of the human synapsin locus relative to the synapsin transcription start site. Other nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater sequence identity) relative to this nucleic acid sequence.

[0079] The aforementioned nucleic acid regulatory elements are summarized in Table 2 below.

[0080] Table 2. Exemplary nucleic acid regulatory elements

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] In addition to the above-mentioned regulatory elements, regulatory elements as described herein include those regulatory elements formed by combining ApoE-HCR elements or its functional part and desmin promoter and / or synaptic protein promoter or its functional part.For example, the regulatory elements that can be used in combination with compositions and methods as described herein include those regulatory elements containing the SEQ ID NO:1 containing the ApoE-HCR shown in the section containing 193 nucleotides that can be operably connected to desmin promoter or its functional part.The functional part of desmin promoter can be, for example, a nucleic acid spanning nucleotides -984 to -644 relative to desmin transcription start site or a nucleic acid spanning nucleotides -269 to +76 relative to desmin transcription start site.In some embodiments, ApoE-HCR elements or its functional part are operably connected to desmin promoter, and the desmin promoter contains nucleotides -984 to nucleotides -644 and nucleotides -269 to nucleotides +76 relative to desmin transcription start site of people's desmin locus. The transcriptional regulatory elements described herein may also include a combination of the synapsin promoter or a functional portion thereof with ApoE-HCR and / or desmin regulatory elements.

[0087] Table 3 below shows exemplary combinations of transcriptional regulatory elements.

[0088] Table 3. Combination of transcriptional regulatory elements

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] Other nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or greater sequence identity) relative to the nucleic acid sequences shown in Table 3.

[0095] Methods for delivering exogenous nucleic acids to target cells

[0096] Transfection technology

[0097] Techniques that can be used to introduce transgenes (e.g., transgenes operably linked to transcriptional regulatory elements described herein) into target cells are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the relevant cells. Mammalian cells (e.g., human cells) subjected to an external electric field in this way are subsequently susceptible to uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. Similar techniques Nucleofection TM The use of an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. TM The techniques and protocols that can be used to perform this technique are described in detail in, for example, Distler et al., Experimental Dermatology 14:315 (2005) and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0098] Other techniques that can be used to transfect target cells include extrusion perforation methods. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied stress. The advantage of this technique is that a vector is not necessary for delivering nucleic acids to cells (e.g., human target cells). Extrusion perforation is described in detail, for example, in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0099] Lipofection represents another technique that can be used to transfect target cells. This method involves loading nucleic acid into liposomes, which generally have cationic functional groups (e.g., quaternary amines or protonated amines) toward the outside of the liposomes. This promotes electrostatic interactions between liposomes and cells due to the anionic nature of the cell membrane, ultimately causing the uptake of exogenous nucleic acids, such as by direct fusion of liposomes with cell membranes or by endocytosis of complexes. Lipofection is described in detail in, for example, U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques for causing exogenous nucleic acid uptake using ionic interactions with cell membranes include contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that facilitates interaction with cell membranes are activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, e.g., in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner because this method utilizes an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, e.g., in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0100] Another tool that can be used to induce uptake of exogenous nucleic acids by target cells is laser transfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0101] Microvesicles represent another potential medium that can be used to modify the target cell genome according to the methods described herein. For example, microvesicles caused by co-expression of glycoprotein VSV-G and, for example, genome modification proteins (e.g., nucleases) can be used to effectively deliver proteins to cells, followed by catalysis of site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of cells for covalent inclusion of related polynucleotides (e.g., genes or regulatory sequences). The use of such vesicles (also referred to as Gesicles) for genetic modification of eukaryotic cells is described in detail in, for example, Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract]. Methylation changes in early embryonic genes in cancer [Abstract], Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, Abstract No. 122.

[0102] Incorporation of target genes through gene editing technology

[0103] In addition to the above tools, a variety of tools that can be used to include related genes in target cells (such as human cells) have been developed. A kind of such method that can be used to include polynucleotides encoding target genes in target cells involves the use of transposons. Transposons are polynucleotides that encode transposases and contain related polynucleotide sequences or genes that are 5' and 3' excision sites on both sides. Once the transposon is delivered to the cell, the expression of the transposase gene immediately begins and produces an active enzyme from the transposon cracking related genes. This activity is mediated by the site-specific recognition of the transposase excision site of the transposon. In some cases, these excision sites can be terminal repeats or reverse terminal repeats. Once excised from the transposon, the related gene can be immediately integrated into the genome of the mammalian cell by the cleavage catalyzed by the transposase of the similar excision site present in the nuclear genome. This allows the related gene to be inserted into the cracked nuclear DNA at the complementary excision site, and the subsequent covalent connection of the phosphodiester bond of the DNA linked to the mammalian cell genome completes the inclusion process. In some cases, the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA before being incorporated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail, e.g., WO 2010 / 085699) and the sleeping beauty transposon (described in detail, e.g., US 2005 / 0112764), each of which is incorporated herein by reference for its disclosure of the use of transposons in gene delivery to relevant cells.

