AAV gene therapy for mucopolysaccharidosis IIIB

Abstract

Provided herein is a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome, wherein the vector genome comprises an engineered nucleic acid sequence encoding a functional human N-acetyl-alpha-glucosaminidase (hNAGLUcoV3) and a regulatory sequence leading to the expression of hNAGLU in target cells. Also provided are a pharmaceutical composition comprising the rAAV described herein in a formulation buffer, nucleic acid molecules, packaging host cells, an rAAV production system, and a method for treating a human subject diagnosed with MPS IIIB.

Description

[Technical Field]

[0001] Reference to electronic sequence listings The electronic sequence listing filed with this specification, known as "UPN-23-10402PCT_ST26_Sequence Listing.xml," which was created on June 10, 2024, and has a size of 104,796 bytes, and the contents of the electronic sequence listing (e.g., sequences and text within it), are incorporated herein by reference in their entirety. [Background technology]

[0002] Mucopolysaccharidosis type IIIB (MPS IIIB, or Sanfilippo syndrome type B, Sanfilippo disease type B) is an autosomal recessive genetic disorder caused by a deficiency of N-acetyl-alpha-D-glucosaminidase (NAGLU), an enzyme involved in the lysosomal catabolism of glycosaminoglycan (GAG) heparan sulfate. This deficiency leads to the intracellular accumulation of undegraded heparan sulfate, as well as gangliosides GM2 and GM3, in the central nervous system, causing neurological dysfunction and neuroinflammation.

[0003] MPS IIIB is a neurodegenerative disorder characterized by an initial asymptomatic period, followed by progressive intellectual decline, and ultimately leading to severe dementia. Severe behavioral problems are a prominent symptom in most patients, and these are primarily characterized by extreme hyperactivity. Other symptoms include sleep problems, recurrent diarrhea, frequent ear, nose, and throat infections, hearing and visual impairments, and epilepsy. Patients typically die in their late teens or early twenties, although longer survival has been reported in patients with attenuated forms of MPS IIIB.

[0004] There is no specific treatment for MPS IIIB. Clinical management of patients with MPS IIIB currently still consists mainly of supportive care aimed at symptom remission and prevention of complications. Medications (such as anticonvulsants for seizures) are used to alleviate symptoms and improve quality of life. Hematopoietic stem cell transplantation, e.g., bone marrow transplantation or umbilical cord blood transplantation, does not appear to significantly alleviate neuropsychological deterioration. Enzyme replacement therapy (ERT) for MPS IIIB via intravenous administration and intraventricular infusion has shown elevated NAGLU enzyme activity in mouse models and is currently being investigated in clinical trials in MPS IIIB patients. However, ERT still requires multiple doses, significantly impacts the patient's quality of life, and is expensive. For example, Aoyagi-Scharber M et al, Clearance of Heparan Sulfate and Attenuation of CNS Pathology by Intracerebroventricular BMN 250 in Sanfilippo Type B Mice, Mol See Ther Methods Clin Dev.2017 Jun 6;6:43-53.doi:10.1016 / j.omtm.2017.05.009.eCollection 2017 Sep 15, and WO2017 / 132675A1.

[0005] There is a need for compositions and methods for the effective treatment of MPS IIIB in the relevant technical field. [Overview of the Initiative]

[0006] In one embodiment, recombinant AAV (rAAV) comprises an AAV capsid and a vector genome within the AAV capsid, wherein the vector genome is a nucleic acid molecule containing an expression cassette, and the expression cassette is functional human N-acetyl-alpha-glucosaminidase (hNAG). Provided herein is an rAAV comprising a manipulated nucleic acid sequence encoding hNAGLU, which is operably linked to a regulatory sequence for hNAGLU that leads to the expression of hNAGLU in target cells, wherein the hNAGLU coding sequence is sequence number 1 or a sequence at least 99% identical to sequence number 1 (hNAGLUcoV3). In one embodiment, the hNAGLU coding sequence is sequence number 1 (hNAGLUcoV3). In one embodiment, the regulatory sequence comprises a hybrid promoter, which comprises a cytomegalovirus early-stage (CMV IE) enhancer, a chicken beta-actin promoter, and a chimeric intron containing a chicken beta-actin intron. In another embodiment, the regulatory sequence comprises a promoter element, which comprises a chicken beta-actin promoter having the sequence of sequence number 4 or a sequence at least 99.9% identical thereto. In yet another embodiment, the regulatory sequence comprises an enhancer element, which comprises a CMV IE enhancer having the sequence of sequence number 3 or a sequence at least 99.9% identical thereto. In one embodiment, the regulatory sequence includes an intron, the intron includes a chicken beta-actin intro having the sequence of SEQ ID NO: 5 or a sequence that is at least 99.9% identical thereto. In another embodiment, the regulatory sequence further includes rabbit beta-globin polyA having the sequence of SEQ ID NO: 6 or a sequence that is at least 99.9% identical thereto. In a particular embodiment, the expression cassette includes the sequence of SEQ ID NO: 7 (CB7.CI.hNAGLUcoV3.RBG). In a particular embodiment, the vector genome includes the sequence of SEQ ID NO: 8 (CB7.CI.hNAGLUcoV3.RBG). In one embodiment, the AAV capsid is the AAVhu68 capsid. In another embodiment, the AAV capsid is the AAVhu95 capsid, the AAVhu96 capsid, the AAV9 capsid, or the AAVrh91 capsid.

[0007] In one embodiment, a recombinant AAV (rAAV) is provided herein, comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises an AAV 5' inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding functional human N-acetyl-alpha-glucosaminidase (hNAGLU) operably linked to a regulatory sequence that enables the expression of hNAGLU in target cells (e.g., by leading transcription, translation, and / or expression), and an AAV 3'ITR, wherein the hNAGLU coding sequence is sequence number 1 or sequence at least 99% identical to sequence number 1 (hNAGLUcoV3). In one embodiment, rAAV is intended for use in the treatment of impairments associated with mucopolysaccharidosis III B (MPS IIIB), defects in hNAGLU, and / or in subjects with hNAGLU-related impairments to improve gait or mobility, reduce tremors, reduce spasms, improve posture, or reduce the progression of vision loss.

[0008] In another embodiment, a pharmaceutical composition comprising rAAV in a formulation buffer is provided herein, wherein the rAAV comprises recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising an AAV 5' inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding functional human N-acetyl-alpha-glucosaminidase (hNAGLU), a regulatory sequence leading to the expression of hNAGLU in target cells, and an AAV 3'ITR, the hNAGLU coding sequence being sequence number 1 or a sequence at least 99% identical to sequence number 1 (hNAGLUcoV3). In certain embodiments, the pharmaceutical composition is suitable for co-administration with a functional hNAGLU protein. In one embodiment, the pharmaceutical composition is formulated for delivery via intraventricular (ICV), intrathecal (IT), intracisional (ICM), or intravenous (IV) injection. In another embodiment, the pharmaceutical composition is 1 × 10⁶ per gram of brain mass 9 The number of GCs per gram of brain mass is approximately 1 x 10⁻¹⁶. 13It can be administered in doses of one GC. In another embodiment, the pharmaceutical composition is formulated to have a pH of 6 to 8.

[0009] In another embodiment, a method for treating a human subject diagnosed with MPS IIIB, a disorder related to a defect in hNAGLU, and / or for improving gait or motor function, reducing tremor, reducing spasms, improving posture, or reducing the progression of vision loss in a subject with hNAGLU-related disorder, comprising: a suspension of rAAV in a formulation buffer, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, the vector genome comprising an AAV 5' inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding functional human N-acetyl-alpha-glucosaminidase (hNAGLU), a regulatory sequence leading to the expression of hNAGLU in target cells, and AAV A suspension containing a 3'ITR and whose hNAGLU coding sequence is at least 99% identical to sequence number 1 or sequence number 1 (hNAGLUcoV3) is administered at a rate of 1 × 10⁶ per gram of brain mass. 9 The number of GCs per gram of brain mass is approximately 1 x 10⁻¹⁶. 13 Methods are provided herein that include administering GC in doses of a certain number of units. In one embodiment, the suspension is suitable for co-administration with a functional hNAGLU protein. In another embodiment, the suspension is delivered intraventricular, intrathecal, or intravenously to a subject in need. In a particular embodiment, the suspension is delivered to a subject in need via an Omaya device. In a particular embodiment, the suspension has a pH of 6–8. In yet another embodiment, the subject receives enzyme replacement therapy at a reduced dose or at a lower frequency compared to standard treatment via enzyme replacement therapy alone, and / or the subject demonstrates improvement in biomarkers associated with MPS IIIB. In a particular embodiment, rAAV is administered once to the subject in need. In one embodiment, rAAV is administered two or more times to the subject in need.

[0010] Other aspects and advantages of the present invention will be readily apparent from the following detailed description of the invention. [Brief explanation of the drawing]

[0011] [Figure 1A] This paper provides a comparison of differently engineered sequences in WT C57BL6 mice after IV administration, based on enzyme activity readout. Figure 1A demonstrates NAGLU activity in the liver, and Figure 1B demonstrates NAGLU activity in plasma. Co-variants from two naturally occurring common variants (reference protein and R737G missense variant) are evaluated for transgene expression. AAVhu68.hNAGLUcoV3 administered at a dose of 1 × 10¹¹ GCs demonstrates the highest enzyme activity and is the variant selected for further study. Figure 1C provides a comparison of immunostaining of liver cells comparing the activity of AAVhu68.hNAGLUcoV3 at two different doses (1 × 10¹⁰ GCs and 1 × 10¹¹ GCs) administered to WT mice, the comparison demonstrating greater transgene expression at the 1 × 10¹¹ GC dose. [Figure 1B] This paper provides a comparison of differently engineered sequences in WT C57BL6 mice after IV administration, based on enzyme activity readout. Figure 1A demonstrates NAGLU activity in the liver, and Figure 1B demonstrates NAGLU activity in plasma. Co-variants from two naturally occurring common variants (reference protein and R737G missense variant) are evaluated for transgene expression. AAVhu68.hNAGLUcoV3 administered at a dose of 1 × 10¹¹ GCs demonstrates the highest enzyme activity and is the variant selected for further study. Figure 1C provides a comparison of immunostaining of liver cells comparing the activity of AAVhu68.hNAGLUcoV3 at two different doses (1 × 10¹⁰ GCs and 1 × 10¹¹ GCs) administered to WT mice, the comparison demonstrating greater transgene expression at the 1 × 10¹¹ GC dose. [Figure 1C]This paper provides a comparison of differently engineered sequences in WT C57BL6 mice after IV administration, based on enzyme activity readout. Figure 1A demonstrates NAGLU activity in the liver, and Figure 1B demonstrates NAGLU activity in plasma. Co-variants from two naturally occurring common variants (reference protein and R737G missense variant) are evaluated for transgene expression. AAVhu68.hNAGLUcoV3 administered at a dose of 1 × 10¹¹ GCs demonstrates the highest enzyme activity and is the variant selected for further study. Figure 1C provides a comparison of immunostaining of liver cells comparing the activity of AAVhu68.hNAGLUcoV3 at two different doses (1 × 10¹⁰ GCs and 1 × 10¹¹ GCs) administered to WT mice, the comparison demonstrating greater transgene expression at the 1 × 10¹¹ GC dose. [Figure 2A] This shows the dose-dependent expression of AAVhu68.hNAGLUcoV3 in WT and MPS IIIB mice after ICV administration. NAGLU activity in serum (Figure 2A), brain (Figure 2C), and liver (Figure 2D) is measured in WT and MPS IIIB mice after ICV administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC, compared to control (PBS). Figure 2B shows the anti-NAGLU titer in WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC. NAGLU activity is detected in a dose-dependent manner in both the brain and liver, compared to control. Figure 2E shows immunohistochemistry of brain tissue from MPS IIIB mice comparing administration of a control (PBS) with administration of AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. The study demonstrates dose-dependent expression of the AAVhu68.hNAGLUcoV3 transgene in the brain, a key target organ. [Figure 2B]This shows the dose-dependent expression of AAVhu68.hNAGLUcoV3 in WT and MPS IIIB mice after ICV administration. NAGLU activity in serum (Figure 2A), brain (Figure 2C), and liver (Figure 2D) is measured in WT and MPS IIIB mice after ICV administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC, compared to control (PBS). Figure 2B shows the anti-NAGLU titer in WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC. NAGLU activity is detected in a dose-dependent manner in both the brain and liver, compared to control. Figure 2E shows immunohistochemistry of brain tissue from MPS IIIB mice comparing administration of a control (PBS) with administration of AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. The study demonstrates dose-dependent expression of the AAVhu68.hNAGLUcoV3 transgene in the brain, a key target organ. [Figure 2C] This shows the dose-dependent expression of AAVhu68.hNAGLUcoV3 in WT and MPS IIIB mice after ICV administration. NAGLU activity in serum (Figure 2A), brain (Figure 2C), and liver (Figure 2D) is measured in WT and MPS IIIB mice after ICV administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC, compared to control (PBS). Figure 2B shows the anti-NAGLU titer in WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC. NAGLU activity is detected in a dose-dependent manner in both the brain and liver, compared to control. Figure 2E shows immunohistochemistry of brain tissue from MPS IIIB mice comparing administration of a control (PBS) with administration of AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. The study demonstrates dose-dependent expression of the AAVhu68.hNAGLUcoV3 transgene in the brain, a key target organ. [Figure 2D]This shows the dose-dependent expression of AAVhu68.hNAGLUcoV3 in WT and MPS IIIB mice after ICV administration. NAGLU activity in serum (Figure 2A), brain (Figure 2C), and liver (Figure 2D) is measured in WT and MPS IIIB mice after ICV administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC, compared to control (PBS). Figure 2B shows the anti-NAGLU titer in WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC. NAGLU activity is detected in a dose-dependent manner in both the brain and liver, compared to control. Figure 2E shows immunohistochemistry of brain tissue from MPS IIIB mice comparing administration of a control (PBS) with administration of AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. The study demonstrates dose-dependent expression of the AAVhu68.hNAGLUcoV3 transgene in the brain, a key target organ. [Figure 2E] This shows the dose-dependent expression of AAVhu68.hNAGLUcoV3 in WT and MPS IIIB mice after ICV administration. NAGLU activity in serum (Figure 2A), brain (Figure 2C), and liver (Figure 2D) is measured in WT and MPS IIIB mice after ICV administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC, compared to control (PBS). Figure 2B shows the anti-NAGLU titer in WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC. NAGLU activity is detected in a dose-dependent manner in both the brain and liver, compared to control. Figure 2E shows immunohistochemistry of brain tissue from MPS IIIB mice comparing administration of a control (PBS) with administration of AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. The study demonstrates dose-dependent expression of the AAVhu68.hNAGLUcoV3 transgene in the brain, a key target organ. [Figure 3A]This study demonstrates the reduction of lysosomal pathology in the brains of WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 compared to controls. MPS IIIB mice are administered AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GCs or 5 × 10¹⁰ GCs. The area percentage of lysosomal-associated membrane protein 1 (LAMP-1) in brain tissue is measured. The LAMP-1 area percentage in the cortex (Figure 3A) and hippocampus (Figure 3B) is reduced in MPS IIIB mice administered AAVhu68.hNAGLUcoV3 compared to controls. Figure 3C shows immunostaining of LAMP-1 in WT mice, MPS IIIB mice administered PBS, and MPS IIIB mice administered AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. These studies demonstrate a dose-dependent reduction in LAMP 1 staining in the brains of MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3, which indicates an improvement in lysosomal pathology. [Figure 3B] This study demonstrates the reduction of lysosomal pathology in the brains of WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 compared to controls. MPS IIIB mice are administered AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GCs or 5 × 10¹⁰ GCs. The area percentage of lysosomal-associated membrane protein 1 (LAMP-1) in brain tissue is measured. The LAMP-1 area percentage in the cortex (Figure 3A) and hippocampus (Figure 3B) is reduced in MPS IIIB mice administered AAVhu68.hNAGLUcoV3 compared to controls. Figure 3C shows immunostaining of LAMP-1 in WT mice, MPS IIIB mice administered PBS, and MPS IIIB mice administered AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. These studies demonstrate a dose-dependent reduction in LAMP 1 staining in the brains of MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3, which indicates an improvement in lysosomal pathology. [Figure 3C]This study demonstrates the reduction of lysosomal pathology in the brains of WT and MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3 compared to controls. MPS IIIB mice are administered AAVhu68.hNAGLUcoV3 at doses of 1 × 10¹⁰ GCs or 5 × 10¹⁰ GCs. The area percentage of lysosomal-associated membrane protein 1 (LAMP-1) in brain tissue is measured. The LAMP-1 area percentage in the cortex (Figure 3A) and hippocampus (Figure 3B) is reduced in MPS IIIB mice administered AAVhu68.hNAGLUcoV3 compared to controls. Figure 3C shows immunostaining of LAMP-1 in WT mice, MPS IIIB mice administered PBS, and MPS IIIB mice administered AAVhu68.hNAGLUcoV3 at a dose of 1 × 10¹⁰ GCs. These studies demonstrate a dose-dependent reduction in LAMP 1 staining in the brains of MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3, which indicates an improvement in lysosomal pathology. [Figure 4A] This study demonstrates a reduction in the heparan sulfate (HS) substrate in MPS IIIB mice administered with AAVhu68.hNAGLUcoV3 compared to controls. Heparan sulfate (HS) is a biomarker substrate associated with the disease MPS IIIB. The amount of HS is measured in the brain (Figure 4A) and liver (Figure 4B) of WT and MPS IIIB mice. AAVhu68.hNAGLUcoV3 is administered at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC and compared to MPS IIIB mice administered with PBS. The study demonstrates a dose-dependent reduction of HS in brain (a key target organ) tissue of MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3, indicating targeted involvement of the transgene. [Figure 4B]This study demonstrates a reduction in the heparan sulfate (HS) substrate in MPS IIIB mice administered with AAVhu68.hNAGLUcoV3 compared to controls. Heparan sulfate (HS) is a biomarker substrate associated with the disease MPS IIIB. The amount of HS is measured in the brain (Figure 4A) and liver (Figure 4B) of WT and MPS IIIB mice. AAVhu68.hNAGLUcoV3 is administered at doses of 1 × 10¹⁰ GC or 5 × 10¹⁰ GC and compared to MPS IIIB mice administered with PBS. The study demonstrates a dose-dependent reduction of HS in brain (a key target organ) tissue of MPS IIIB mice after administration of AAVhu68.hNAGLUcoV3, indicating targeted involvement of the transgene. [Figure 5A] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5B] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5C]The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5D] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5E] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5F] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5G]The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5H] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5I] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5J] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5K]The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5L] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 5M] The following are alignments of the transgene coding sequences: SEQ ID NO: 1 (hNAGLUcoV3), SEQ ID NO: 20 (WT hNAGLU), SEQ ID NO: 26 (hNAGLUcoV1), SEQ ID NO: 27 (hNAGLUcoV1-R737G), SEQ ID NO: 28 (hNAGLUcoV2), SEQ ID NO: 29 (hNAGLUcoV2-R737G), SEQ ID NO: 30 (hNAGLUcoV3-R737G), and SEQ ID NO: 32 (hNAGLU from US2020 / 0289675 (SEQ ID NO: 1)). [Figure 6A] This report provides a comparison of different engineered sequences in WT C57BL6 mice after IV administration, based on enzyme activity readout. Figure 6A demonstrates NAGLU activity in the liver, and Figure 6B demonstrates NAGLU activity in plasma. Wild-type cDNA (hNAGLUwt) is compared to three engineered sequences (hNAGLUco, hNAGLUcoV3, and hNAGLUcoV1) and evaluated for transgene expression. The wt sequence and the three engineered sequences were administered at a dose of 3 × 10¹¹ GCs. hNAGLUcoV3 demonstrated the highest enzyme activity and performed superiorly to the other constructs and native cDNA. [Figure 6B] This report provides a comparison of different engineered sequences in WT C57BL6 mice after IV administration, based on enzyme activity readout. Figure 6A demonstrates NAGLU activity in the liver, and Figure 6B demonstrates NAGLU activity in plasma. Wild-type cDNA (hNAGLUwt) is compared to three engineered sequences (hNAGLUco, hNAGLUcoV3, and hNAGLUcoV1) and evaluated for transgene expression. The wt sequence and the three engineered sequences were administered at a dose of 3 × 10¹¹ GCs. hNAGLUcoV3 demonstrated the highest enzyme activity and performed superiorly to the other constructs and native cDNA. [Figure 7] Clinical scores in male and female WT and MPS IIIB mice are shown compared to MPS IIIB mice treated with AAVhu68.hNAGLUcoV3 at doses of 1.3 × 10¹⁰ GCs, 4.5 × 10¹⁰ GCs, or 1.3 × 10¹¹ GCs. Higher clinical scores indicate a worse phenotype. All MPS IIIB mice treated with AAVhu68.hNAGLUcoV3, regardless of dose, showed similar clinical scores to WT mice. [Figure 8] Survival curves for WT and MPS IIIB mice are shown, compared to MPS IIIB mice treated with AAVhu68.hNAGLUcoV3 at doses of 1.3 × 10¹⁰ GCs, 4.5 × 10¹⁰ GCs, or 1.3 × 10¹¹ GCs. The probability of survival is shown. Survival rescue is shown in all MPS IIIB mice treated with AAVhu68.hNAGLUcoV3, regardless of dose, compared to untreated MPS IIIB mice. [Figure 9A]This study demonstrates dorsal root ganglion (DRG) pathogenesis in non-human primates (NHPs). Figure 9A shows a representative image of brain slice "5" taken from the cortex and periventricular region. Figure 9B shows a representative image of brain slice "9" taken from the occipital lobe cortex. Figures 9C-9E show hNAGLU expression by in situ hybridization. Three NHPs were administered AAVhu68.hNAGLUcoV3 at a dose of 3.3 × 10¹¹ GC / brain g. The treatment was well tolerated in two animals necropped at 90 days. These two NHPs demonstrated typical mild to moderate DRG pathogenesis. One animal experienced an immune-mediated non-autologous cytotoxic T cell response to hNAGLU and was necropped at 42 days. [Figure 9B] This study demonstrates dorsal root ganglion (DRG) pathogenesis in non-human primates (NHPs). Figure 9A shows a representative image of brain slice "5" taken from the cortex and periventricular region. Figure 9B shows a representative image of brain slice "9" taken from the occipital lobe cortex. Figures 9C-9E show hNAGLU expression by in situ hybridization. Three NHPs were administered AAVhu68.hNAGLUcoV3 at a dose of 3.3 × 10¹¹ GC / brain g. The treatment was well tolerated in two animals necropped at 90 days. These two NHPs demonstrated typical mild to moderate DRG pathogenesis. One animal experienced an immune-mediated non-autologous cytotoxic T cell response to hNAGLU and was necropped at 42 days. [Figure 9C]This study demonstrates dorsal root ganglion (DRG) pathogenesis in non-human primates (NHPs). Figure 9A shows a representative image of brain slice "5" taken from the cortex and periventricular region. Figure 9B shows a representative image of brain slice "9" taken from the occipital lobe cortex. Figures 9C-9E show hNAGLU expression by in situ hybridization. Three NHPs were administered AAVhu68.hNAGLUcoV3 at a dose of 3.3 × 10¹¹ GC / brain g. The treatment was well tolerated in two animals necropped at 90 days. These two NHPs demonstrated typical mild to moderate DRG pathogenesis. One animal experienced an immune-mediated non-autologous cytotoxic T cell response to hNAGLU and was necropped at 42 days. [Figure 9D] This study demonstrates dorsal root ganglion (DRG) pathogenesis in non-human primates (NHPs). Figure 9A shows a representative image of brain slice "5" taken from the cortex and periventricular region. Figure 9B shows a representative image of brain slice "9" taken from the occipital lobe cortex. Figures 9C-9E show hNAGLU expression by in situ hybridization. Three NHPs were administered AAVhu68.hNAGLUcoV3 at a dose of 3.3 × 10¹¹ GC / brain g. The treatment was well tolerated in two animals necropped at 90 days. These two NHPs demonstrated typical mild to moderate DRG pathogenesis. One animal experienced an immune-mediated non-autologous cytotoxic T cell response to hNAGLU and was necropped at 42 days. [Figure 9E]This study demonstrates dorsal root ganglion (DRG) pathogenesis in non-human primates (NHPs). Figure 9A shows a representative image of brain slice "5" taken from the cortex and periventricular region. Figure 9B shows a representative image of brain slice "9" taken from the occipital lobe cortex. Figures 9C-9E show hNAGLU expression by in situ hybridization. Three NHPs were administered AAVhu68.hNAGLUcoV3 at a dose of 3.3 × 10¹¹ GC / brain g. The treatment was well tolerated in two animals necropped at 90 days. These two NHPs demonstrated typical mild to moderate DRG pathogenesis. One animal experienced an immune-mediated non-autologous cytotoxic T cell response to hNAGLU and was necropped at 42 days. [Figure 10A] Further research is presented in one NHP that experienced an immune-mediated non-autologous cytotoxic T cell response to hNAGLU and was necropped at 42 days. Responses to individual peptides were evaluated within a subpool that produced a positive IFN-γ response. The IFN-γ response to individual peptides identified a single immunodominant epitope within peptide pool B. Figures 10A and 10B show spot-forming units (SFUs) per million cells for the tested sample. [Figure 10B] Further research is presented in one NHP that experienced an immune-mediated non-autologous cytotoxic T cell response to hNAGLU and was necropped at 42 days. Responses to individual peptides were evaluated within a subpool that produced a positive IFN-γ response. The IFN-γ response to individual peptides identified a single immunodominant epitope within peptide pool B. Figures 10A and 10B show spot-forming units (SFUs) per million cells for the tested sample. [Modes for carrying out the invention]