[0104] Another tool for integrating target genes into the target cell genome is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, which originally evolved as an adaptive defense mechanism for bacteria and archaea to resist viral infection. The CRISPR / Cas system includes palindromic repeat sequences and related Cas9 nucleases in plasmid DNA. This DNA and protein ensemble guides site-specific DNA cleavage of target sequences by first incorporating exogenous DNA into the CRISPR locus. The polynucleotides containing these exogenous sequences and the repeating spacer elements of the CRISPR locus are then transcribed in the host cell to produce guide RNAs, which can then anneal to the target sequence and localize the Cas9 nuclease to this site. In this way, highly site-specific cas9-mediated DNA cleavage can be caused in exogenous polynucleotides, because the interaction that brings cas9 close to the target DNA molecule is controlled by RNA:DNA hybridization. Therefore, the CRISPR / Cas system can be designed to cleave any relevant target DNA molecule. This technology has been used to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an effective way to site-specifically edit the genome of a target cell to cleave DNA before incorporating the gene encoding the target gene. The use of CRISPR / Cas to regulate gene expression has been described, for example, in U.S. Patent No. 8,697,359, which is incorporated herein by reference for disclosures relating to genome editing using the CRISPR / Cas system. Alternative methods for site-specific cleavage of genomic DNA prior to incorporating the gene of interest into the target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain guide polynucleotides for localization to a specific target sequence. Target specificity is controlled by the DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al., Nature Reviews Genetics 11:636 (2010); and Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), each of which is incorporated herein by reference for its disclosure of compositions and methods for genome editing.

[0105] Other genome editing techniques that can be used to incorporate a polynucleotide encoding a target gene into the genome of a target cell include the use of ARCUS, which can be rationally designed to site-specifically cleave genomic DNA. TMMeganuclease. According to the defined structure-activity relationship established for such enzymes, it is advantageous to use these enzymes to include genes encoding target genes into the genome of mammalian cells. Single-chain meganucleases can be modified at certain amino acid positions to produce nucleases that selectively cleave DNA at the desired position, so that the target gene site is specifically incorporated into the nuclear DNA of the target cell. These single-chain nucleases have been widely described in, for example, U.S. Patent Nos. 8,021,867 and US 8,445,251, each of which is incorporated herein by reference for the disclosure of compositions and methods for genome editing.

[0106] Vectors for delivering exogenous nucleic acids to target cells

[0107] Viral vectors for nucleic acid delivery

[0108] Viral genomes provide a rich source of vectors that can be used to effectively deliver relevant genes to the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained in such genomes are typically incorporated into the genome of target cells by generalized or specialized transduction. These processes are performed as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include AAV, retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses that can be used to deliver polynucleotides encoding antibody light chains and heavy chains of the present invention or antibody fragments include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus. Examples of retroviruses include: avian leukosis sarcoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV group, slow virus, foamy virus (Coffin, JM, Retroviridae:The viruses and their replication, Fundamental Virology, 3rd edition, BNFields et al., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, which is incorporated herein by reference for its disclosure of viral vectors for gene therapy.

[0109] AAV vectors for nucleic acid delivery

[0110] In some embodiments, the nucleic acid of the compositions and methods described herein is incorporated into rAAV vectors and / or viral particles to facilitate the introduction of the nucleic acid into cells. The rAAV vectors that can be used in the present invention are recombinant nucleic acid constructs that include (1) a transgene to be expressed (e.g., a polynucleotide encoding a GAA protein) and (2) a viral nucleic acid that promotes heterologous gene integration and expression. The viral nucleic acid may include those sequences of AAV that are required for DNA cis replication and packaging (e.g., functional ITRs) into viral particles. In a typical application, the transgene encodes GAA, which can be used to correct GAA deficiency in patients with lysosomal storage diseases (e.g., Pompe disease). Such rAAV vectors may also contain markers or reporter genes. Useful rAAV vectors have one or more AAV WT genes that are deleted in whole or in part, but retain functional flanking ITR sequences. AAVITRs may have any serotype suitable for a particular application (e.g., derived from serotype 2). Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000) and Monahan and Samulski, Gene Delivery 7:24-30 (2000), each of which is incorporated herein by reference for its disclosure relating to AAV vectors for gene delivery.

[0111] The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate the introduction of nucleic acids or vectors into cells. The capsid protein of AAV constitutes the external non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins VP1, VP2, and VP3 required for the assembly of virions. The construction of rAAV virions has been described in, for example, U.S. Patent Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; and Rabinowitz et al., J. Virol. 76: 791-801 (2002) and Bowles et al., J. Virol. 77: 423-432 (2003), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

[0112] rAAV virions that can be used in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes, including AAV 1, AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, and AAV 9. For targeting muscle cells, rAAV virions comprising at least one serotype 1 capsid protein can be particularly useful. rAAV virions comprising at least one serotype 6 capsid protein can also be particularly useful because the structure of the serotype 6 capsid protein is similar to that of the serotype 1 capsid protein, and therefore it is expected that GAA is also highly expressed in muscle cells. rAAV serotype 9 has also been found to be an effective transducer of muscle cells. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol. 72:2224-2232 (1998); Halbert et al., J. Virol. 74:1524-1532 (2000); Halbert et al., J. Virol. 75:6615-6624 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

[0113] Pseudotyped rAAV vectors can also be used in combination with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) that are pseudotyped because the capsid gene is derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV8 vector or an AAV9 vector encoding a therapeutic protein that is pseudotyped because the capsid gene is derived from AAV serotype 2. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001).

[0114] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations that promote AAV targeting to specific cell types. The construction and characterization of AAV capsid mutants (including insertion mutants, alanine screening mutants, and epitope tag mutants) are described in Wu et al., J. Virol. 74: 8635-45 (2000). Other rAAV virions that can be used in the methods of the present invention include those capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25: 436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19: 423-428 (2001).

[0115] Treatment

[0116] Pompe disease

[0117] Pompe disease (also known as glycogen storage disease type II or GSD II) is caused by a deficiency of the lysosomal enzyme GAA. The disease is an inborn error of metabolism in which GAA deficiency ultimately leads to glycogen accumulation in all tissues, especially skeletal muscle cells. In addition, the role of glycogen accumulation within the central nervous system and the effects of glycogen accumulation on skeletal muscle function have been documented.

[0118] Three clinical forms of this disease are known: pediatric Pompe disease, juvenile Pompe disease, and adult Pompe disease. Pediatric Pompe disease develops soon after birth and presents with progressive muscle weakness and heart failure. The pediatric form of Pompe disease is also characterized by the rapid development of cardiomyopathy, and patients typically show myopathy and neuropathy, leading to death usually in the first year of life. Symptoms in adult and adolescent patients appear later in life and primarily involve skeletal muscle and neurons. Patients who exhibit this form of Pompe disease eventually die of respiratory insufficiency. Patients may survive abnormally for more than 60 years. The severity of the disease is associated with residual acid alpha-glucosidase activity, which is 10-20% of normal activity in the late onset form of the disease and less than 2% of normal activity in the early onset form of the disease.

[0119] Human acid α-glucosidase

[0120] The amino acid sequence of wild-type GAA is shown below in SEQ ID NO: 14:

[0121]

[0122] Exemplary genes encoding GAA polypeptides that can be used in conjunction with the compositions and methods described herein include genes encoding the wild-type GAA protein set forth in SEQ ID NO: 14 and functional GAA enzymes having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identity) to the amino acid sequence of SEQ ID NO: 14. Genes encoding GAA polypeptides that can be used in conjunction with the compositions and methods described herein further include those having one or more amino acid substitutions, such as those having one or more conservative amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 14. For example, GAA polypeptides useful in conjunction with the compositions and methods described herein include those having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO:14.

[0123] Transcriptional regulatory elements as described herein can be operably linked to transgenes, such as GAA, which is lacking in patients with lysosomal storage diseases (e.g., those suffering from Pompe disease). Constructs containing lysosomal enzymes under the transcriptional control of regulatory elements as described herein can be incorporated into vectors (or other transfection agents as described herein) and applied to patients to treat lysosomal storage diseases. Advantageously, transcriptional regulatory elements as described herein can promote the transcription of genes encoding lacking lysosomal enzymes (e.g., GAA) in those cells (e.g., muscle cells and central nervous system cells) attacked by the disease. In addition, regulatory elements as described herein bring the additional benefit of reducing or eliminating immune responses, which may originally be accompanied by the introduction of genes encoding enzymes that patients lack. The advantageous properties of transcriptional regulatory elements as described herein are further reported in detail in Example 1 below.

[0124] Pharmaceutical compositions, routes of administration and unit doses

[0125] The transcriptional regulatory elements described herein can be operably linked to a transgene, such as a lysosomal enzyme, such as GAA, and incorporated into a vehicle for administration to a patient, such as a human patient with a lysosomal storage disease, such as Pompe disease. Pharmaceutical compositions containing vectors (e.g., viral vectors) containing the transcriptional regulatory elements described herein operably linked to a therapeutic transgene can be prepared using methods known in the art. For example, such compositions can be prepared using, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Science 16th edition, Osol, A. ed. (1980); incorporated herein by reference) and in a desired form, such as a lyophilized formulation or an aqueous solution.