[0012] Compositions useful for the treatment of mucopolysaccharidosis type IIIb (MPS IIIB) and / or for the alleviation of symptoms of MPS IIIB are provided herein. These compositions comprise a nucleic acid sequence encoding a functional human N-acetyl-alpha-D-glucosaminidase (hNAGLU), which is operably linked to a regulatory sequence that enables its expression in target cells, wherein the hNAGLU encoding sequence is at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3).

[0013] In one embodiment, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, other compositions, and methods for the expression of functional human NAGLU (hNAGLU). In another embodiment, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, host cells, other compositions, and methods for producing a composition comprising a nucleic acid sequence encoding functional hNAGLU. In yet another embodiment, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, other compositions, and methods for treating MPS IIIB by delivering a nucleic acid sequence encoding functional hNAGLU to a target. In one embodiment, the compositions and methods described herein are useful for providing therapeutic levels of NAGLU to the central nervous system (CNS). Additionally or alternatively, the compositions and methods described herein include The compositions and methods described herein are useful for providing therapeutic levels of NAGLU in peripheral areas, such as the blood, liver, kidney, or peripheral nervous system. In certain embodiments, the adeno-associated virus (AAV) vector-based methods described herein provide novel therapeutic options that, by providing the NAGLU protein to subjects requiring its expression, restore desired function of NAGLU, alleviate symptoms associated with MPS IIIB, improve biomarkers associated with MPS IIIB, or help facilitate other treatments for MPS IIIB.

[0014] As used herein, the term “therapeutic level” means an enzyme activity of at least about 5%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, over 100%, about 2x, about 3x, or about 5x compared to a healthy control. A suitable assay for measuring NAGLU enzyme activity is described herein. In some embodiments, such therapeutic levels of NAGLU may result in: alleviation of MPS IIIB-related symptoms; improvement of MPS IIIB-related biomarkers of the disease; or facilitation of other treatments for MPS IIIB, e.g., GAG levels in the brain, liver, cerebrospinal fluid (CSF), serum, urine, or any other biological sample; prevention of neurocognitive decline; reduction of lysosomal pathology; reversal of certain MPS IIIB-related symptoms; and / or prevention of progression of certain MPS IIIB-related symptoms; or any combination thereof.

[0015] As used herein, “healthy control” means a subject or a biological sample thereof in which the subject does not have MPS disorder. A healthy control may come from a single subject. In another embodiment, a healthy control is a pool of multiple subjects.

[0016] As used herein, the term “biological sample” refers to any cell, biological fluid, or tissue. Suitable samples for use in the present invention may include, but are not limited to, whole blood, leukocytes, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid, and skin cells. Such samples may be further diluted with saline, buffer, or a physiologically acceptable diluent. Alternatively, such samples may be concentrated by conventional means.

[0017] With regard to the description of these inventions, each of the compositions described herein is intended to be useful in a different embodiment in the methods of the present invention. In addition, each of the compositions described herein as useful in a method is also intended to be an embodiment of the present invention in a different embodiment.

[0018] Unless otherwise defined herein, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains, and by referring to published documents that provide general guidance to those skilled in the art for many of the terms used herein.

[0019] As used herein, “disease,” “disorder,” and “condition” refer to mucopolysaccharidosis type IIIb (also known as MPS IIIB, MPS IIIb, Sanfilippo syndrome type B, or Sanfilippo disease type B).

[0020] As used herein, the terms “symptoms associated with MPS IIIB” or “symptoms” refer to the symptoms found in patients with MPS IIIB and in animal models of MPS IIIB. Such symptoms include delayed speech; social interaction Difficulty using and communicating; sleep disorders; progressive intellectual disability and loss of previously acquired skills (developmental regression); epileptic seizures and motor impairments; large head; slight hepatomegaly (mild hepatomegaly); umbilical hernia or inguinal hernia; short stature, stiff joints, mild multiple dysostosis, multiple skeletal malformations; chronic diarrhea; recurrent upper respiratory tract infections; recurrent ear infections; hearing impairment; visual problems; asymmetrical ventricular septal hypertrophy; coarse facial features; coarse hair; dense cranial vault; multiple dysostosis; growth abnormalities; urinary heparan sulfate excretion; cerebrospinal fluid (CSF) This includes, but is not limited to, GAG accumulation in serum, urine, and / or other biological samples; abnormal expression and / or enzymatic activity of N-sulfoglycosamine sulfohydrolase (SGSH) or N-sulfoglycosamine sulfohydrolase (IDUA); accumulation of GM2 and GM3; altered activity of lysosomal enzymes; accumulation of free unesterified cholesterol in the CNS; inflammatory responses in the CNS and skeletal tissues; hypertrichosis (excessive hair growth); hyperactivity; oval thoracolumbar vertebrae; splenomegaly; eyebrow fusion; rib thickening; hernias; and swaying and vagrantic gait.

[0021] As used herein, “patient” or “subject” means a male or female human, dog, or animal model used in clinical research. In one embodiment, the subject of these methods and compositions is a human diagnosed with MPS IIIB. In certain embodiments, the human subject of these methods and compositions is a prenatal infant, neonatal, infant, toddler, preschooler, elementary school child, teenager, young adult, or adult. In further embodiments, the subject of these methods and compositions is a pediatric MPS IIIB patient.

[0022] Clinical tests and urinalysis (which detects the excretion of excess mucopolysaccharides in the urine) are the first steps in diagnosing MPS disease. Enzyme assays measuring enzyme activity levels in blood, skin cells, or various cells are also used to provide a definitive diagnosis of MPS IIIB. See ncbi.nlm.nih.gov / gtr / all / tests / ?-term=4669[geneid] and ncbi.nlm.nih.gov / gtr / all / tests / ?term=C0086648-[DISCUI]&filter=method:1_2;testtype:clinical. Various genetic tests are available to detect NAGLU mutations associated with MPS IIIB. See, for example, ncbi.nlm.nih.gov / gtr / conditions / C0086648 / , ncbi.nlm.nih.gov / gtr / all / -tests / ?term=C0086648[DISCUI]&filter=method:2_7;testtype:clinical, and www.ncbi.nlm.nih.gov / gtr / tests / 506481 / . Prenatal diagnosis using amniocentesis and chorionic villus sampling can verify whether a fetus is affected by a disorder. Genetic counseling can help parents with a family history of mucopolysaccharidosis determine whether they carry the mutated gene that causes the disorder. See, for example, A Guide to Understanding MPS III, National MPS Society, 2008, mpssociety.org / learn / diseases / mps-iii / .

[0023] As used throughout this specification and the claims, the terms “comprise” and “contain,” and their variations, particularly those including “comprises,” “comprising,” “contains,” and “containing,” include other components, elements, components, and steps, etc. The terms “consists of” or “consisting of” exclude other components, elements, components, and steps, etc. Various embodiments in this specification are presented using the language of “comprise,” but in various circumstances, the relevant embodiments may also be described using the language of “consisting of” or “essentially consisting of.” It should be understood that this will be done.

[0024] As stated above, when the term "approximately" is used to qualify a number, unless otherwise specified, it means a variation of ±10% (for example, ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values ​​in between) from the given reference.

[0025] In certain cases, the terms "E+ number" or "e+ number" are used to refer to exponents. For example, "5E10" or "5e10" is 5 × 10¹⁰. These terms may be used interchangeably.

[0026] Please note that the terms "a" or "an" refer to one or more; for example, "a vector" is understood to represent one or more vectors. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0027] 1. N-acetyl-alpha-glucosaminidase (NAGLU) As used herein, the terms “N-acetyl-alpha-glucosaminidase,” “NAGLU,” and “NaGlu” are used interchangeably with “alpha-N-acetylglucosaminidase.” The present invention includes variants or functional fragments of any of the NAGLU proteins expressed from nucleic acid sequences provided herein, which, when delivered in compositions or by methods provided herein, restore a desired function, alleviate symptoms, improve symptoms associated with an MPS IIIB-related biomarker, or facilitate other treatments for MPS IIIB. Examples of suitable biomarkers for MPS III include those described in WO2017 / 136533, which are incorporated herein by reference.

[0028] As used herein, the term “functional NAGLU” means an enzyme having the amino acid sequence of full-length wild-type (natural) human NAGLU (hNAGLU) (as shown in SEQ ID NO: 19 and UniProtKB acceptance number: P54802), variants thereof, mutants thereof having conserved amino acid substitutions, fragments thereof, full-length or fragments of any combination of variants and mutants having conserved amino acid substitutions, providing at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or nearly the same as or greater than 100% of the biological activity level of normal human NAGLU. In one embodiment, functional NAGLU refers to the wild-type NAGLU protein having the sequence of SEQ ID NO: 19.

[0029] Examples of NAGLU variants include, but are not limited to, E705K, which consists of the amino acid sequence of SEQ ID NO: 19, having lysine (Lys, K) at the 705th amino acid instead of glutamic acid (Glu, E) in the wild type, and R737G, which consists of the amino acid sequence of SEQ ID NO: 19, having glycine (Gly, G) at the 737th amino acid instead of arginine (Arg, R) in the wild type.