[0126] Viral vectors (e.g., AAV vectors and other vectors described herein) containing transcriptional regulatory elements that can be operably connected to therapeutic transgenes can be administered to patients (e.g., human patients) by a variety of administration routes. The administration route can vary with, for example, the onset and severity of the disease, and can include, for example, intradermal administration, transdermal administration, parenteral administration, intravenous administration, intramuscular administration, intranasal administration, subcutaneous administration, transdermal administration, intratracheal administration, intraperitoneal administration, intraarterial administration, intravascular administration, inhalation, perfusion, lavage, and oral administration. Intravascular administration includes delivery to the patient's vascular system. In some embodiments, administration is administered to a blood vessel that is considered a vein (intravenous), and in some administrations, administration is administered to a blood vessel that is considered an artery (intraarterial). Veins include, but are not limited to, internal jugular veins, peripheral veins, coronary veins, hepatic veins, portal veins, great saphenous veins, pulmonary veins, superior vena cava, inferior vena cava, gastric veins, splenic veins, inferior mesenteric veins, superior mesenteric veins, cephalic veins, and / or femoral veins. Arteries include, but are not limited to, coronary arteries, pulmonary arteries, brachial arteries, internal carotid arteries, aortic arches, femoral arteries, peripheral arteries, and / or ciliary arteries. It is contemplated that delivery may be through or to arterioles or capillaries.

[0127] Treatment regimens may vary and generally depend on the severity of the disease and the age, weight, and sex of the patient. Treatment may include administration of vectors (e.g., viral vectors) or other agents described herein that can be used to introduce relevant genes into target cells at different unit doses. Each unit dose will generally contain a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route of administration and formulation are within the skill of those skilled in the clinical field. The unit dose need not be administered as a single injection, but may include continuous infusions over a set time period. The unit dose of the viral vectors described herein can be conveniently described in terms of plaque forming units (pfu) of the viral construct. The unit dose may be, for example, between 10 3 pfu, 10 4 pfu, 10 5 pfu, 10 6pfu, 10 7 pfu, 10 8 pfu, 10 9 pfu, 10 10 pfu, 10 11 pfu, 10 12 pfu to 10 13 Additionally or alternatively, depending on the type of virus and the titer that can be achieved, 1 to 100, 10 to 50, 100-1,000, or up to about or at least about 1×10 4 pcs, 1×10 5 pcs, 1×10 6 pcs, 1×10 7 pcs, 1×10 8 pcs, 1×10 9 pcs, 1×10 10 pcs, 1×10 11 pcs, 1×10 12 pcs, 1×10 13 pcs, 1×10 14 or 1×10 15 or more infectious virus particles (vp), including all values ​​and ranges therebetween.

[0128] The mixture of nucleic acid and viral vector described herein can be prepared in water appropriately mixed with one or more excipients, carriers or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oil. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the preparation can be sterile and can be fluid to the extent that there is easy injectability. The preparation can be stable under manufacturing and storage conditions and can be preserved to resist the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and / or vegetable oils. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0129] For example, if necessary, the solution containing the pharmaceutical composition described herein can be appropriately buffered, and the liquid diluent can be first made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous administration, intramuscular administration, subcutaneous administration, and intraperitoneal administration. In this regard, according to the present disclosure, the sterile aqueous medium that can be adopted will be known to those skilled in the art. For example, a dose can be dissolved in 1ml isotonic NaCl solution and added to 1000ml subcutaneous infusion fluid or injected at the proposed infusion site. Depending on the disease of the treated subject, a certain dose change will necessarily occur. In either case, the person responsible for administration will determine the dosage applicable to individual subjects. In addition, for human administration, the preparation can meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biologics standards.

[0130] Example

[0131] The following examples are put forth so as to provide one of ordinary skill in the art with a description of how to use, prepare, and evaluate the compositions and methods described herein and are intended solely as exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0132] Example 1. Establishing Optimal Tissue Expression for Acid Alpha-Glucosidase Gene Delivery in Pompe Disease Patients

[0133] Target

[0134] Several adeno-associated virus (AAV) vectors were designed to target expression in different tissues (e.g., muscle and liver) within a range of doses in a mouse model of Pompe disease. Results were compared after systemic administration of 6 adeno-associated virus (AAV) vectors in a mouse model of Pompe disease, which were designed to direct expression of the human acid alpha-glucosidase gene (hGAA) to different tissues and / or combinations of tissues within a range of doses. The vector with the best multi-tissue expression profile was selected to translate to a clinical trial of systemic administration of AAV-GAA to treat Pompe disease.

[0135] In this study, vector dose and target tissue expression profiles were examined to determine how they affect multiple endpoints relevant to Pompe disease in patients in this model, including respiratory, cardiac, and skeletal muscle function as well as GAA activity and glycogen accumulation in tissues. The findings described herein support clinical translation of optimized hGAA vectors for AAV gene therapy.