[0030] As used herein, “conservative amino acid substitution” or “conservative amino acid substitution” refers to an amino acid change, substitution, or substitution to a different amino acid known to those skilled in the art, having similar biochemical properties (e.g., charge, hydrophobicity, and size). See, for example, French et al. What is a conservative substitution? Journal of Molecular Evol See ution, March 1983, Volume 19, Issue 2, pp 171-175, and YAMPOLSKY et al. The Exchangeability of Amino Acids in Proteins, Genetics. 2005 Aug;170(4):1459-1472, each of which is incorporated herein by reference in its entirety.

[0031] A variety of assays exist for measuring NAGLU expression and activity levels using conventional methods. For example, Example 1 described herein, ncbi_nlm_nih_gov / gtr / all / tests / ?term=C0086648[DISCUI]&filter=method:1_2;testtype:clinical, ncbi_nlm_nih_gov / gtr / all / tests / ?term=C0086648[DISCUI]&filter=method:1_1;testtype:clinical, Kan SH et al, Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB. Proc Natl Acad Sci US A. 2014 Oct 14;111(41):14870-5. Doi:10.1073 / pnas.1416660111. Epub 2014 Sep 29, US2017 / 0088859. See also, each of these is incorporated herein by reference in whole.

[0032] In one embodiment, a nucleic acid sequence encoding a functional NAGLU protein is provided. In one embodiment, the nucleic acid sequence is the wild-type coding sequence reproduced by SEQ ID NO: 20. In one embodiment, the nucleic acid sequence is approximately 80% or less identical to the wild-type human NAGLU sequence of SEQ ID NO: 20.

[0033] Nucleic acids refer to polymeric forms of nucleotides and include RNA, mRNA, cDNA, genomic DNA, peptide nucleic acids (PNA), as well as the synthetic and mixed polymers described above. A nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of any type of nucleotide (e.g., a peptide nucleic acid oligomer). The term also includes single- and double-stranded DNA. It will be understood by those skilled in the art that functional variants of these nucleic acid molecules are also intended to be part of the present invention. A functional variant is a nucleic acid sequence that can be directly translated using a standard genetic code to provide an amino acid sequence identical to that translated from the parent nucleic acid molecule.

[0034] In certain embodiments, nucleic acid molecules and other constructs encoding functional human NAGLU (hNAGLU), which are encompassed by the present invention and useful in the generation of expression cassettes and vector genomes, may be engineered for expression in yeast cells, insect cells, or mammalian cells, e.g., human cells. Methods are known and previously described (e.g., WO96 / 09378). A sequence is considered engineered if at least one undesirable codon is replaced by a more preferred codon compared to the wild-type sequence. In this specification, an undesirable codon is a codon that is used less frequently in the organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in the organism than the undesirable codon. The frequency of codon use for a specific organism can be found in a codon frequency table, e.g., Kazusa_jp / codon. Preferably, two or more undesirable codons, preferably most or all of the undesirable codons, are replaced by more preferred codons. Preferably, the most frequently used codon in the organism is used in the engineered sequence. Substitution with a preferred codon generally results in higher expression. Furthermore, as a result of gene coding degeneracy, multiple different nucleic acid molecules encode the same polypeptide. It will also be understood by those skilled in the art that this is possible. Furthermore, it will be understood that, using conventional techniques, nucleotide substitutions that do not affect the amino acid sequence encoded by a nucleic acid molecule can be made to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Therefore, unless otherwise specified, “nucleic acid sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleic acid sequences can be cloned using conventional molecular biology techniques or newly generated by DNA synthesis, which can be carried out using conventional procedures by service companies with business in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins).

[0035] In one embodiment, the NAGLU coding sequence is an engineered sequence. In one embodiment, the engineered sequence is useful for improving production, transcription, expression, or safety in a subject. In another embodiment, the engineered sequence is useful for increasing the efficacy of the resulting therapeutic composition or treatment. In a further embodiment, the engineered sequence is useful for increasing the efficacy of the expressed functional NAGLU protein and may also increase safety by allowing lower doses of the therapeutic reagent delivering the functional protein.

[0036] In one embodiment, the manipulated NAGLU coding sequence is characterized by an improved translation rate compared to the wild-type NAGLU coding sequence. In one embodiment, the NAGLU coding sequence has less than 83% identity with the wild-type hNAGLU sequence of SEQ ID NO: 20. In one embodiment, a manipulated nucleic acid sequence is provided, comprising the sequence of SEQ ID NO: 1 (hNAGLUcoV3). In one embodiment, nucleic acid sequences encoding a functional hNAGLU, which are the manipulated nucleic acid sequence of SEQ ID NO: 1 (hNAGLUcoV3) or a nucleic acid sequence that is at least about 99% identical thereto, are provided herein. Although not currently in high demand, other manipulated nucleic acid sequences are provided herein as SEQ ID NOs: 26 (hNAGLUcoV1), SEQ ID NOs: 27 (hNAGLUcoV1-R737G), SEQ ID NOs: 28 (hNAGLUcoV2), SEQ ID NOs: 29 (hNAGLUcoV2-R737G), SEQ ID NOs: 30 (hNAGLUcoV3-R737G), and SEQ ID NOs: 32 (hNAGLU (SEQ ID NOs: 1) in US2020 / 0289675). Table 1 shows the percentage identity of wild-type NAGLU, the manipulated sequences described above, and the hNAGLU sequence described in US2020 / 0289675 compared to hNAGLUcoV3 (SEQ ID NOs: 1). Nucleotide sequences were aligned using CLUSTAL multiplexed sequence alignment by MUSCLE(3.8). Nucleotide sequences were entered using FASTA format with default settings. [Table 1]

[0037] "Engineered" means that a nucleic acid sequence encoding a functional NAGLU protein as described herein is assembled and placed on any suitable gene factor, e.g., naked DNA, phage, transposon, cosmid, episome, etc., and that the NAGLU sequence supported on it is introduced into a host cell, for example, to generate a non-viral delivery system (e.g., an RNA-based system, naked DNA, etc.) or to generate a viral vector in a packaging host cell and / or for delivery to a host cell of interest. In one embodiment, the gene factor is a vector. In one embodiment, the gene factor is a plasmid. Methods used to construct such engineered constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor. See Press, Cold Spring Harbor, NY (2012).

[0038] In the context of nucleic acid sequences and / or amino acid sequences, the terms “percent (%) identity,” “sequence identity,” “percent sequence identity,” or “percent identity” refer to the fact that residues in two sequences are identical when aligned for similarity, and often have corrections for missing or additional bases or amino acids compared to a reference sequence. With respect to nucleic acids, the length of sequence identity may also be specified as spanning the full length of the genome, the full length of the gene coding sequence. In certain embodiments, a fragment of at least about 500–5000 nucleotides or a smaller fragment, e.g., at least about 9 nucleotides, typically at least about 20–24 nucleotides, at least about 28–32 nucleotides, or at least about 36 or more nucleotides, may also be selected. Similarly, with respect to amino acids, identity may span the full length of the protein, or It may be a specified peptide, polypeptide, or region. A suitable amino acid fragment may be at least about 7 amino acids long and may contain up to about 700 amino acids.

[0039] Where used herein, the phrase “at least X% identity” includes values ​​of X and greater values ​​of X. For example, at least 95% identity includes values ​​of 95% or greater, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9%, up to 100%, or 95% to 100%, and values ​​in between. In this example, 95% may also include values ​​where the decimal point is rounded to the nearest value of 95% according to the principle of direct rounding to an integer, which includes, but is not limited to, rounding to zero, rounding from zero, rounding to the nearest integer, rounding up, and rounding down. For example, if an integer's decimal point starts at 5, 6, 7, 8, or 9, the integer is rounded up to the next perfect integer (i.e., from 95.7% to 96%), and if an integer's decimal point starts at 0, 1, 2, 3, or 4, the integer is rounded down to the next perfect integer (i.e., from 95.3% to 95%).

[0040] Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal Omega," "Clustal W," "MUSCLE," "CAP Sequence Assembly," "BLAST," "MAP," and "MEME," which are accessible via web servers on the Internet. Other sources for such programs are known to those skilled in the art. Alternatively, the Vector NTI utility can also be used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs mentioned above. As another example, polynucleotide sequences can be compared using Fasta®, a program in GCG version 10.1. Fasta® provides alignment and percent sequence identity of the best overlap region between query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta® with its default parameters provided in GCG version 10.1 (word size of 6 and NOPAM factor for scoring matrix), which are incorporated herein by reference.

[0041] Percent identity may be readily determined for the full length of a protein, a polypeptide, about 32 amino acids, about 330 amino acids, or an amino acid sequence spanning a peptide fragment thereof, or for a corresponding nucleic acid sequence encoding the sequence. A suitable amino acid fragment may be at least about 8 amino acids long and may be up to about 700 amino acids. Generally, when referring to “identity,” “homology,” or “similarity” between two different sequences, “identity,” “homology,” or “similarity” is determined with respect to “aligned” sequences. An “aligned” sequence or “alignment” is a set of nucleic acid sequences or protein (amino acid) sequences that often contain corrections for missing or additional bases or amino acids compared to a reference sequence.

[0042] Identity may be determined by preparing a sequence alignment, which may be determined by using various algorithms and / or computer programs known or commercially available in the art (e.g., BLAST, ExPASy, Clustal Omega, FASTA, e.g., those using the Needleman-Wunsch algorithm, Smith-Waterman algorithm). Alignment is performed using one of various publicly available or commercially available multiplex sequence alignment programs. Multiplex sequence alignment programs are also available for nucleic acid sequences. An example of such a program is one that can be performed via a web server on the internet. Accessible programs include "Clustal Omega," "Clustal W," "MUSCLE," "CAP Sequence Assembly," "BLAST," "MAP," and "MEME." Other sources for such programs are known to those skilled in the art. Alternatively, the Vector NTI utility can also be used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using the program Fasta® in GCG version 10.1. Fasta® provides alignment of the best overlap region and percent sequence identity between query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta® with its default parameters provided in GCG version 10.1 (word size of 6 and NOPAM factor for scoring matrix), which are incorporated herein by reference. For amino acid sequences, sequence alignment programs such as "Clustal Omega," "Clustal X," "MUSCLE," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box" are available. Generally, any of these programs are used with their default settings, but those skilled in the art can modify these settings as needed. Alternatively, those skilled in the art can utilize other algorithms or computer programs that provide at least a level of identity or alignment as provided by the referenced algorithms and programs. See, for example, JDThomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

[0043] As used herein, “desired function” means NAGLU enzyme activity at at least 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more than 100% of a healthy control.

[0044] As used herein, the terms “relieve symptoms,” “improve symptoms,” or grammatical variations thereof refer to the reversal of symptoms associated with MPS IIIB, the resolution or prevention of the progression of symptoms associated with MPS IIIB. In one embodiment, relief or improvement refers to a reduction of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% in the total number of symptoms in a patient after administration of the composition(s) or use of the method described, compared to before administration or use. In another embodiment, relief or improvement refers to a reduction of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% in the severity or progression of symptoms after administration of the composition(s) or use of the method described, compared to before administration or use.

[0045] It should be understood that the compositions of functional NAGLU proteins and NAGLU coding sequences described herein are intended to be applied to other compositions, regimens, aspects, embodiments, and methods described herein.

[0046] 2. Expression cassette In one embodiment, an expression cassette is provided comprising an engineered nucleic acid sequence encoding a functional hNAGLU and a regulatory sequence operably ligated thereto, which leads to the expression of the hNAGLU. In one embodiment, an expression cassette is provided comprising an engineered nucleic acid sequence described herein encoding a functional hNAGLU and a regulatory sequence that enables its expression. In certain embodiments, the regulatory sequence includes a promoter. In certain embodiments, the regulatory sequence includes one or more introns, one or more enhancers, and a polyadenylation (poly-A) signal sequence.

[0047] In one embodiment, the promoter is a chicken β-actin (also known as chicken beta-actin, CB, or CBA) promoter. Various chicken beta-actin promoters have been described, either alone or in combination with various enhancer elements (for example, CB7 is a chicken beta-actin promoter with a cytomegalovirus enhancer element, a CAG promoter comprising the first exon and first intron of chicken beta-actin and the splice acceptor of the rabbit beta-globin gene, and the CBh promoter [SJ Gray et al, Hu Gene Ther, 201 1 Sep;22(9):143-1 153]). In a particular embodiment, the promoter is a CMV promoter. In a particular embodiment, the CB promoter includes the nucleic acid sequence of SEQ ID NO: 4.

[0048] In further embodiments, the promoter is a CB7 (also called a hybrid CB7) promoter comprising a cytomegalovirus early-stage (CMV IE) enhancer and a chicken β-actin promoter, optionally having a spacer sequence, comprising a chicken β-actin intron, and optionally having a chimeric intron further comprising a chicken β-actin splicing donor (containing an exon sequence, a chicken β-actin intron) and a rabbit beta-globin splicing acceptor. In certain embodiments, the CMV IE enhancer comprises the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the promoter is a CB7 hybrid promoter, which comprises a CMV IE enhancer, a chicken β-actin promoter, and a chimeric intron (CI) comprising a chicken β-actin intron. In certain embodiments, the regulatory sequence further comprises a chicken β-actin intron. In certain embodiments, the chicken β-actin intron comprises the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the hybrid CB7 promoter includes the nucleic acid sequence of Sequence ID No. 31. See, for example, the cytomegalovirus (CMV) early enhancer (260 bp, C4, GenBank number K03104.1). Chicken beta-actin promoter (281 bp; CB; GenBank number X00182.1). In certain embodiments, the regulatory sequence further includes rabbit globin polyA (also referred to as rabbit beta-globin or RBG polyA). In certain embodiments, rabbit globin polyA includes the nucleic acid sequence of Sequence ID No. 6.

[0049] In certain embodiments, the expression cassette contains the nucleic acid sequence of SEQ ID NO: 7, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99-100% identical thereto. In certain embodiments, the expression cassette contains the nucleic acid sequence of SEQ ID NO: 7, or a sequence that is at least 99% identical thereto. In one embodiment, the hNAGLU coding sequence is at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3). In further embodiments, the hNAGLU coding sequence is SEQ ID NO: 1 (hNAGLUcoV3). In certain embodiments, the hNAGLU coding sequence has the nucleic acid sequence of SEQ ID NO: 1, or a sequence that is at least 99-100% identical thereto.

[0050] As used herein, the terms “expression” or “gene expression” refer to the process by which information from a gene is used to synthesize a functional gene product. The gene product may be a protein, a peptide, or a nucleic acid polymer (e.g., RNA, DNA, or PNA).

[0051] As used herein, "expression cassette" refers to a biologically useful nucleic acid sequence (for example) A nucleic acid molecule includes a gene (cDNA, mRNA, etc.) encoding a protein, enzyme, or other useful gene product, and a regulatory sequence operably ligated thereto that controls, guides, enables, or regulates the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, a “operably ligated” sequence includes both regulatory sequences that are adjacent to or not adjacent to the nucleic acid sequence, and one or more regulatory sequences act with the nucleic acid sequence in cis or trans. Such regulatory sequences may include, for example, one or more promoters, enhancers, introns, Kozak sequences, polyadenylation sequences, and TATA signals. Regulatory sequences may include, for example, transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance nucleic acid or protein stability; and, when desired, sequences that enhance protein processing and / or secretion. Many diverse expression regulatory sequences, including those native and non-native, constitutive, inducible, and / or tissue-specific, are known in the art and may be utilized herein depending on the desired type of expression. Among other elements, an expression cassette may include, among other elements, one or more upstream (5'~) regulatory sequences of the gene sequence, such as promoters, enhancers, and introns, and one or more enhancers or downstream (3'~) regulatory sequences of the gene sequence, such as a 3' untranslated region (3'UTR) containing a polyadenylation site. In certain embodiments, a regulatory sequence is operably linked to the nucleic acid sequence of a gene product, and the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intervening nucleic acid sequence, i.e., a 5' untranslated region (5'UTR). In certain embodiments, the expression cassette contains the nucleic acid sequences of one or more gene products. In some embodiments, the expression cassette can be a monocistronic or bicistronic expression cassette.In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source that are inserted into a target cell. Typically, such an expression cassette for generating a viral vector contains a coding sequence for the gene product described herein, adjacent to the packaging signal of the viral genome, and other expression regulatory sequences, such as those described herein. In certain embodiments, the vector genome may contain two or more expression cassettes.

[0052] As used herein, the terms “regulatory sequence,” “regulatory control sequence,” or “expression control sequence” refer to nucleic acid sequences, such as initiator sequences, enhancer sequences, promoter sequences, intron sequences, and poly(A) signal sequences, that lead, enable, induce, repress, or otherwise control the transcription, translation, and / or expression of protein-coding nucleic acid sequences to which they are operably linked.

[0053] When used to describe nucleic acid sequences or proteins, the term "exogenous" means that the nucleic acid or protein does not spontaneously arise at its location within a chromosome or host cell. Furthermore, an exogenous nucleic acid sequence refers to a sequence that originates from the same host cell or subject and is inserted within them, but exists in an unnatural state, for example, at different copy numbers or under the control of different regulatory elements.