[0136] Materials and methods

[0137] Vectors and vector manufacturing

[0138] Vector 2 (AAV9-Des-hGAAco), vector 3 (AAV8-LDes-hGAAco), vector 4 (AAV8-LNDes-hGAAco), vector 5 (AAV8-LDes2-hGAAco) and vector 6 (AAV8-Des3-hGAAco) were cloned by standard molecular biology techniques and transiently transfected into mammalian cells based on the 2 plasmids to produce them in a scaled production method. The genomic titer of each vector was determined by ddPCR. The candidate vectors are summarized in Table 4 below. Figure 1 Schematic diagram showing the general organization of the inverted terminal repeat (ITR) regions of the constructs.

[0139] Table 4. Candidate vectors

[0140]

[0141]

[0142] *To normalize for strong AAV8 liver tropism and expression, vector 1 was administered at a 3-fold lower dose than vectors 2-6.

[0143] Transcriptional regulatory elements

[0144] The nucleic acid sequence of the transcriptional regulatory element operably linked to the GAA transgene in vector No. 1 contains a 193-nucleotide segment of ApoE-HCR from 5' to 3' (shown in SEQ ID NO: 1), which is operably linked to a human alpha-1 antitrypsin promoter (shown in SEQ ID NO: 13 below). The nucleic acid sequence of the combined ApoE-HCR / human alpha-1 antitrypsin regulatory element used in this construct is shown in SEQ ID NO: 9.

[0145]

[0146] The nucleic acid sequence of the transcriptional regulatory element operably linked to the GAA transgene in vector No. 2 is a shortened desmin promoter. This shortened desmin promoter contains a 5' region having a nucleic acid sequence of SEQ ID NO: 5 (containing nucleotides -984 to -644 relative to the desmin transcription start site), which is fused to a 3' region having a nucleic acid sequence of SEQ ID NO: 6 (containing nucleotides -269 to +70 relative to the desmin transcription start site). The nucleic acid sequence of the transcriptional regulatory element used in this construct is shown in SEQ ID NO: 7.

[0147] The nucleic acid sequence of the transcriptional regulatory element of the GAA transgenic operably connected to the vector number 3 contains a 193-nucleotide segment (shown in SEQ ID NO:1) of ApoE-HCR from 5' to 3', and the segment containing 193 nucleotides is operably connected to a shortened desmin promoter. This shortened desmin promoter contains a 5' region with a nucleic acid sequence of SEQ ID NO:5 (containing nucleotides -984 to -644 relative to the desmin transcription start site), and the 5' region is fused to a 3' region with a nucleic acid sequence of SEQ ID NO:6 (containing nucleotides -269 to +70 relative to the desmin transcription start site). The nucleic acid sequence of the combined desmin promoter used in this construct is shown in SEQ ID NO:7. The nucleic acid sequence of the combined ApoE-HCR / desmin transcriptional regulatory element used in this construct is shown in SEQ ID NO:10.

[0148] The nucleic acid sequence of the transcriptional regulatory element of the GAA transgene operably connected to the vector number 4 contains a synapsin promoter from 5' to 3', and the synapsin promoter is operably connected to a segment containing 193 nucleotides of ApoE-HCR, and the segment containing 193 nucleotides is operably connected to a shortened desmin promoter. The nucleic acid sequence of the synapsin promoter used in this construct is shown in SEQ ID NO:8 (containing nucleotides -465 to -90 relative to the synapsin transcription start site). The nucleic acid sequence of the segment of the ApoE-HCR region used in this construct is shown in SEQ ID NO:1. The shortened desmin promoter used in this construct contains a 5' region with a nucleic acid sequence of SEQ ID NO:5 (containing nucleotides -984 to -644 relative to the desmin transcription start site), and the 5' region is fused to a 3' region with a nucleic acid sequence of SEQ ID NO:6 (containing nucleotides -269 to +70 relative to the desmin transcription start site). The nucleic acid sequence of the combined desmin promoter used in this construct is shown in SEQ ID NO:7. The nucleic acid sequence of the combined synapsin / ApoE-HCR / desmin transcriptional regulatory elements used in this construct is shown in SEQ ID NO:11.

[0149] The nucleic acid sequence of the transcriptional regulatory element of the GAA transgenic operably connected to the vector number 5 contains a 50-nucleotide segment (shown in SEQ ID NO:4) of ApoE-HCR from 5' to 3', and the segment containing 50 nucleotides is operably connected to the shortened desmin promoter. This shortened desmin promoter contains a 5' region with a nucleic acid sequence of SEQ ID NO:5 (containing nucleotides -984 to -644 relative to the desmin transcription start site), and the 5' region is fused to a 3' region with a nucleic acid sequence of SEQ ID NO:6 (containing nucleotides -269 to +70 relative to the desmin transcription start site). The nucleic acid sequence of the combined desmin promoter used in this construct is shown in SEQ ID NO:7. The nucleic acid sequence of the combined ApoE-HCR / desmin transcriptional regulatory element used in this construct is shown in SEQ ID NO:12.

[0150] The nucleic acid sequence of the transcriptional regulatory element operably linked to the GAA transgene in vector No. 6 is a shortened desmin promoter. This shortened desmin promoter contains a 5' region having a nucleic acid sequence of SEQ ID NO: 5 (containing nucleotides -984 to -644 relative to the desmin transcription start site), which is fused to a 3' region having a nucleic acid sequence of SEQ ID NO: 6 (containing nucleotides -269 to +70 relative to the desmin transcription start site). The nucleic acid sequence of the transcriptional regulatory element used in this construct is shown in SEQ ID NO: 7.

[0151] In vivo studies

[0152] All animal procedures were approved by the Institutional Animal Care and Use Committee of Jackson Laboratories, Bar Harbor, ME and performed at Jackson Laboratories, Bar Harbor, ME. B6.129-Gaa was obtained from Jackson Laboratories wt Mice and B6.129-Gaa tm1Rabn Homozygous mutant mice. AAV batches were prepared for injection by dilution in vehicle and after weighing the mice, 3 × 10 12 vg / kg, 1×10 13 vg / kg, 3×10 13 vg / kg or 1×10 14vg / kg was administered by a single intravenous administration of a carrier or vehicle. Clinical observations and mortality observations were performed daily from administration to the end of the study. At 4 weeks or 12 weeks after administration, mice were killed and autopsied. The harvested tissues were processed and sent to a third-party contract research organization for subsequent analysis.

[0153] GAA activity

[0154] The activity of alpha-glucosidase (GAA) was evaluated in mouse tissues (e.g., liver, heart, brain, and spine) and serum. Serum or tissue homogenate was cultivated with the fluorescent substrate 4-methylumbelliferyl α-D-glucose pyranoside (4-MUG), which was hydrolyzed by GAA to produce the hydrolyzate 4-methylumbelliferyl α-D-glucose pyranoside (4-MU). 4-MU was detected with a fluorescence plate reader, and the enzyme activity in the sample was quantified using a 4-MU standard curve.

[0155] Anti-GAA antibody analysis

[0156] Maxisorp 96-well plates (Thermo Fisher Scientific) were coated with recombinant hGAA protein (R&D Systems). After blocking, plasma samples diluted 1:200 were added to the plates and incubated at 37°C for 1 hr. Anti-mouse secondary antibodies conjugated to HRP were used for detection. Then, fluorescent substrates were added to the wells and light intensity was evaluated on a plate reader.

[0157] RNA-Seq analysis

[0158] Total RNA was isolated from tissues and sequencing libraries were prepared using standard Illumina strand-specific protocols and poly A selection. Libraries were indexed for each sample, pooled by tissue type, and sequenced across 4 lanes (2×150 bp reads) on an Illumina HiSeq. Adapter sequences were first trimmed from FASTQ files using Skewer, then transcript abundance including hGAA was quantified from trimmed FASTQ files using Salmon (accounting for GC bias and strand information), and transcript counts were finally normalized for library size using the DESeq2 package from Bioconductor.

[0159] Pathological evaluation

[0160] The isopentane frozen tissue was sliced ​​and stained with H&E and PAS according to standard procedures. The sections were then analyzed by a trained pathologist in a blinded manner and scored according to the following convention (see also Table 5): 1. Normal; 2. Large and small glycogen aggregates were visible in 10-49% of the fibers; 3. Large and small glycogen aggregates were visible in 50-90% of the fibers; 4. Small glycogen aggregates were visible in >90% of the fibers, and large glycogen aggregates were only visible in a few fibers; 5. Small glycogen aggregates were visible in >90% of the fibers, and large glycogen aggregates were visible in >30% of the fibers.

[0161] result

[0162] A hybrid promoter tunes the balance of GAA expression in liver and muscle

[0163] GAA activity was evaluated in muscle and liver tissue. 13 GAA activity was evaluated in quadriceps muscle tissue of male (black dots) and female (grey triangles) control-treated and vehicle-treated mice dosed with 100 vg / kg of dapoxetine at one month post-dose (see Materials and Methods). Figure 2 ). To determine the GAA activity in liver tissue, 1×10 13 GAA activity was evaluated in liver tissues of male (black dots) and female (grey triangles) mice treated with control and vehicle at 1 month post-dose. Figure 4 ). In addition, for 1×10 13 hGAA transcripts were measured by RNA-seq analysis in liver tissues of male (dots) and female (triangles) mice treated with control and vehicle-treated mice dosed with 100 vg / kg of dapoxetine. Figure 3 ). The horizontal line indicates the median expression level of endogenous murine GAA.