[0054] When used to describe nucleic acid sequences or proteins, the term “heterogeneous” means that the nucleic acid or protein originates from a different organism or a different species of the same organism from the host cell or target in which it is expressed. When used in relation to proteins or nucleic acids in plasmids, expression cassettes, or vectors, the term “heterogeneous” means that the protein or nucleic acid exists in a different sequence or subsequence, and that the different sequence or subsequence is in another sequence or subsequence, and that the aforementioned proteins or nucleic acids are naturally related to each other. To show that they are not found in the same relationship.

[0055] In one embodiment, the regulatory sequence includes a promoter. In one embodiment, the promoter is a chicken β-actin promoter. In a further embodiment, the promoter is a hybrid of a cytomegalovirus early enhancer and a chicken β-actin promoter. In a further embodiment, the promoter is a cytomegalovirus early enhancer (CMV IE) A CB7 (also called hybrid CB7) promoter containing an enhancer and a chicken β-actin promoter, optionally having a spacer sequence, containing a chicken β-actin intron, and optionally having a chimeric intron further containing a chicken β-actin splicing donor (containing an exon sequence and a chicken β-actin intron) and a rabbit beta-globin splicing acceptor.In another embodiment, preferred promoters include, but are not limited to, the elongation factor 1 alpha (EF1 alpha) promoter (see, e.g., Kim DW et al, Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23), the synapsin 1 promoter (see, e.g., Kuegler S et al, Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47), and the neuron-specific enolase (NSE) promoter (see, e.g., Kim J et al, Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004). It may include a CB6 promoter (see Feb;145(2):613-9.Epub 2003 Oct 16), or a CB6 promoter (see, for example, Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol Biotechnol.2016 Jan;58(1):30-6.Doi:10.1007 / s12033-015-9899-5).

[0056] In one embodiment, the expression cassette is designed for expression and secretion in human subjects. In one embodiment, the expression cassette is designed for expression in the central nervous system (CNS), including cerebrospinal fluid and the brain. In further embodiments, the expression cassette is useful for expression in both the CNS and the liver. Preferred promoters may include, but are not limited to, constitutive promoters, tissue-specific promoters, or inducible / regulatory promoters. An example of a constitutive promoter is the chicken beta-actin promoter. Various chicken beta-actin promoters have been described alone or in combination with various enhancer elements (e.g., CB7 is a chicken beta-actin promoter with a cytomegalovirus enhancer element; the CAG promoter includes the promoter, the first exon and first intron of chicken beta-actin, and the splice acceptor of the rabbit beta-globin gene; the CBh promoter, SJ Gray et al, Hu Gene Ther, 2011 Sep;22(9):1143-1153). Examples of tissue-specific promoters include liver (albumin, Miyatake et al., (1997) J. Virol., 71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) G Gene Ther., 3:1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503-14), neurons (e.g., neuron-specific enolase (NSE) promoter, Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503-15; neurofilament light chain gene, Piccioli et al. al., (1991) Proc. Natl. Acad. Sci. USA, 88:5611-5; and neuron-specific vgf genes, Piccioli et al., (1995) Neuron, 15:373-84), and other tissues are well known. Alternatively, a moduloable promoter may be selected. See, for example, WO2011 / 126808B2, which is incorporated herein by reference.

[0057] In one embodiment, the regulatory sequence further includes an enhancer. In one embodiment, the regulatory sequence includes one enhancer. In another embodiment, the regulatory sequence includes two or more enhancers. These enhancers may be the same or different. For example, an enhancer may include an alpha mic / bik enhancer or a CMV enhancer (e.g., a CMV IE enhancer). This enhancer may be present in two copies located adjacent to each other. Alternatively, duplicate copies of an enhancer may be separated by one or more sequences.

[0058] In one embodiment, the regulatory sequence further comprises an intron. In a further embodiment, the intron is a chicken beta-actin intron. In a further embodiment, the intron is a chimeric intron comprising a chicken beta-actin intron. In a further embodiment, the intron is a chimeric intron comprising a chicken beta-actin intron, and further comprising a chicken beta-actin splicing donor (containing an exon sequence, a chicken beta-actin intron) and a rabbit beta-globulin splicing acceptor. Other suitable introns include those known in the art, human beta-globulin introns, and / or commercially available chimeric introns from Promega®, and those described in WO2011 / 126808.

[0059] In one embodiment, the regulatory sequence further comprises a polyadenylation signal (PolyA). In a further embodiment, PolyA is rabbit globin PolyA. See, for example, WO2014 / 151341. Alternatively, another PolyA, such as a human growth hormone (hGH) polyadenylation sequence, SV40 PolyA, or synthetic PolyA, may be included in the expression cassette.

[0060] In certain embodiments, the regulatory sequence includes a hybrid promoter, which comprises a CMV IE enhancer, a chicken beta-actin promoter, and a chimeric intron containing a chicken beta-actin intron. In one embodiment, the regulatory sequence includes a promoter element, which comprises a chicken beta-actin promoter having the sequence of SEQ ID NO: 4 or a sequence that is at least 99.9% identical thereto. In one embodiment, the regulatory sequence includes an enhancer element, which comprises a CMV IE enhancer having the sequence of SEQ ID NO: 3 or a sequence that is at least 99.9% identical thereto. In further embodiments, the regulatory sequence includes an intron, which comprises a chicken beta-actin intron having the sequence of SEQ ID NO: 5 or a sequence that is at least 99.9% identical thereto. In one embodiment, the regulatory sequence further comprises rabbit beta-globin polyA having the sequence of SEQ ID NO: 6 or a sequence that is at least 99.9% identical thereto. In certain embodiments, the AAV vector genome includes an expression cassette containing the sequence of SEQ ID NO: 7 (CB7.CI.hNAGLUcoV3.RBG). In certain embodiments, the AAV vector genome includes the sequence of SEQ ID NO: 8 (AAV.CB7 Includes .CI.hNAGLUcoV3.RBG).

[0061] It should be understood that the compositions in the expression cassettes described herein are intended to be applied to other compositions, regimens, aspects, embodiments, and methods described herein.

[0062] 3. Vector In one embodiment, a vector (e.g., a recombinant adeno-associated virus vector having an AAV capsid) is provided herein, comprising an engineered nucleic acid sequence encoding a functional human NAGLU and a regulatory sequence (e.g., in a vector genome packaged within an rAAV capsid) that leads to its expression in target cells. In one embodiment, the hNAGLU coding sequence is at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3). In a further embodiment, the hNAGLU coding sequence is SEQ ID NO: 1 (hNAGLUcoV3).

[0063] As used herein, “vector” is a biological or chemical portion comprising a nucleic acid sequence that can be introduced into a suitable target cell for replication or expression of the nucleic acid sequence. Examples of vectors include, but are not limited to, recombinant viruses, plasmids, lipoplexes, polymerosomes, polyplexes, dendrimers, cell-permeable peptide (CPP) conjugates, magnetic particles, or nanoparticles. In one embodiment, the vector is a nucleic acid molecule, in which an exogenous or heterologous or engineered nucleic acid encoding a functional hNAGLU may be inserted, which can then be introduced into a suitable target cell. Such a vector preferably has one or more origins of replication and one or more sites into which recombinant DNA can be inserted. Vectors often have means by which cells having the vector can be selected from cells that do not have them, for example, the vector encoding a drug resistance gene. Common vectors include plasmids, viral genomes, and “artificial chromosomes.” Conventional methods for generating, producing, characterizing, or quantifying vectors are available to those skilled in the art.

[0064] In one embodiment, the vector is a nonviral plasmid containing its expression cassette as described, e.g., “Naked DNA”, “Naked Plasmid DNA”, RNA, and mRNA, the nonviral plasmid conjugated with various compositions and nanoparticles, the compositions and nanoparticles including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polyglycan compositions, and other polymers, lipid and / or cholesterol-based nucleic acid conjugates, and other constructs, e.g., those described herein. See, for example, X. Su et al, Mol. Pharmaceuticals, 2011, 8(3), pp 774-787; published online: March 21, 2011, WO2013 / 182683, WO2010 / 053572, and WO2012 / 170930, all of which are incorporated herein by reference.

[0065] In certain embodiments, the vectors described herein are “replica-deficient viruses” or “viral vectors,” where “replica-deficient viruses” or “viral vectors” means synthetic or artificial viral particles in which an expression cassette containing a nucleic acid sequence encoding a functional hNAGLU is packaged within a viral capsid or envelope, and any viral genome sequence similarly packaged within the viral capsid or envelope is replication-deficient, i.e., unable to produce progeny virions but retaining the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome contains signals required for amplification and packaging of the artificial genome). These genes can be manipulated to be "gutless," containing only the nucleic acid sequences encoding NAGLU adjacent to the gene, but these genes can be supplied during production. Therefore, replication and infection by the progeny virions cannot occur without the presence of the viral enzymes required for replication, and thus they are considered safe for use in gene therapy.

[0066] As used herein, recombinant viral vectors are adeno-associated viruses (AAV), adenoviruses, bocaviruses, hybrid AAV / bocaviruses, herpes simplex viruses, or lentiviruses.

[0067] As used herein, the term “host cell” may refer to a packaging cell line on which a vector (e.g., recombinant AAV) is produced. A host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect, or yeast) containing exogenous or heterologous DNA, wherein the exogenous or heterologous DNA is introduced into the cell by any of the following means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, fast DNA-coated pellets, viral infection, and protoplast fusion. Examples of host cells may include, but are not limited to, isolated cells, cell cultures, Escherichia coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK-293 cells, liver cells, kidney cells, central nervous system cells, neurons, glial cells, or stem cells.

[0068] As used herein, the term “target cells” refers to any target cells on which the expression of functional NAGLU is desired. In certain embodiments, the term “target cells” is intended to refer to the target cells being treated with MPS IIIB. Examples of target cells include, but are not limited to, liver cells, kidney cells, central nervous system cells, neurons, glial cells, and stem cells. In certain embodiments, the vector is delivered to the target cells ex vivo. In certain embodiments, the vector is delivered to the target cells in vivo.

[0069] It should be understood that the compositions in the vectors described herein are intended to be applied to other compositions, regimens, aspects, embodiments, and methods described herein.

[0070] 4. Adeno-associated virus (AAV) In one embodiment, a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein is provided herein. The rAAV is intended for use in mucopolysaccharidosis III B (MPS IIIB). In one embodiment, a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome is provided herein, wherein the vector genome is a nucleic acid molecule comprising an expression cassette, the expression cassette comprising an engineered nucleic acid sequence encoding functional human N-acetyl-alpha-glucosaminidase (hNAGLU) and operably linked to a regulatory sequence that leads to the expression of hNAGLU in target cells, the hNAGLU coding sequence being sequence number 1 or a sequence at least 99% identical to sequence number 1 (hNAGLUcoV3). In certain embodiments, rAAV comprises a vector genome, the vector genome comprising an AAV 5' inverted end repeat (ITR), an expression cassette comprising an engineered nucleic acid sequence encoding the functional hNAGLU described herein, a regulatory sequence that leads to the expression of hNAGLU in target cells, and an AAV 3'ITR. The present cassette comprises a regulatory sequence operably linked thereto, which induces the expression of hNAGLU in target cells, and an AAV 3'ITR.

[0071] In another embodiment, a recombinant nucleic acid molecule comprising a vector genome is provided herein, wherein the vector genome comprises an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), an expression cassette, and an AAV 3'ITR, and the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 7. In a particular embodiment, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 8. In a particular embodiment, the recombinant nucleic acid molecule is a plasmid.

[0072] In one embodiment, the hNAGLU coding sequence is at least 99% identical to SEQ ID NO: 1 (hNAGLUcoV3). In a further embodiment, the hNAGLU coding sequence is SEQ ID NO: 1 (hNAGLUcoV3). In one embodiment, the regulatory sequence includes a promoter. In a further embodiment, the regulatory sequence further includes an enhancer. In one embodiment, the regulatory sequence further includes an intron. In one embodiment, the regulatory sequence further includes poly(A). In a particular embodiment, the AAV vector genome includes an expression cassette containing the sequence of SEQ ID NO: 7 (CB7.CI.hNAGLUcoV3.RBG) encoding the hNAGLU protein of SEQ ID NO: 2. In a particular embodiment, the AAV vector genome includes the sequence of SEQ ID NO: 8 (AAV.CB7.CI.hNAGLUcoV3.RBG) encoding the hNAGLU protein of SEQ ID NO: 2. In one embodiment, the AAV capsid is the AAVhu68 capsid, which has coding sequence sequence number 9 and / or amino acid sequence number 10. In another embodiment, the AAV capsid is the AAVhu95 capsid, which has coding sequence sequence number 13 and / or amino acid sequence number 14. In yet another embodiment, the AAV capsid is the AAVhu96 capsid, which has coding sequence sequence number 15 and / or amino acid sequence number 16. In yet another embodiment, the AAV capsid is the AAV9 capsid, which has coding sequence sequence number 17 and / or amino acid sequence number 18. In yet another embodiment, the AAV capsid is the AAVrh91 capsid, which has coding sequence sequence number 11 and / or amino acid sequence number 12. In one embodiment, the rAAV described herein is for use in mucopolysaccharidosis III B (MPS IIIB).Furthermore, see also International Patent Application PCT / US2021 / 055436, filed on 18 October 2021 and currently International Publication No. 2022 / 082109; International Patent Application PCT / US2022 / 077315, filed on 30 September 2022 and currently International Publication No. 2023 / 056399; and International Patent Application PCT / US2021 / 045945, filed on 13 August 2021 and currently International Publication No. 2022 / 036220, which are incorporated herein by reference in their entirety.

[0073] In one embodiment, the regulating array is as described above.

[0074] In one embodiment, an rAAV is provided comprising an AAV serotype hu68 (AAVhu68) capsid and a vector genome, wherein the vector genome comprises a CB7 promoter expressing an engineered version of hNAGLU and a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (CB7.CI.hNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAVhu68 capsid and a vector genome comprising an expression cassette containing the sequence of SEQ ID NO: 7, and the rAAV is represented as AAVhu68.CB7.CI.hNAGLUcoV3.rBG. In one embodiment, the rAAV comprises an AAVhu68 capsid and a vector genome containing the sequence of SEQ ID NO: 8, and the rAAV is represented as AAVhu68.CB7.CI.hNAGLUcoV3.rBG.

[0075] In one embodiment, an rAAV is provided comprising an AAV serotype hu95 (AAVhu95) capsid and a vector genome, wherein the vector genome comprises a CB7 promoter expressing an engineered version of hNAGLU and a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (AAV.CB7.CI.hNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAVhu95 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, and the rAAV is represented as AAVhu95.CB7.CI.hNAGLUcoV3.rBG.

[0076] In one embodiment, an rAAV is provided comprising an AAV serotype hu96 (AAVhu96) capsid and a vector genome, wherein the vector genome comprises a CB7 promoter expressing an engineered version of hNAGLU and a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (.CB7.CI.hNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAVhu96 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, and the rAAV is represented as AAVhu96.CB7.CI.hNAGLUcoV3.rBG.

[0077] In one embodiment, an rAAV is provided comprising an AAV serotype 9 (AAV9) capsid and a vector genome, wherein the vector genome comprises a CB7 promoter expressing an engineered version of hNAGLU and a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (CB7.CI.hSNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAV9 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, and the rAAV is represented as AAV9.CB7.CI.hSNAGLUcoV3.rBG.

[0078] In one embodiment, an rAAV is provided comprising an AAV serotype rh91 (AAVrh91) capsid and a vector genome, wherein the vector genome comprises a CB7 promoter expressing an engineered version of hNAGLU and a rabbit beta-globin (rBG) polyA sequence. In a further embodiment, the rAAV vector genome comprises the sequence of SEQ ID NO: 8 (CB7.CI.hSNAGLUcoV3.rBG). In one embodiment, the rAAV comprises an AAVrh91 capsid and a vector genome comprising the sequence of SEQ ID NO: 8, and the rAAV is represented as AAVrh91.CB7.CI.hSNAGLUcoV3.rBG.

[0079] As used herein, the term “vector genome” refers to a nucleic acid molecule packaged within a viral capsid, such as an AAV capsid, that can be delivered to a host cell or a patient cell. In certain embodiments, the vector genome includes terminal repeat sequences (e.g., AAV inverted terminal repeat sequences (ITRs)) at the 5' and 3' ends, which are necessary to package the vector genome within the capsid and contain an expression cassette between them, the expression cassette containing a nucleic acid sequence that encodes a functional NAGLU as described herein and is operably linked to a regulatory sequence that leads to its expression. In one example, the vector genome contains, at a minimum, an AAV2 5'ITR, a nucleic acid sequence encoding a functional NAGLU, and an AAV2 3'ITR, from 5' to 3'. However, ITRs may be selected from AAVs of different sources other than AAV2. Furthermore, other ITRs may be used. In addition, the vector genome contains a regulatory sequence that leads to the expression of the functional NAGLU.