[0164] These results show that the hybrid promoter construct is expressed in liver and muscle. GAA activity is maintained in quadriceps with muscle-directed hybrid promoter constructs. Consistent with the engineered liver-enhanced promoter design, vector 1 as well as hybrid vectors 3 and 4 exhibit the highest expression and activity levels in liver.

[0165] The effect of hepatic expression on antibody reactivity to hGAA was also evaluated. 13 vg / kg( Figure 5A ) or 3×10 13 vg / kg( Figure 5BAnti-GAA antibody levels were evaluated in the sera of male (black dots) and female (grey triangles) mice treated with 1×10 13 vg / kg( Fig. 6A ) or 3×10 13 vg / kg( Figure 6B )-treated control mice and vehicle-treated mice, GAA activity was evaluated in the sera of male mice (black dots) and female mice (grey triangles) one month after dosing.

[0166] Increased liver expression resulted in reduced hGAA antibody reactivity. Reduced hGAA antibody reactivity resulted in increased serum GAA in Vector 1, Vector 3, and Vector 4. In conclusion, increased promoter liver activity resulted in reduced anti-GAA immunogenicity in a dose-dependent manner and increased serum GAA activity levels.

[0167] Improved pathology in quadriceps muscle from the hybrid promoter relative to the liver promoter-only vector

[0168] Muscle pathology has traditionally been difficult to correct in humans with enzyme replacement therapy. To determine the impact of each vector in improving muscle pathology, 1 × 10 13 vg / kg( Fig. 7A ) or 3×10 13 vg / kg( Figure 7B )-treated control mice and vehicle-treated mice, and pathological scores in sectioned quadriceps muscle tissues of male mice (black dots) and female mice (grey triangles) were evaluated (Table 5).

[0169] GAA activity, glycogen levels, and repair of muscle pathology were evaluated in mice administered a vector containing the hGAA transgene directed by a hybrid promoter. 13 GAA activity in serum and quadriceps muscle and glycogen accumulation in quadriceps muscle of representative control-treated mice and vehicle-treated mice dosed with 50 vg / kg, and their values ​​are reported in Figure 8 Glycogen accumulation scores were evaluated in isopentane frozen quadriceps tissue sections sectioned according to standard procedures and stained with H&E and PAS, and the values ​​are reported in Figure 8 Images of H&E and PAS-stained sections of representative mice are also shown in Figure 8 middle.

[0170] Muscle expression is required for the improvement of muscle pathology in vivo. In the GAA mouse model, mice dosed with the heterozygous promoter achieved improved muscle pathology scores. Several vectors were expressed in male and female mice at 3×10 13vg / kg dose resulted in improved pathology scores.

[0171] Table 5. Pathological scores

[0172]

[0173] Evidence for association of GAA activity and de novo synthesis of GAA transcripts with vector 3 in the spinal cord

[0174] To evaluate the ability of heterozygous vectors to restore CNS-based Pompe disease manifestations, 3 × 10 13 GAA activity was evaluated in the spinal cords of male (black dots) and female (grey triangles) mice treated with control and vehicle at 400 vg / kg of 1% dapoxetine. Fig. 9 ). In addition, for 1×10 13 hGAA transcripts were measured by RNA-seq analysis in spinal cord tissue of male (dots) and female (triangles) mice treated with control and vehicle-treated mice dosed with 100 vg / kg of dapoxetine. Fig.10 ). The horizontal line indicates the median expression level of endogenous murine GAA. The ratio of per vector expression level in liver or CNS tissue was estimated by dividing the RNA expression level in liver and spinal cord as determined by RNA-Seq analysis by the average vector copy number (VCN) in liver or brain tissue, respectively ( Fig.11 ).

[0175] Results show evidence of GAA activity and de novo synthesis of GAA transcripts in spinal cord tissue from vector 3. Expression of vector 3 in the spinal cord was equivalent to expression in the liver on a per-VCN basis.

[0176] in conclusion

[0177] As shown in this example, an engineered synthetic hybrid promoter (e.g., the engineered synthetic hybrid promoter in vector 3) directs true expression of hGAA in muscle, liver, and CNS tissues. Liver contribution results in a favorable immunogenicity profile, e.g., for vector 3. Muscle contribution also results in favorable GAA activity, glycogen recovery, and pathological observations in muscle, e.g., for vector 3. In addition, there is evidence of CNS activity (e.g., for vector 3), as based on GAA activity in the spinal cord and de novo synthetic transcripts. These findings support the clinical translation of the vectors described herein as optimized hGAA vectors for AAV gene therapy of Pompe disease.