[0080] The AAV sequence of the vector typically contains cis-acting AAV5' and AAV3' inverted terminal repeat (ITR) sequences (e.g., B.J. Carter, in “Handbook”). See "of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990). The ITR sequence is approximately 145 base pairs (bp) in length. Preferably, a substantially complete sequence encoding the ITR is used within the molecule, but some minor modifications to these sequences are permissible. The ability to modify these ITR sequences is within the scope of the art. (e.g., Sambrook et al.) See K. Fisher et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989), and K. Fisher et al., J. Virol., 70:520 532 (1996). An example of such a molecule used in the present invention is a “cis-acting” plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are adjacent to 5' and 3' AAV ITR sequences (also referred to as “AAV 5'ITR”, “5'ITR”, “AAV 5'ITR”, or “5'ITR”, “AAV 3'ITR”, “3'ITR”, “AAV 3'ITR”, or “3'ITR”). In one embodiment, the ITR is from a different AAV than the one supplying the capsid. In one embodiment, the ITR sequence is from AAV2. However, ITRs from other AAV sources may be selected. A shortened version of the 5' ITR, referred to as ΔITR, has been described, in which the D sequence and terminal separation sites (trs) are deleted. In certain embodiments, the vector genome contains a shortened AAV2 ITR of 130 base pairs, in which the outer A element is deleted. While we do not wish to be bound by theory, it is thought that the shortened ITR is restored to the 145 base pair wild-type (WT) length during vector DNA amplification using the inner (A') element as a template. In other embodiments, full-length AAV 5' and 3' ITRs are used. If the ITR source is from AAV2 and the AAV capsid is from a different AAV source, the resulting vector may be referred to as pseudotyped. However, other configurations of these elements may be preferred.

[0081] In certain embodiments, rAAVs are provided herein that include a nucleic acid molecule comprising a vector genome, which is a vector genome and an expression cassette, which comprises a nucleic acid molecule having at least one AAV ITR at its 5' and / or 3' ends. In certain embodiments, the vector genome is a nucleic acid molecule comprising a 5'-AAV ITR, an expression cassette, and a 3'-AAV ITR.

[0082] In certain embodiments, rAAV comprises a vector genome containing a nucleic acid molecule, the nucleic acid molecule comprising, from 5' to 3', an AAV-5'ITR-optional enhancer-promoter-optional intron-coding sequence-polyadenylation (polyA) signal sequence-AAV3'-ITR. In other embodiments, the orientation of the ITRs may vary from the orientation presented to the vector genome of the nucleic acid (e.g., plasmid) used for production. Thus, in certain embodiments, rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs, respectively. In certain embodiments, rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, the rAAV provided herein may be partially cleaved such that the 5' AAV ITR and / or 3' AAV ITR are not detectable within the vector genome packaged in the final rAAV product.

[0083] As used herein, the term “AAV” means naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or available in view of the compositions and methods described herein, and artificial AAVs. Adeno-associated virus (AAV) viral vectors contain an AAV protein capsid. The AAV DNase-resistant particle, in which the expression cassette is packaged, is adjacent to the inverted terminal repeat (ITR) of the AAV for delivery to target cells. The AAV capsid consists of 60 capsid protein subunits, VP1, VP2, and VP3, arranged icosahedral symmetrically in a ratio of approximately 1:1:10 to 1:1:20, depending on the selected AAV. Various AAVs can be selected as the source of the capsid for the AAV viral vector described above. See, for example, U.S. Patent Application Publications 2007 / 0036760-A1, 2009 / 0197338-A1, and EP1310571. See also WO2003 / 042397 (AAV7 and other monkey AAVs), U.S. Patents 7,790449 and 7,282199 (AAV8), WO2005 / 033321 and U.S. 7,906,111 (AAV9), and WO2006 / 110689 and WO2003 / 042397 (rh.10). These documents also describe and are incorporated by reference other AAVs that may be selected to generate AAVs. Of the AAVs isolated or manipulated from humans or non-human primates (NHPs) and well-characterized, human AAV2 was the first AAV developed as a gene transfer vector and is widely used in efficient gene transfer experiments in various target tissues and animal models. Unless otherwise specified, the AAV capsids, ITRs, and other selected AAV components described herein may be readily selected from any of the AAVs, and the AAVs include, but are not limited to, AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, any variant of any known or referred AAV or AAV discovered herein, or any of those variants or mixtures thereof. See, for example, WO2005 / 033321, which is incorporated herein by reference.

[0084] In a particular embodiment, the AAV capsid is clade F AAV, and clade F AAV capsids include AAVhu68 capsid [for example, see US2020 / 0056159, PCT / US21 / 55436, and PCT / US18 / 19992 filed on 27 February 2018, which is now International Publication No. 2018 / 160582, and is incorporated herein by reference], AAVhu95 capsid [for example, U.S. Patent Provisional Application No. 63 / filed on 2 October 2201] The capsid is selected from [see U.S. Patent Application No. 251,599, International Patent Application PCT / US2022 / 077315 filed September 30, 2022], or AAVhu96 capsid [see, for example, U.S. Provisional Patent Application No. 63 / 251,599 filed October 2, 2201, and International Patent Application PCT / US2022 / 077315 filed September 30, 2022], or AAV9 capsid. In certain embodiments, the AAV capsid is a clade A capsid, for example, AAVrh91 capsid. Please refer to PCT / US20 / 030266, filed on 29 April 2020, currently International Publication No. 2020 / 223231, which is incorporated herein by reference, and please refer to International Application PCT / US21 / 45945, filed on 13 August 2021, which is incorporated herein by reference.

[0085] In certain embodiments, the AAV capsid is AAVhu68 capsid. In certain embodiments, the AAV capsid is AAV9 capsid. In certain embodiments, the AAV capsid is AAVhu95 capsid. In certain embodiments, the AAV capsid is AAVhu96 capsid.

[0086] In certain embodiments, the AAV capsid for the compositions and methods described herein is selected based on target cells. In certain embodiments, the AAV capsid transduces CNS cells and / or PNS cells. In certain embodiments, other AAV capsids may be selected. AAV capsids include cy02 capsid, rh43 capsid, AAV8 capsid, rh01 capsid, AAV9 capsid, rh8 capsid, rh The capsid is selected from 10-capsid, bb01-capsid, hu37-capsid, rh02-capsid, rh20-capsid, rh39-capsid, rh64-capsid, AAV6-capsid, AAV1-capsid, hu44-capsid, hu48-capsid, cy05-capsid, hu11-capsid, hu32-capsid, pi2-capsid, or variations thereof. In certain embodiments, the AAV capsid is a clade F-capsid, such as AAV9-capsid, AAVhu68-capsid, hu31-capsid, hu32-capsid, or variations thereof. For example, see WO2005 / 033321, WO2018 / 160582, and US2015 / 0079038, published on April 14, 2015, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV capsid is a non-clade F capsid, e.g., a clade A, B, C, D, or E capsid. In certain embodiments, the non-clade F capsid is AAV1 or a variation thereof. In certain embodiments, the AAV capsid transduces target cells other than neural cells. In certain embodiments, the AAV capsid is a clade A capsid (e.g., AAV1, AAV6, AAVrh91), a clade B capsid (e.g., AAV2), a clade C capsid (e.g., hu53), a clade D capsid (e.g., AAV7), or a clade E capsid (e.g., rh10).

[0087] AAV capsids are aggregates of heterogeneous populations of vp1 protein, vp2 protein, and vp3 protein. As used herein, the term “heterogeneous” or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a population consisting of non-identical elements, e.g., a population having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0088] As used herein, when used to refer to vp capsid proteins, the term “heterogeneous” or any grammatical variation thereof refers to a group of non-identical elements having vp1, vp2, or vp3 (also referred to as VP1, VP2, VP3, or Vp1, Vp2, Vp3) monomers (proteins) with different modified amino acid sequences, for example. The term “heterogeneous group” as used in relation to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to the differences in amino acid sequences of vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1 protein, vp2 protein, and vp3 protein, which have modifications from predicted amino acid residues. These subpopulations contain, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, a particular subpopulation may have at least one, two, three, or four highly deamidated asparagine-glycine pairs, and optionally include an asparagine(N) position further containing other deamidated amino acids, where deamide results in amino acid changes and other optional modifications.

[0089] In certain embodiments, an AAV capsid is provided having a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3), wherein the AAV capsid isoforms contain a plurality of highly deamidated "NG" positions. In certain embodiments, the highly deamidated positions are located at positions specified below with respect to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that a referenced "NG" is removed, and the mutant "NG" is manipulated to a different position.

[0090] As used herein, with respect to AAV, the term “variant” means any AAV sequence derived from a known AAV sequence that has a conserved amino acid substitution, and at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least This means AAV sequences that share 97%, at least 99%, or more sequence identity. In another embodiment, an AAV capsid may include variants that may contain up to about 10% variation from any described or known AAV capsid sequence. That is, an AAV capsid shares about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with any AAV capsid provided herein and / or known in the art. In one embodiment, an AAV capsid shares at least 95% identity with any AAV capsid. When determining the identity percentage of an AAV capsid, the comparison may be made across any of the variable proteins (e.g., vp1, vp2, or vp3).

[0091] ITRs or other AAV components may be readily isolated or manipulated from AAVs using techniques available to those skilled in the art. Such AAVs may be isolated, manipulated, or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences may be manipulated by synthesis or other suitable means, or by referencing publicly available sequences, e.g., in the literature or databases, e.g., GenBank or PubMed. AAV viruses may be manipulated by conventional molecular biology techniques to enable optimization of these particles for cell-specific delivery of nucleic acid sequences, minimization of immunogenicity, adjustment of stability and particle lifetime, efficient degradation, and precise delivery to the nucleus.

[0092] As used herein, the terms “rAAV” and “artificial AAV” are used interchangeably and, non-limitingly, mean an AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprises a nucleic acid heterologous to the AAV. In one embodiment, the capsid protein is a non-spontaneously occurring capsid. Such an artificial capsid may be produced by any preferred technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence, the heterologous sequence may be obtained from a different selected AAV, a non-adjacent portion of the same AAV, a non-AAV viral source, or a non-viral source. The artificial AAV may, non-limitingly, be a pseudotyped AAV capsid, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. A pseudotyped vector in which the capsid of one AAV is replaced with a heterologous capsid protein is useful in the present invention. In one embodiment, AAV2 / 5 and AAV2 / 8 are exemplary pseudotype vectors. Selected gene elements may be delivered by any preferred method, including transfection, electroporation, liposome delivery, membrane fusion techniques, rapid DNA coating pellets, viral infection, and protoplast fusion. Methods used to construct such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombination, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0093] In one embodiment, the rAAV described herein is a self-complementary AAV. “Self-complementary AAV” refers to a construct in which the coding region held by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV associate to form a single double-stranded DNA (dsDNA) unit readily available for immediate replication and transcription. For example, DM McCarty et al., “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficacy.” See “ent transduction independently of DNA synthesis”, Gene Therapy, (August 2001), Vol. 8, No. 16, pp. 1248–1254. Self-complementary AAVs are described, for example, in U.S. Patents 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in whole.

[0094] In certain embodiments, the rAAVs described herein are nuclease-resistant. Such nucleases may be single nucleases or mixtures of nucleases, and may be endonucleases or exonucleases. Nuclease-resistant rAAVs indicate that the AAV capsid is fully assembled and these packaged genomic sequences are protected from degradation (digestion) during a nuclease incubation step designed to remove any contaminating nucleic acids from the production process. In many cases, the rAAVs described herein are DNase-resistant.

[0095] The recombinant adeno-associated viruses (AAVs) described herein may be generated using known techniques. See, for example, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, US7588772B2, and WO2017 / 136500, which are incorporated herein by reference. Such a method involves culturing a host cell to contain a nucleic acid sequence encoding an AAV capsid, a functional rep gene, an expression cassette described herein adjacent to an AAV inverted terminal repeat (ITR), and sufficient helper function to enable packaging of the expression cassette into the AAV capsid protein. A host cell containing a nucleic acid sequence encoding an AAV capsid, a functional rep gene, a described vector genome, and sufficient helper function to enable packaging of the vector genome into the AAV capsid protein is also provided herein. In one embodiment, the host cells are HEK293 cells. These methods are described in further detail in WO2017 / 160360A2, which is incorporated herein by reference.

[0096] In one embodiment, a cell culture useful for producing recombinant AAV is provided, wherein the recombinant AAV has a capsid selected from AAVhu68, AAVrh91, AAVhu95, or AAVhu96. Such a cell culture contains a nucleic acid molecule, e.g., a vector genome, which is a nucleic acid molecule suitable for packaging within the AAVhu68 capsid, and includes an AAV ITR and a non-AAV nucleic acid sequence that encodes a gene and is operably linked to a regulatory sequence that leads to gene expression in the host cell, and also contains sufficient AAV rep and adenovirus helper functions to enable packaging of the vector genome into recombinant AAVhu68 or AAVrh91 capsid (e.g., SEQ ID NO: 11 or SEQ ID NO: 12), AAVhu95 capsid (e.g., SEQ ID NO: 13 or SEQ ID NO: 14), or AAVhu96 capsid (e.g., SEQ ID NO: 15 or SEQ ID NO: 16). In one embodiment, the cell culture consists of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., Spodoptera frugiperda (Sf9) cells). In certain embodiments, the baculovirus provides the necessary helper functions for packaging the vector genome within a recombinant AAVhu68, AAVrh91, AAVhu95, or AAVhu96 capsid.

[0097] Other methods for producing rAAV, available to those skilled in the art, may be utilized. Preferred methods may, but are not limited to, baculovirus expression systems or yeast-mediated production. For example, Robert M. Kotin, Large-scale recombinant adeno-associated virus production.Hum M ol Genet. 2011 Apr 15; 20(R1): R2 - R6. Online publication on April 29, 2011. Doi: 10.1093 / hmg / ddr141, Aucoin MG et al., Production of adeno - associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios. Biotechnol Bioeng. 2006 Dec 20; 95(6): 1081 - 92, SAMI S. THAKUR, Production of Recombinant Adeno - associated viral vectors in yeast. Thesis presented to the Graduate School of the University of Florida, 2012, Kondratov O et al. Direct Head - to - Head Evaluation of Recombinant Adeno - associated Viral Vectors Manufactured in Human versus Insect Cells, Mol Ther. 2017 Aug 10. Pii: S1525 - 0016(17)30362 - 3. Doi: 10.1016 / j.ymthe.2017.08.003. [Epub before print], Mietzsch M et al, OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA. Hum Gene Ther Methods. 2017 Feb; 28(1): 15 - 22. Doi: 10.1089 / hgtb.2016.164., Li L et al.Production and characterization of novel recombinant adeno-associated virus replicative-form genomes:a eukaryotic source of DNA for gene transfer.PloS One.2013 Aug 1;8(8):e69879.Doi:10.1371 / journal.pone J Invertebr Pathol.2011 Jul;107 Suppl:S80-93.Doi:10.1016 / j.jip doi:10.1093 / hmg / ddr141.Epub 2011 Apr 29. Hum Mol Genet.2011 Apr 15;20(R1):R2-6.

[0098] A two-step affinity chromatography purification at high salt concentrations, followed by anion exchange resin chromatography, is used to purify the vector drug product and remove empty capsids. These methods are described in detail in International Patent Application PCT / US2016 / 065970 and US11,098,286B2 (incorporated herein by reference), filed on 9 December 2016, entitled "Scalable Purification Method for AAV9". A purification method for AAV8 is described in International Patent Application PCT / US2016 / 065976 and US11,015,174B2 (incorporated herein by reference), filed on 9 December 2016, entitled "Scalable Purification Method for AAV8". A method for purifying rh10, international patent applications PCT / US16 / 066013 and US11,028,372B2 filed on 9 December 2016, entitled "Scalable Purification Method for AAVrh10" (as incorporated herein by reference). (Incorporated herein) A method for purifying AAV1 is described in International Patent Application PCT / US2016 / 065974 and US11,015,173B2, filed on 9 December 2016, entitled "Scalable Purification Method for AAV1" (incorporated herein by reference). See also International Patent Application PCT / US2018 / 019992, filed on 27 February 2018, now International Publication No. 2018 / 160582, and International Patent Application PCT / US2021 / 055436, filed on 18 October 2021, now International Publication No. 2022 / 082109, both of which are incorporated herein by reference in their entirety. Other preferred methods may be selected.

[0099] Conventional methods for characterizing or quantifying rAAV are available to those skilled in the art. To calculate the content of empty and packed particles, the VP3 band volume for a selected sample (e.g., in the example herein, an iodixanol gradient-purified preparation, where the number of GCs equals the number of particles) is plotted against the loaded GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. The ratio of particles to genome copies (pt / GC) is obtained by dividing Pt / mL by GC / mL. Empty pt / mL is obtained by Pt / mL - GC / mL. The percentage of empty particles is obtained by dividing empty pt / mL by pt / mL and multiplying by 100. In general, methods for assaying AAV vector particles having empty capsids and packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330 and Sommer et al., Molec.Ther. (2003) 7:122-128. To test for denatured capsids, the method comprises subjecting a treated AAV stock to SDS-polyacrylamide gel electrophoresis, wherein the SDS-polyacrylamide gel electrophoresis consists of any gel capable of separating three capsid proteins, for example, a gradient gel containing 3-8% trisacetic acid in buffer; then running the gel until the sample material is separated; and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. Next, the anti-AAV capsid antibody is used as a primary antibody that binds to the denatured capsid protein, preferably as an anti-AAV capsid monoclonal antibody, and most preferably as a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J.Viral. (2000) 74:9281-9293).Next, a secondary antibody is used, which contains means for binding to the primary antibody and detecting the binding to the primary antibody, more preferably an anti-IgG antibody with a covalently bound detection molecule, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine the binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation, or colorimetric change, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, a sample may be taken from the column fraction and heated in an SDS-PAGE load buffer containing a reducing agent (e.g., DTT). Capsid proteins were separated on a precast gradient polyacrylamide gel (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions, or using other suitable staining methods, namely SYPRO ruby ​​or Coomassie staining. In one embodiment, the concentration of the AAV vector genome (vg) in the column fraction may be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, the sample is further diluted and amplified using a TaqMan® fluorescence-generating probe specific to the DNA sequence between primers and primers. A specified level of fluorescence (threshold cycle, Ct) is obtained. The number of cycles required to reach the target is measured for each sample on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. Alternatively, an endpoint assay based on digital PCR may be used.