[0178] Example 2. Treatment of Pompe disease by administration of a vector containing a GAA transgene operably linked to transcriptional control elements

[0179] Using conventional molecular biology techniques known in the art, a gene encoding a therapeutic protein (e.g., GAA) can be operably linked to a transcriptional regulatory element (e.g., the transcriptional regulatory element described in Example 1 above). The gene can then be incorporated into a vector (e.g., a viral vector) and administered to a patient suffering from a disease associated with the deficiency of the gene. For example, a viral vector containing a GAA gene under the control of a transcriptional regulatory element can be administered to a patient suffering from Pompe disease (a lysosomal storage disease characterized by a deficiency of GAA) that promotes the expression of GAA in muscle cells, neurons, and hepatocytes. For example, an AAV vector (e.g., a pseudotyped AAV2 / 8 or AAV2 / 9 vector) can be generated that incorporates the GAA gene between the 5' inverted terminal repeat and the 3' inverted terminal repeat of the vector, and the gene can be placed under the control of the transcriptional regulatory element described in Example 1 above. The AAV vector can be administered to a subject by a variety of routes of administration (e.g., intravenous, intramuscular, or subcutaneous, among others).

[0180] After the vector is administered to the patient, those skilled in the art can monitor the expression of the GAA gene and the improvement of the patient's response to therapy by various methods. For example, the physician can monitor the patient's muscle function (e.g., myocardial function) and / or glycogen accumulation to determine the patient's response to therapy. The finding that the patient's muscle function has improved and / or the level of glycogen accumulation has decreased after the administration of therapy can indicate that the patient has responded favorably to the treatment. Subsequent doses can be determined and administered as needed.

[0181] Other Implementations

[0182] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0183] Although the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention which generally follow from the principles of the invention and which include such departures from the invention as come within known or customary practice in the art to which the invention pertains and which may be applied to the basic features shown above and which fall within the scope of the appended claims.

[0184] Other embodiments are within the claims.

Claims

1. A nucleic acid regulatory element, which consists of the nucleic acid sequence of SEQ ID NO:

12.

2. A vector comprising the nucleic acid regulatory element according to claim 1, wherein the nucleic acid regulatory element is operably linked to a transgene, and wherein the nucleic acid regulatory element induces expression of the transgene after the vector is introduced into a cell.

3. The vector according to claim 2, wherein the transgene is acid alpha-glucosidase (GAA).

4. The vector according to claim 2, wherein the cell is a muscle cell, a neuron or a hepatocyte.

5. The vector according to claim 2, wherein the vector is a viral vector.

6. The vector of claim 5, wherein the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, poxvirus, baculovirus, herpes simplex virus and vaccinia virus. The vector according to claim 5 , wherein the viral vector is a lentivirus.

8. The vector of claim 6, wherein the viral vector is AAV.

9. The vector of claim 8, wherein the AAV is an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, or an AAVrh74 serotype.

10. The vector of claim 5, wherein the viral vector is a pseudotyped AAV.

11. The vector of claim 10, wherein the pseudotyped AAV is rAAV2 / 8 or rAAV2 / 9.

12. A composition comprising a nucleic acid molecule comprising the nucleic acid regulatory element according to claim 1, wherein the composition is a liposome, a vesicle, an exosome or a dendrimer.

13. A composition comprising a nucleic acid molecule comprising the nucleic acid regulatory element according to claim 1, wherein the composition is a synthetic vesicle or a synthetic exosome.

14. A composition comprising a nucleic acid molecule comprising the nucleic acid regulatory element according to claim 1, wherein the composition is a nanoparticle.

15. The composition of any one of claims 12-14, wherein the nucleic acid regulatory element is operably linked to a transgene, and wherein the nucleic acid regulatory element induces expression of the transgene upon introduction of the composition into a cell.

16. Use of the vector of claim 2 or the composition of any one of claims 12 to 14 in the preparation of a medicament for treating Pompe disease that expresses a transgene in a cell, wherein the vector or composition is contacted with the cell for a time sufficient to simulate the transcription of the transgene in the cell, wherein the transgene is GAA.

17. Use of a therapeutically effective amount of a vector according to claim 2 or a composition according to any one of claims 12-14 in the preparation of a medicament for treating Pompe disease in a human patient in need thereof, wherein the vector comprises the nucleic acid regulatory element according to claim 1, the composition comprises a nucleic acid molecule comprising the nucleic acid regulatory element according to claim 1, wherein the nucleic acid regulatory element is operably linked to a transgene, wherein the transgene is GAA.

18. A kit comprising the vector of claim 2 or the composition of any one of claims 12-14, wherein the kit further comprises a package insert instructing a user of the kit to contact the vector or the composition with a cell, thereby expressing a transgene operably linked to the regulatory control element.

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