[0100] In one embodiment, an optimized q-PCR method is used, which utilizes a broad-spectrum serine protease, such as proteinase K (e.g., commercially available from Qiagen). More specifically, the optimized qPCR genome titer assay is similar to a standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer, treated with proteinase K, and subsequently thermally inactivated. Preferably, the sample is diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated more than 2-fold. Typically, the proteinase K treatment is about 0.2 mg / mL, but can vary from 0.1 mg / mL to about 1 mg / mL. The processing steps are generally carried out at approximately 55°C for approximately 15 minutes, but may be carried out at lower temperatures (e.g., approximately 37°C to approximately 50°C) for longer periods (e.g., approximately 20 to approximately 30 minutes), or at higher temperatures (e.g., up to approximately 60°C) for shorter periods (e.g., approximately 5 to 10 minutes). Similarly, thermal inactivation is generally carried out at approximately 95°C for approximately 15 minutes, but the temperature may be lower (e.g., approximately 70 to approximately 90°C) and the time may be extended (e.g., approximately 20 to approximately 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in standard assays.

[0101] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, a method for determining single-stranded and self-complementary AAV vector genome titers by ddPCR has been described. See, for example, M. Lock et al, Hu Gene Therapy Methods, Hu Gene Ther Methods. 2014 Apr;25(2):115-25. Doi:10.1089 / hgtb.2013.131.Epub 2014 Feb 14.

[0102] Furthermore, methods are available for determining the ratios between the capsid proteins vp1, vp2, and vp3. For example, Vamseedhar Rayaprolu et al, Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics, J Virol. 2013 Dec;87(24):13150-13160, Buller See RM, Rose JA. 1978. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J. Virol. 25:331-338, and Rose JA, Maizel JV, Inman JK, Shatkin AJ. 1971. Structural proteins of adenovirus-associated viruses. J. Virol. 8:766-770.

[0103] As used herein, the terms “treatment” or “to treat” refer to a composition(s) and / or method(s) for the purpose of achieving remission of one or more symptoms of MPS IIIB, restoration of desired function of NAGLU, or improvement of a biomarker of the disease. In some embodiments, the terms “treatment” or “to treat” encompass administering one or more compositions described herein to a subject for the purposes described herein. It is defined as follows. Therefore, “treatment” may include one or more of the following in a given subject: reducing the onset or progression of MPS IIIB, preventing the disease, reducing the severity of disease symptoms, delaying their progression, eliminating disease symptoms, slowing the progression of the disease, or increasing the effectiveness of the therapy.

[0104] It should be understood that the compositions in rAAV described herein are intended to be applied to other compositions, regimes, aspects, embodiments, and methods described herein.

[0105] 5. Pharmaceutical composition In one aspect, a pharmaceutical composition comprising the vectors described herein in a formulation buffer is provided herein. In one embodiment, the pharmaceutical composition is suitable for co-administration with a functional hNAGLU protein or a protein comprising functional hNAGLU. In one embodiment, a pharmaceutical composition comprising the rAAV described herein in a formulation buffer is provided. In one embodiment, the rAAV is about 1×10 9 genome copies (GC) / mL to about 1×10 14 GC / mL. In a further embodiment, the rAAV is about 3×10 9 GC / mL to about 3×10 13 GC / mL. In still a further embodiment, the rAAV is about 1×10 9 GC / mL to about 1×10 13 GC / mL. In one embodiment, the rAAV is formulated at at least about 1×10 11 GC / mL.

[0106] Also provided herein is a composition comprising the rAAV described herein and an aqueous suspension medium. In certain embodiments, the suspension is formulated for intravenous delivery, intrathecal administration, or intraventricular administration. In one aspect, the composition contains at least one rAAV stock and an optional carrier, excipient, and / or preservative.

[0107] As used herein, a “stock” of rAAVs refers to a population of rAAVs. Despite the heterogeneity of capsid proteins resulting from deamidation, rAAVs within a stock are expected to share the same vector genome. A stock may include, for example, rAAVs having a capsid with a selected AAV capsid protein and a heterogeneous deamidation pattern characteristic of a selected production system. A stock may be produced from a single production system or pooled from multiple runs of a production system. A variety of production systems may be selected, including but not limited to those described herein.

[0108] In one embodiment, the formulation further comprises a surfactant, a preservative, an excipient, and / or a buffer dissolved in an aqueous suspension. In one embodiment, the buffer is PBS. In another embodiment, the buffer is artificial cerebrospinal fluid (aCSF), for example, Eliott's formulation buffer, or Harvard apparatus perfusion fluid (artificial CSF having a final ion concentration (mM) of Na 150, K 3.0, Ca 1.4, Mg 0.8, P 1.0, Cl 155). A variety of suitable solutions are known, and the solutions include buffered saline, a surfactant, and one or more physiologically suitable salts or mixtures of salts adjusted to an equivalent ion strength of about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or physiologically suitable salts adjusted to an equivalent ion concentration.

[0109] Preferably, the formulation is adjusted to a physiologically acceptable pH range, for example, pH 6–8, or pH 6.5–7.5, pH 7.0–7.7, or pH 7.2–7.8. Since the pH of cerebrospinal fluid is approximately 7.28–7.32, a pH within this range may be desired for intrathecal delivery, and a pH of 6.8–7.2 may be desired for intravenous delivery. However, other pH ranges within the broadest range and these sub-ranges may be selected for other delivery routes.

[0110] Suitable surfactants or combinations of surfactants may be selected from among non-toxic nonionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated at a primary hydroxyl group, such as Pluronic® F68 [BASF], also known as poloxamer 188, is selected, having a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, namely nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) adjacent to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), such as SOLUTOL HS 15 (macrogol-15 hydroxystearic acid), LABRASOL (polyoxycaprylic glyceride), polyoxy-10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer), followed by three digits, where the first two digits × 100 indicate the approximate molecular mass of the polyoxypropylene core, and the last digit × 10 indicates the percentage polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension.

[0111] For example, the formulation may contain, for instance, a buffered saline solution, which comprises one or more of the following in water: sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate·7H₂O), potassium chloride, calcium chloride (e.g., calcium chloride·2H₂O), sodium hydrogen phosphate, and mixtures thereof. Preferably, for intrathecal delivery, the molar osmotic concentration is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290), see, for example, emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent may be used as a suspension, or in combination with another suspension and other optional excipients. See, for example, Elliotts B® solution [Lukare Medical].

[0112] In other embodiments, the formulation may contain one or more penetration enhancers. Examples of suitable penetration enhancers include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA.

[0113] Additionally, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a vector containing a nucleic acid sequence encoding a functional NAGLU as described herein. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agent, isotonic and absorption retardant, buffer, carrier solution, suspension, and colloid. The use of such media and agents for pharmaceutically active substances is well known in the art. Furthermore, supplemental active ingredients may be incorporated into the composition. Delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles, may be used for the introduction of the composition of the present invention into suitable host cells. In particular, the rAAV vector may be formulated for delivery encapsulated within any of lipid particles, liposomes, vesicles, nanospheres, or nanoparticles. In one embodiment, a therapeutically effective amount of the vector is contained within the pharmaceutical composition. The choice of carrier is not limited to the present invention. Other conventional pharmaceutically acceptable carriers, such as preservatives or chemical stabilizers, may also be used. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, and phenol. , and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0114] The term "pharmaceutically acceptable" means that a molecular entity or composition does not cause an allergic reaction or similar adverse reaction when administered to a host.

[0115] As used herein, the terms “dosage” or “amount” may refer to the total amount or quantity of medication delivered to a subject over the course of treatment, or the amount or quantity of medication delivered in a single unit (or multiple units or divided doses).

[0116] Furthermore, the replication-defective virus composition is approximately 1.0 × 10⁻⁶. 9 The number of GCs is approximately 1.0 × 10⁻¹⁰. 16It can be formulated in dosage units to contain an amount of replication-defective virus in the range of GCs (to treat an average subject weighing 70 kg), the amount including all integers or fractions within the range, preferably 1.0 × 10 for a human patient. 12 GC of individual items ~ 1.0 × 10 14 It contains GCs. In one embodiment, the composition contains at least 1 × 10 per dose. 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9×10 9 The formulation contains GCs, the content of which includes all integer or fractional amounts within the range. In another embodiment, the composition contains at least 1 × 10 per dose. 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10 The formulation contains GCs, the content of which includes all integer or fractional amounts within the range. In another embodiment, the composition contains at least 1 × 10 per dose. 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9×10 11 The formulation contains GCs, the content of which includes all integer or fractional amounts within the range. In another embodiment, the composition contains at least 1 × 10 per dose. 12 , 2×10 12 , 3 x 10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×1012 , 8×10 12 , or 9×10 12 The formulation contains GCs, the content of which includes all integer or fractional amounts within the range. In another embodiment, the composition contains at least 1 × 10 per dose. 13 , 2×10 13 , 3 x 10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , or 9×10 13 The formulation contains GCs, the content of which includes all integer or fractional amounts within the range. In another embodiment, the composition contains at least 1 × 10 per dose. 14 , 2×10 14 , 3 x 10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9×10 14 The formulation contains GCs, the content of which includes all integer or fractional amounts within the range. In another embodiment, the composition contains at least 1 × 10 per dose. 15 , 2×10 15 , 3 x 10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9×10 15 The formulation contains GCs, and the content includes all integer or fractional amounts within the range. In one embodiment, for human use, the dose is 1 × 10⁶ per dose. 10 ~Approx. 1×10 12 The range of a garbage collector can be a number of integers or fractions, and it can include all integers or fractions within that range.

[0117] In a particular embodiment, the rAAV composition is approximately 1 × 10⁶ per gram of brain mass. 9about 1×10 per gram (g) of brain mass 13 and can be formulated into dosage units for containing an amount of rAAV within the range of about 1×10 10 genomic copies (GC) per gram of brain mass, the amount including all integer or fractional amounts and endpoints within the range. In another embodiment, the dosage is 1×10 13 GCs per gram of brain mass to about 1×10 9 GCs per gram of brain mass. In a specific embodiment, the dosage of the vector administered to the patient is at least about 1.0×10 9 GCs / g of brain mass, about 1.5×10 9 GCs / g of brain mass, about 2.0×10 9 GCs / g of brain mass, about 2.5×10 9 GCs / g of brain mass, about 3.0×10 9 GCs / g of brain mass, about 3.5×10 9 GCs / g of brain mass, about 4.0×10 .5×10 9 GCs / g of brain mass, about 5.0×10 9 GCs / g of brain mass, about 5.5×10 9 GCs / g of brain mass, about 6.0×10 9 GCs / g of brain mass, about 6.5×10 9 GCs / g of brain mass, about 7.0×10 9 GCs / g of brain mass, about 7.5×10 9 GCs / g of brain mass, about 8.0×10 9 GCs / g of brain mass, about 8.5×10 9 GCs / g of brain mass, about 9.0×10 9 GCs / g of brain mass, about 9.5×10 9 GCs / g of brain mass, about 1.0×10 10 GCs / g of brain mass, about 1.5×10 10 GCs / g of brain mass, about 2.0×10 10 GCs / g of brain mass, about 2.5×10 10 GCs / g of brain mass, about 3.0×10 10 GCs / g of brain mass, about 3.5×10 10 GCs / g of brain mass, about 4.0×10 10 GCs / g of brain mass, about 4.5×1010 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 10 The number of GCs per brain mass in g is approximately 1.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 1.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 11 The number of GCs per brain mass in g is approximately 1.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 1.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 12The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 12 The number of GCs per brain mass in g is approximately 1.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 1.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams, or approximately 1.0 × 10⁻⁶. 14 This is calculated as the number of GCs per brain mass in g.

[0118] In one embodiment, the pharmaceutical composition containing rAAV as described herein is approximately 1 × 10⁶ per gram of brain mass. 9 The number of GCs per gram of brain mass is approximately 1 x 10⁻¹⁶. 14 It can be administered in doses of this GC.

[0119] The aqueous suspensions or pharmaceutical compositions described herein are designed for delivery to a subject requiring them via any preferred route or a combination of different routes. In one embodiment, the pharmaceutical composition is formulated for delivery via intraventricular (ICV), intrathecal (IT), or intracisional injection. In one embodiment, the compositions described herein are designed for delivery to a subject requiring them by intravenous infusion. Alternative Other routes of administration may be selected (e.g., oral, inhalation, intranasal, intratracheal, intra-arterial, intraocular, intramuscular, and other parenteral routes). In certain embodiments, the pharmaceutical composition is delivered intrathecally, optionally via cisterna magna (ICM) injection. In certain embodiments, the composition is delivered via intraparenchymal administration. In certain embodiments, the composition is delivered via the Omaya reservoir delivery system.

[0120] As used herein, the terms “intrathecal delivery” or “intrathecal administration” refer to a route of administration for a drug in which the drug is administered via injection into the spinal canal, more specifically, into the subarachnoid space, thereby allowing the drug to reach the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular, suboccipital / cisterna magna, and / or C1-2 puncture. For example, the material may be introduced by lumbar puncture to diffuse throughout the subarachnoid space. In another example, the injection may be into the cisterna magna. Intracisterna magna delivery may increase vector diffusion and / or reduce toxicity and inflammation caused by administration. For example, Christian Hinderer et al., Widespread gene transfer in the central nervous system of cynomolgus macaques following See "Delivery of AAV9 into the cisterna magna," Mol Ther Methods Clin Dev. 2014;1:14051. Published online December 10, 2014. doi:10.1038 / mtm.2014.51. In certain embodiments, the rAAV, vector, or composition described herein is administered to the subject requiring it via intrathecal administration. In certain embodiments, intrathecal administration is carried out as described in U.S. Patent Publication 2018 / 0339065A1, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, CNS administration is carried out using an Omaya reservoir (also referred to as an Omaya device or Omaya system).

[0121] As used herein, the terms “intracisternal delivery” or “intracisternal administration” refer to a route of administration of a drug in which the drug is administered directly into the cerebrospinal fluid of the ventricles or into the cerebellomedulla oblongata, more specifically, by suboccipital puncture, by direct injection into the cisterna magna, or by a permanently positioned tube.

[0122] As used herein, the terms “intraparum,” “dentate nucleus,” or “IDN” refer to a route for direct administration of the composition to the dentate nucleus. IDN enables targeting of the dentate nucleus and / or cerebellum. In certain embodiments, IDN administration is performed using a ClearPoint® Neuro Navigation System (MRI Interventions, Inc., Memphis, TN) and a ventricular cannula, which enable MRI-guided visualization and administration. Alternatively, other devices and methods may be selected.

[0123] It should be understood that the compositions in the pharmaceutical compositions described herein are intended to be applied to other compositions, regimens, aspects, embodiments, and methods described herein.

[0124] 6.Treatment method In one embodiment, a method for treating a human subject diagnosed with MPS IIIB is provided herein. In another embodiment, a method for treating a human subject having hNAGLU-related disorder or disorder related to a defect in hNAGLU is provided herein. Currently, if there is a clinical suspicion of MPS III, the first step requires a quantitative test to detect the presence of GAG in urine by spectrophotometric analysis using dimethylmethylene blue (DMB). The DMB test involves the binding of GAG to dimethylmethylene blue, and spectral analysis. The diagnosis is based on the quantification of the GAG-DMB complex using a photometer. The sensitivity of this test is 100%, and the specificity is 75-100%. Due to the fact that in some patients with attenuated forms of the disease, the levels of GAG excretion may overlap with those of healthy controls, and the increased excretion of heparan sulfate in MPS III may be negligible, a negative result when detecting GAG in urine does not rule out the presence of MPS III. The golden rule technique for current diagnosis is the determination of enzyme activity in cultured dermal fibroblasts, leukocytes, plasma, or serum. A specific diagnosis of MPS IIIB is confirmed by showing a decrease or absence of one of the NAGLU enzyme activities involved in the degradation of heparan sulfate in the patient's leukocytes or fibroblasts, and the reduction should be less than 10% compared to the activity in healthy individuals, while other sulfatases are normal. Diseases resulting from deficiencies in multiple sulfatases also show reduced activity in heparan N-sulfatase, N-acetylglucosamine 6-sulfatase, and other sulfatases; therefore, biochemical analysis of at least other sulfatases is required to confirm the diagnosis of MPS III and thus rule out multiple sulfatase deficiency. However, the diagnostic method is not limited to the present invention, and other suitable methods may be selected.

[0125] The method comprises administering to a subject a suspension of the vector described herein. In one embodiment, the method involves administering to a subject about 1 × 10⁶ per gram of brain mass. 9 GC ~ Approximately 1 x 10⁻¹⁶ units per gram of brain mass 14 The rAAV described herein in the formulation buffer is administered at the GC dose.

[0126] The compositions(s) and methods(s) provided achieve efficacy in treating subjects having MPS IIIB and requiring treatment. The efficacy of the methods in subjects can be demonstrated by evaluating (a) an increase in NAGLU enzyme activity, (b) alleviation of MPS IIIB symptoms, (c) improvement in MPS IIIB-related biomarkers, e.g., GAG levels and polyamine (e.g., spermine) levels in cerebrospinal fluid (CSF), serum, urine, and / or other biological samples, or (e) enhancement of any treatment(s) for MPS IIIB. In certain embodiments, efficacy can be determined by monitoring cognitive improvements and / or correction of anxiety, improvements in gait and / or mobility, reduction in tremor frequency and / or severity, reduction in spasticity / convulsions, improvements in posture, and improvements in corneal opacity. Examples of preferred scoring incorporated in this section of the Spec. Additionally or alternatively, the efficacy of the methods may be predicted based on animal models. An example of a preferred mouse model is described in Example 1. In another embodiment, a multi-parameter grading scale was developed in an animal model to evaluate disease correction and response to the MPSIIIA vector therapy described herein. Animals were assigned scores based on an evaluation of combinations of tremor, posture, hair texture, spasticity, corneal opacity, and gait / mobility. In certain embodiments, any combination of one or more of these factors may be used alone or in combination with other factors to demonstrate efficacy. See Burkholder et al. Curr Protoc Mouse Biol. June 2012, 2:145-65, Tumpey et al. J Virol. May 1998, 3705-10, and Guyenet et al. J Vis Exp. May 2010, 39;1787).Cognitive improvements and anxiety reductions in treated animals are assessed by motor evaluation in open fields (i.e., beam-breaking measurements, e.g., as described in Tatem et al. J Vis Exp, 2014, (91):51785) and elevated plus maze assays (e.g., as described in Walf and Frye, Nat Protoc, 2007, 2(2):322-328).

[0127] As used herein, “promotion of any treatment(s) for MPS IIIB” or any grammatical variation thereof means the administration(s) of the described composition(s) and the described method(s) This refers to a reduction in the dosage or frequency of treatment for MPS IIIB in a subject, other than the compositions or methods first disclosed in this invention, compared to standard treatment without the use of (multiple) drugs.

[0128] Examples of preferred treatments facilitated by the compositions or methods described herein include: (a) Pharmacological treatments used to alleviate symptoms (e.g., epileptic seizures and sleep disorders) and improve quality of life. (b) Hematopoietic stem cell transplantation, e.g., bone marrow transplantation or umbilical cord blood transplantation (e.g., Vellodi A,Young E,New M,Pot-Mees C,Hugh-Jones K.Bone marrow transplantation for Sanfilippo disease type BJ Inherit Metab Dis.1992;15:911-8,Garbuzova-Davis,S,Willing,AE,Desjarlais,T,et al.Transplantation of human umbilical cord cells blood benefits an animal model of Sanfilippo syndrome type B. Stem Cells Dev. 2005;14:384-394, and Garbuzova-Davis, S, Klasko, SK, and Sanberg, PR. Intravenous administration of human umbilical cord blood cells in an animal See model of MPS III BJ Comp Neurol. 2009;515:93-101. (c) Enzyme replacement therapy (ERT) (e.g., via intravenous administration or intraventricular infusion, e.g., Aoyagi-Scharber M et al, Clearance of Heparan Sulfate and Attenuation of CNS Pathology by Intracerebroventricular BMN 250(NAGLU-IGF2)in Sanfilippo Type B Mice,Mol Ther Methods Clin Dev.2017 Jun 6;6:43-53.doi:10.1016 / j.omtm.2017.05.009.eCollection 2017 Sep 15, and Alexion Pharmaceuticals.Safety,Pharmacokinetics,and Pharmacodynamics / Efficacy of SBC-103 in MPS IIIB. Available from ClinicalTrialsgov [Internet]. Bethesda: National Library of Medicine (US). 2000, clinicaltrials.gov / show / NCT02324049. See NLM identifier: NCT02324049. (d) Substrate synthesis inhibitory therapy (e.g., treatment with genistein, Delgadillo V) et al.Genistein supplementation in patients affected by Sanfilippo disease.J Inherit Metab Dis.2011 Oct;34(5):1039-44.doi:10.1007 / s10545-011-9342-4.Epub 2011 May 10、Piotrowska E et al,Two-year follow-up of Sanfilippo Disease patients treated with a genistein-rich isoflavone extract:assessment of effects on cognitive functions and general status of patients.Med Sci Monit.2011 Apr;17(4):CR196-202、およびPiotrowska,E et al,Genistin-rich soy isoflavone extract in substrate reduction therapy for Sanfilippo syndrome:an open label,pilot study in 10 pediatric patients.Curr.Ther.Res.Clin.Exp.2008;69:166-179)、 (e) Chaperone therapy (Kan SH,Troitskaya LA,Sinow CS,Haitz K,Todd AK,Di Stefano A,et al.Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of alpha-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.Biochem IGF2 in J.2014;458:281-9, Boado RJ,Lu JZ,Hui EK,Lin H,Pardridge WM.Insulin Receptor Antibody alpha-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B See HIRMAb in Fibroblasts. Mol Pharm. 2016;13:1385-92, CpGH89 inhibitors in Ficko-Blean, E, Stubbs, KA, Nemirovsky, O, et al. Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci US A. 2008;105:6560-6565, and Zhao, KW and Neufeld, EF. Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells. Protein Expr Purif. 2000;19:202-211, and (f) Any combination of these may be, but is not limited to, these.

[0129] In one embodiment, the described method results in improvement of the MPS IIIB-related biomarker in the subject.

[0130] The term "increase in NAGLU enzyme activity" is used interchangeably with the term "increase in desired NAGLU function" and refers to NAGLU activity of at least about 5%, 10%, 15%, 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the NAGLU enzyme range of a healthy patient. NAGLU enzyme activity can be measured by assays described herein. In one embodiment, NAGLU enzyme activity may be measured in serum, plasma, blood, urine, CSF, or another biological sample. In one embodiment, administration of a composition described herein, or use of a method described herein, results in an increase in NAGLU enzyme activity in serum, plasma, saliva, urine, or another biological sample. Alternatively, other CSF biomarkers, such as CSF GAG levels and spermine levels, may be measured to determine the therapeutic effect. See, for example, WO2017 / 136533.

[0131] Neurocognition can be determined by conventional methods, see, for example, WO2017 / 136500A1, which is incorporated herein by reference in its entirety. Prevention of neurocognitive decline is at least about 5%, at least about 20%, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% of the neurocognitive decline in subjects administered with the compositions described herein or treated with the methods described herein, compared to patients with MPS IIIB. This refers to a deceleration of approximately 85%, 90%, 95%, or 100%.

[0132] As used herein, the terms “biomarker” or “biomarker associated with MPS IIIB” refer to the presence, concentration, expression level, or activity of a biological or chemical molecule in the biological sample of interest that correlates positively or negatively with the progression or onset of MPS IIIB. In one embodiment, the biomarker is the GAG ​​level in cerebrospinal fluid (CSF), serum, urine, cutaneous fibroblasts, leukocytes, plasma, or any other biological sample. In another embodiment, the biomarker is assessed using clinical chemistry. In yet another embodiment, the biomarker is liver volume or spleen volume. In one embodiment, the biomarker is the activity of heparan N-sulfatase, N-acetylglucosamine 6-sulfatase, and other sulfatases. In another embodiment, the biomarker is the spermine level in CSF, serum, or another biological sample. In yet another embodiment, the biomarker is lysosomal enzyme activity in serum, CSF, or another biological sample. In one embodiment, the biomarker is assessed via magnetic resonance imaging (MRI) of the brain. In another embodiment, the biomarker is a neurocognitive score measured by a neurocognitive development test. As used herein, the term “improvement of biomarker” means a reduction in a biomarker positively correlated with disease progression, or an increase in a biomarker negatively correlated with disease progression, where the reduction or increase is at least about 5%, at least about 20%, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to before administration of the composition described herein or before use of the method described herein.

[0133] In one embodiment, the method further includes detecting or monitoring biomarkers associated with MPS IIIB in a subject before initiating therapy with the therapy provided herein. In one embodiment, the method includes detecting a biomarker that is a polyamine (e.g., spermine) in a sample from a subject (see WO / 2017 / 136533, which is incorporated herein by reference). Thus, in one embodiment, the method includes detecting spermine in a patient sample for the purpose of diagnosing the patient with MPSIIIB. In another embodiment, spermine concentration levels in a patient sample are detected to monitor the effectiveness of therapy for MPSIIIB using the vector described herein. Currently, patients with MPSIIIB are not considered candidates for bone marrow transplantation (BMT), substrate synthesis suppression therapy (SRT), or enzyme replacement therapy (ERT). However, in certain embodiments, gene therapy patients treated with the vector expressing NAGLU described herein have at least sufficient enzyme expression levels below any normal range to be treated with ERT or SRT. Such ERT may be co-therapy, in which the dose of ERT is monitored and adjusted over several months or years after vector administration. Additionally or alternatively, SRT may be co-therapy, in which the dose of SRT is monitored and adjusted over several months or years after vector administration. Additionally or alternatively, chaperone therapy may be co-therapy, in which the dose of chaperone therapy is monitored and adjusted over several months or years after vector administration.

[0134] Therefore, in one embodiment, the suspension is suitable for co-administration with a functional hNAGLU protein or a recombinant protein containing functional NAGLU. In one embodiment, the recombinant protein is NAGLU fused with insulin-like growth factor 2 (IGF2).

[0135] In one embodiment, the suspension is delivered to the target area in the ventricle, intrathecal cavity, cisterna magna, or intravenously.

[0136] In one embodiment, the suspension has a pH of about 6 to about 8.

[0137] As used herein, enzyme replacement therapy (ERT) is a medical treatment consisting of supplementing an enzyme in a patient who is deficient in or lacking a particular enzyme. The enzyme is usually produced as a recombinant protein and administered to the patient. In one embodiment, the enzyme is functional NAGLU. In another embodiment, the enzyme is a recombinant protein containing functional NAGLU. In one embodiment, the enzyme is a recombinant protein containing functional NAGLU and insulin-like growth factor 2 (IGF2). Aoyagi-Scharber M et al. al, Clearance of Heparan Sulfate and Attenuation of CNS Pathology by Intracerebroventricular BMN 250 in Sanfilippo Type B Mice, Mol Ther Methods Clin Dev. 2017 Jun 6;6:43-53.doi:10.1016 / j.omtm.2017.05.009.eCollection 2017 Sep 15, and WO2017132675A1. Systemic, intrathecal, intraventricular, or intracisional delivery may be used for ERT or SRT co-therapy.

[0138] As used herein, substrate synthesis suppression therapy (SRT) refers to therapy that uses small molecule drugs to partially inhibit the biosynthesis of compounds that accumulate in the absence of NAGLU. In one embodiment, SRT is genistein-mediated therapy. See, for example, Ritva Tikkanen et al, Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders. Int J Mol Sci. 2016 Jul;17(7):1065. Published online July 4, 2016. doi:10.3390 / ijms17071065, Delgadillo V et al, Epub 2011 May 10, and de Ruijter J et al, Genistein in Sanfilippo disease: a randomized controlled crossover trial. Ann Neurol. 2012 Jan;71(1):110-20. doi:10.1002 / ana.22643. NAGLU.

[0139] As used herein, chaperone therapy refers to therapy that uses small molecule drugs to promote the folding and / or secretion of naglu. In one embodiment, chaperone therapy is IGF2-mediated therapy. For example, Kan SH, Troitskaya LA, Sinow CS, Haitz K, Todd AK, Di Stefano A, et al. Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of alpha-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.Biochem J.2014;458:281-9, and Boado RJ,Lu JZ,Hui EK,Lin H,Pardridge WM.Insulin Receptor Antibodyalpha-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo See HIRMAb in Type B Fibroblasts. Mol Pharm. 2016;13:1385-92. In another embodiment, chaperone therapy is therapy mediated by a CpGH89 inhibitor. For example, see Ficko-Blean, E, Stubbs, KA, Nemirovsky, O, et al. Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Aca See d Sci US A.2008;105:6560-6565. In another embodiment, the chaperone therapy is the therapy disclosed in Zhao, KW and Neufeld, EF. Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells. Protein Expr Purif.2000;19:202-211.

[0140] Suitable volumes for delivering these doses and concentrations may be determined by those skilled in the art. For example, volumes of about 1 μL to 150 mL may be selected, with higher volumes being selected for adults. Typically, for neonates, suitable volumes are about 0.5 mL to about 10 mL, and for infants, about 0.5 mL to about 15 mL may be selected. For toddlers, volumes of about 0.5 mL to about 20 mL may be selected. For children, volumes up to about 30 mL may be selected. For pre-teens and teenagers, volumes up to about 50 mL may be selected. In other embodiments, patients may accept intrathecal administration in volumes of about 5 mL to about 15 mL, or about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined. Doses are adjusted to balance the therapeutic benefits with any side effects, and such dosages may vary depending on the therapeutic use in which the recombinant vector is used.

[0141] In one embodiment, the rAAV described herein is approximately 1 × 10⁶ per gram of brain mass. 9 GC ~ Approximately 1 x 10⁻¹⁶ units per gram of brain mass 14 It is administered in GC doses. In certain embodiments, rAAV is approximately 1 × 10⁶ per kg of body weight. 9 GC ~ Approximately 1 x 10 per kg of body weight 13 It is administered simultaneously to the body at the GC dose.

[0142] In one embodiment, a subject is delivered a therapeutically effective dose of the vector described herein. As used herein, “therapeutically effective dose” means an amount of a composition comprising a nucleic acid sequence encoding a functional NAGLU that delivers and expresses a sufficient amount of the enzyme in target cells to achieve efficacy. In one embodiment, the dose of the vector is about 1 × 10¹⁶ per gram of brain mass, including all integers or decimals within its range and endpoint. 9 GC ~ Approximately 1 x 10⁻¹⁶ units per gram (g) of brain mass 13 This is a genome copy (GC). In another embodiment, the dosage is 1 × 10⁶ per gram of brain mass. 10 GC ~ Approximately 1 x 10⁻¹⁶ units per gram of brain mass 13It is GC. In a specific embodiment, the dose of the vector administered to the patient is at least about 1.0 × 10⁻⁶ 9 The number of GCs per brain mass in grams is approximately 1.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 9 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 9 The number of GCs per brain mass in g is approximately 1.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 1.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 10The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 10 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 10 The number of GCs per brain mass in g is approximately 1.0 × 10⁻⁶. 11 Individual GC / Brain mass in grams, approximately 1.5 × 10⁻⁶ 11 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 11 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 11 The number of GCs per brain mass in g is approximately 1.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 1.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 12The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 12 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 12 The number of GCs per brain mass in g is approximately 1.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 1.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 2.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 2.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 3.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 3.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 4.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 4.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 5.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 5.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 6.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 6.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 7.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 7.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 8.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 8.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 9.0 × 10⁻⁶. 13 The number of GCs per brain mass in grams is approximately 9.5 × 10⁻⁶. 13 The number of GCs per brain mass in grams, or approximately 1.0 × 10⁻⁶. 14 This is calculated as the number of GCs per brain mass in g.

[0143] Prior to treatment, MPS IIIB patients can be evaluated for neutralizing antibodies (Nabs) against the AAV serotypes used to deliver the hNAGLU gene. Such Nabs can interfere with transduction efficiency and reduce treatment efficacy. In one embodiment, the method further comprises the subject receiving an immunosuppressive co-therapy. Without wishing to be bound by theory, the immunosuppressive co-therapy, hereinafter, induces anergy or immune tolerance to rAAV and / or the transgene, blocks the immune response to optimize efficacy, minimizes a new immune response to the transgene, minimizes the impact of an existing immune response to the transgene, minimizes the impact of an existing immune response to AAV, prevents immune-mediated toxicity, minimizes the destruction of TG-expressing cells, reduces axonal damage / DRG neurotoxicity in NHPs, one or more of the above.

[0144] In certain embodiments, the method further comprises combination therapy, e.g., pre-treatment with rAAV and / or transient co-treatment with an immunosuppressive agent therein. Optionally, the immunosuppressive co-therapy may be used as a prophylactic means without an upfront assessment of neutralizing antibodies against the AAV vector capsid and / or other components of the formulation. Pre-immunosuppressive therapy may be desirable to prevent potential harmful immune responses to the hNAGLU transgene product, i.e., may be desirable if the transgene product may be recognized as "foreign". The results of the non-clinical studies in NHPs described below are consistent with the development of an immune response to hNAGLU (see Example 6). Similar responses may not occur in human subjects, but as a precaution, immunosuppressive therapy may be recommended or used for all recipients of rAAV containing the vector genome encoding hNAGLU.

[0145] Immunosuppressive agents for such combination therapies include, but are not limited to, glucocorticoids, steroids or corticosteroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalogs), and inhibitors of cell division including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents to immunophilins. Immunosuppressive agents can include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mycophenolate mofetil, methotrexate, leflunomide (Arava), cyclophosphamide, chlorambucil (Leukeran), chloroquine (e.g., hydroxychloroquine), quinidine sulfate, mefloquine, a combination of atovaquone and proguanil, sulfasalazine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, antibodies directed to the IL-2 receptor (CD25) or CD3, anti-IL-2 antibodies, abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), tocilizumab (Actemra) and tofacitinib (Xeljanz), cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-alpha) binders, as well as combinations of these drugs.

[0146] In certain embodiments, immunosuppressive therapy may be initiated 0, 1, 2, 7 days before or earlier than the administration of gene therapy. Such therapy may involve co-administration of two or more drugs on the same day, the (e.g., prednerisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)). One or more of these drugs may be continued after the administration of gene therapy at the same or adjusted dose. Such therapy may last, if necessary, about 1 week (7 days), about 60 days, or longer. In one embodiment, the two or more drugs may be, for example, one or more corticosteroids (e.g., prednerisone or prednisone), and optionally, MMF and / or calcinuerin inhibitors (e.g., tacrolimus or sirolimus (i.e., rapamycin)). In one embodiment, the two or more drugs are mycophenolate mofetil (MMF) and / or sirolimus. In another embodiment, two or more drugs may be, for example, methylprednisolone, prednisone, tacrolimus, and / or sirolimus. In a particular embodiment, the drugs are MMF and tacrolimus for 0–15 days prior to vector delivery, maintained with MMF for about 8 weeks, and / or using tacrolimus through follow-up appointments. One or more of these drugs may be continued after gene therapy administration at the same or adjusted doses. In a particular embodiment, the patient is initially administered an IV steroid (e.g., methylprednisolone) to load the dose, followed by an oral steroid (e.g., prednisolone), which is gradually reduced so that the patient is steroid-free by 12 weeks. Corticosteroid treatment may be supplemented with tacrolimus (24 weeks) and / or sirolimus (12 weeks), and further supplemented with MMF. When using both tacrolimus and sirolimus, each dose should be a low dose adjusted to maintain a blood level of approximately 4 ng / mL to approximately 8 ng / mL, or a trough level of approximately 8 ng / mL to 16 ng / mL.In certain embodiments, when only one of these agents is used, the total dose for tacrolimus and / or sirolimus may range from approximately 16 ng / mL to approximately 24 ng / mL. When only one of the agents is used, the labeled dose (higher dose) should be used, for example, tacrolimus at 0.15–0.20 mg / kg / day and sirolimus at 1 mg / m2 / day should be given as two divided doses every 12 hours, with the load dose being 3 mg / m2. If MMF is added to the regimen, the doses for tacrolimus and / or sirolimus can be maintained because they have different mechanisms of action. Other therapies may be initiated from approximately day -14 to day -1 (e.g., day -2, day 0, etc.) and may be continued, if necessary, for approximately to a maximum of approximately 1 week (7 days), or a maximum of approximately 60 days, or a maximum of approximately 12 weeks, or a maximum of approximately 16 weeks, or a maximum of approximately 24 weeks, or a maximum of approximately 48 weeks, or longer. In certain embodiments, a tacrolimus-free regimen is selected.

[0147] In certain embodiments, the patient accepts immunosuppression (IS) as follows: Corticosteroids: 10 mg / kg IV methylprednisolone once before administration on day 1, and oral prednisone starting on day 2 at 0.5 mg / kg / day and tapering down to discontinue by week 12; Tacrolimus: 1 mg BID orally from day 2 to week 24, and tapering down over 8 weeks between weeks 24 and 32; Sirolimus: (- Loading dose on day 2, then sirolimus 0.5 mg / m2 / day in divided doses in BIDs until week 48. In certain embodiments, IS therapy is discontinued at week 48 after administration of rAAV.

[0148] In certain embodiments, the method further comprises administering an intramuscular steroid or corticosteroid to the subject before and / or after administration of rAAV. In certain embodiments, the method further comprises administering an oral steroid or corticosteroid to the subject before and / or after administration of rAAV.

[0149] In a particular embodiment, the immunosuppressive therapy regimen is as follows: Corticosteroids: On the morning of vector administration (before the first day's medication), the patient receives 10 mg / kg of IV methylprednisolone (maximum 500 mg) over at least 30 minutes. Methylprednisolone is administered before lumbar puncture and intrathecal (IC) injection of rAAV. Premedication with acetaminophen and antihistamines is optional.

[0150] With the goal of discontinuing prednisone by week 12, oral prednisone is started on day 2. The prednisone dosage is as follows: Day 2 to end of week 2: 0.5 mg / kg / day. Weeks 3 and 4: 0.35 mg / kg / day. Weeks 5 to 8: 0.2 mg / kg / day. Weeks 9 to 12: 0.1 mg / kg.

[0151] Prednisone is discontinued after 12 weeks. The precise dose of prednisone can be adjusted to the next highest clinically practical dose.

[0152] Sirolimus: Two days before vector administration (-Day 2): Sirolimus is administered in three doses of 1 mg / m2 load dose every 4 hours. From Day 1 onwards: Sirolimus is divided into two doses of 0.5 mg / m2 / day, with a target blood level of 4-8 ng / ml. Sirolimus is discontinued after the 48-week visit.

[0153] Tacrolimus: Tacrolimus is initiated on day 2 (the day after rAAV administration) with a dose of 1 mg twice daily and adjusted over 24 weeks to achieve a blood level of 4–8 ng / mL. Starting with a visit at week 24, tacrolimus is gradually reduced over 8 weeks. At week 24, the dose is reduced by approximately 50%. At week 28, the dose is further reduced by approximately 50%. Tacrolimus is discontinued at week 32.

[0154] In one embodiment, the method further comprises administering an anti-AAV neutralizing antibody (NAb) to a subject to reduce peripheral transduction and mitigate the potential risk of NAGLU-induced toxicity. In a particular embodiment, the method further comprises detecting the presence of systemic AAV NAb before treatment with anti-AAV NAb, and patients with levels of anti-AAV NAb exceeding a predetermined level against rAAV capsid (or serum cross-reactive capsid) require pretreatment. No. Such levels may be, for example, about 1:10, about 1:20, about 1:50, about 1:100, about 1:250, or higher or lower levels. In certain embodiments, the method further comprises administering to the patient intravenously about 1 day to about 2 hours before treatment with rAAV as described herein.

[0155] In certain embodiments, a combination regimen is provided to prevent off-target delivery of rAAV, the regimen comprising (a) pre-treating the patient systemically with a composition comprising an anti-AAV capsid neutralizing antibody directed against the AAV capsid in a recombinant AAV vector, and (b) administering rAAV (e.g., rAAV) as described herein to the central nervous system (CNS). See also U.S. Provisional Patent Application No. 63 / 328,227 filed on April 6, 2022, and International Patent Application PCT / US2023 / 065422 filed on April 6, 2023, now International Publication No. 2023 / 196892A1, which are incorporated herein by reference in their entirety.

[0156] As used herein, the term “NAb titer” is a measure of how much neutralizing antibody (e.g., anti-AAV Nab) is produced and neutralizes the physiological effect of its target epitope (e.g., AAV). Anti-AAV NAb titer can be measured as described, for example, in Calcedo, R., et al., *Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses*, *Journal of Infectious Diseases*, 2009. 199(3): pp. 381-390, which is incorporated herein by reference.

[0157] In certain embodiments, a combination regimen is provided which is described as useful for depleting anti-AAV antibodies (and thus allowing administration to a patient to test for exceeding a threshold level of antibodies for a selected AAV capsid), as well as the delivery of anti-IgG enzymes and / or anti-FcRN antibodies, and / or one or more of the following: a) a steroid or combination of steroids, and / or (b) an IgG cleavage enzyme, (c) an inhibitor of Fc-IgE binding, (d) an inhibitor of Fc-IgM binding, (e) an inhibitor of Fc-IgA binding, and / or (f) a gamma interferon. Anti-FcRN antibodies include, for example, rozanolixizumab (UCB7665) (UCB SA); IMVT-1401, RVT-1401 (HL161), HBM9161 (all forms HanAll BioPhrma Co.Ltd), Nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (Efgaltigimod) (Argenx SE), olilanorimab (ALXN 1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co.Ltd). In certain embodiments, a combination of anti-FcRN antibodies is administered. In certain embodiments, the anti-FcRN antibody is administered in combination with a suitable anti-FcRn ligand (i.e., a peptide or protein construct that binds to human FcRn to inhibit IgG binding).

[0158] In one embodiment, a combination regimen for treating a patient having MPSIIIB is provided, comprising administering the vector described herein in combination with a ligand that inhibits the binding of human FcRn to an existing patient-neutralizing antibody (e.g., IgG). In certain embodiments, the patient may be unsensitized to any therapeutic treatment with the vector and may have existing immunity resulting from a previous infection with the wild-type virus. In some embodiments, the patient may have neutralizing antibodies as a result of pretreatment or vaccination. In certain embodiments, the patient may have neutralizing antibodies in a ratio of 1:1 to 1:20, or greater than 1:2, greater than 1:5, greater than 1:10, greater than 1:20, greater than 1:50, greater than 1:100, greater than 1:200, greater than 1:300, or higher. In certain embodiments, the patient has neutralizing antibodies in the range of 1:1 to 1:200, or 1:5 to 1:100, or 1:2 to 1:20, or 1:5 to 1:50, or 1:5 to 1:20. In certain embodiments, the patient receives a single anti-FcRn ligand (e.g., an anti-FcRn antibody) as the sole agent that modulates FcRn-IgG binding and enables effective vector delivery. In other embodiments, a patient may accept a combination of one or more anti-FcRn ligands and a second component (e.g., an Fc receptor downmodulator (e.g., interferon gamma), an IgG enzyme, or another preferred component). Such combinations may be particularly desirable for patients with particularly high neutralizing antibody levels (e.g., above 1:200).

[0159] In certain embodiments, an anti-FcRn ligand(s) (e.g., antibodies) is administered to a patient with a neutralizing antibody prior to, and optionally simultaneously with, a selected viral vector. In certain embodiments, continuous expression of the anti-FcRn ligand after administration of the gene therapy vector may be desired on a short-term (transient basis), for example, until the viral vector is cleared from the patient. In certain embodiments, sustained expression of the anti-FcRn ligand may be desired. Optionally, in this embodiment, the ligand may be delivered via a viral vector, for example, within a viral vector expressing a therapeutic transgene. However, this embodiment is undesirable if the therapeutic gene being delivered is an antibody or antibody construct, or another construct containing an IgG chain. In such embodiments, where an antibody construct containing an IgG chain is delivered via a viral vector to a patient with pre-existing immunity, the anti-FcRn ligand is delivered or administered transiently so that the amount of circulating anti-FcRn ligand is cleared from the serum before an effective level of the vector-mediated transgene product is expressed.

[0160] In certain embodiments, the FcRn ligand is delivered 1 to 7 days before administration of the vector (e.g., rAAV). In certain embodiments, the FcRn ligand is delivered daily. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered on the same day as the vector is administered. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered at least 1 to 4 weeks after rAAV administration. In certain embodiments, the ligand is delivered 4 weeks to 6 months after rAAV administration. In certain embodiments, the ligand is administered via a different route of administration than rAAV. In certain embodiments, the ligand is administered orally, intravenously, or intraperitoneally. See also International Patent Application PCT / US2021 / 037575, filed on 16 June 2021, and WO2021 / 257668A1, which is currently published and is incorporated herein by reference in its entirety.

[0161] In certain embodiments, the method includes measuring serum anti-hNAGLU antibodies. Suitable assays for measuring anti-hNAGLU antibodies are available; see, for example, Example 1.

[0162] In one embodiment, the rAAV described herein is administered once to a subject in need thereof. In another embodiment, the rAAV is administered two or more times to a subject in need thereof.

[0163] It should be understood that the compositions in the methods described herein are intended to be applicable to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0164] 7. Kit In certain embodiments, a kit is provided that includes a concentrated vector suspended in a formulation (optionally frozen), an optional dilution buffer, and devices and other components required for intrathecal, intraventricular, or intracisternal administration. In another embodiment, the kit may additionally or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to enable injection. Such buffer may enable dilution of the concentrated vector at about 1:1 to 1:5 or more. In other embodiments, larger or smaller amounts of buffer or sterile water are included, enabling dose titration and other adjustments by the physician performing the treatment. In still other embodiments, the kit includes one or more components of the device. Suitable dilution buffers, such as saline, phosphate buffered saline (PBS), or glycerol / PBS, are available.

[0165] It should be understood that the compositions in the kits described herein are intended to be applicable to the other compositions, regimens, aspects, embodiments, and methods described throughout this specification.

[0166] 8. Device In one embodiment, the vectors provided herein may be administered intrathecally, for example, via methods and / or devices described in WO2017 / 136500 (which is incorporated herein by reference in its entirety). Alternatively, other devices and methods may be selected. In summary, the method includes the steps of advancing a spinal needle into the cisterna magna of a patient; connecting a flexible tube to the proximal hub of the spinal needle and connecting the outlet port of a valve to the proximal end of the flexible tube; after the advancing and connecting steps, and after the tube has been allowed to aspirate the patient's cerebrospinal fluid, connecting a first container containing a certain amount of isotonic solution to the flush inlet port of the valve; and subsequently connecting a second container containing a certain amount of a pharmaceutical composition to the vector inlet port of the valve. After connecting the first and second containers to the valve, a passage for fluid flow is opened between the vector inlet and outlet ports of the valve, and the pharmaceutical composition is injected into the patient via a spinal needle. After the injection of the pharmaceutical composition, a passage for fluid flow is opened across the flush inlet and outlet ports of the valve, and an isotonic solution is injected into the spinal needle to flush the pharmaceutical composition into the patient. This method and this device may, at their discretion, be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices may be used for such intrathecal delivery.

[0167] It should be understood that the compositions in the devices described herein are intended to be applied to other compositions, regimes, aspects, embodiments, and methods described herein. [Examples]

[0168] Herein, the present invention is described with respect to the following embodiments. These embodiments are provided for illustrative purposes only, and the present invention should not be construed as being limited in any way to these embodiments, but rather as encompassing all variations that become apparent as a result of the teachings provided herein.

[0169] Example 1: Method A. Vector genome design A comparison of different manipulated hNAGLU coding sequences was performed in WT C57BL6 mice after IV administration. The mice were given rAAV containing the AAVhu68 capsid and a vector genome, where the vector genome was the native (reference) protein (hNAGLU) and rAAV containing various hNAGLU coding sequences, including the wild-type sequence encoding the R737G missense variant, was administered. These amino acid sequences compared included hNAGLUcoV1 (SEQ ID NO: 21), hNAGLUcoV1-R737G (SEQ ID NO: 22), hNAGLUcoV2 (SEQ ID NO: 23), hNAGLUcoV2-R737G (SEQ ID NO: 24), hNAGLUcoV3 (SEQ ID NO: 2), and hNAGLUcoV3-R737G (SEQ ID NO: 25). The sequence variants tested were measured in 1 × 10⁶ steps. 10 Individual GCs and 1 × 10 11 Administer GC in individual doses. Administer PBS to control mice. Measure NAGLU activity in the liver and plasma.

[0170] The injection will be administered on day 0 of the study. Plasma samples will be collected on day 7. An autopsy will be performed, and liver tissue will be collected during the autopsy. Plasma samples will be collected from day 7 after the injection and stored at -80°C. Autopsy samples will be collected and stored at -80°C. Stored samples, including plasma and liver tissue, will be analyzed for NAGLUE enzyme activity. Additionally, liver tissue samples will be fixed in formalin and transferred to pathology.

[0171] Based on enzyme activity readout, the manipulated sequence that achieves the highest expression of the transgene is selected for further study. As shown in Figures 1A-1C, co-variant 3 (AAVhu68.hNAGLUcoV3) lacking the R737G mutation has an expression of 1 × 10⁻⁶. 11 When administered at a dose of GC, it demonstrates statistically significantly higher transgene expression compared to other covariants based on enzyme activity. Furthermore, covariant 1 (AAVhu68.hNAGLUcoV1) lacking the R737G mutation exhibits 1 × 10⁻⁶ transgene expression.11 When administered at individual GC doses, it demonstrates higher levels of transgene expression compared to other variants, but not as high as covariant 3. Covariant 2, at any dose, results in minimal or no transgene expression, either without or without the R737G mutation. Immunohistochemical microscopy analysis was performed at 1 × 10⁻⁶ 11 The dose of each GC or 1 × 10 10 This was performed on liver tissue collected from mice administered with AAVhu68.hNAGLUcoV3 at individual GC doses or treated with PBS (data not shown). These results confirm hNAGLUcoV3 expression in liver tissue.

[0172] Overall, AAVhu68.hNAGLUcoV3 performs better than all other co-variants, so we select AAVhu68.hNAGLUcoV3 and proceed.

[0173] B.Vector-AAVhu68.CB7.CI.hNAGLUcoV3.rBG The hNAGLU-modified sequence (hNAGLUcoV3) shown in Sequence ID No. 1 is cloned into an expression construct containing the CB7 promoter (a hybrid of the cytomegalovirus early enhancer, the chicken β-actin promoter, and the chicken β-actin intron (CI)) and a rabbit beta-globin (rBG) polyadenylated sequence. The cis-plasmid further contains the complete vector genome, i.e., an expression cassette adjacent to the AAV2 inverted terminal repeat. For triple transfection, a transplasmid encoding the AAV2 rep protein and the AAVhu68 VP1 capsid-coding sequence is used together with a plasmid providing helper function containing the required adenovirus genes not present in the packaging host cell. Packaging is performed in HEK293 cells expressing adenovirus E1a and E1b gene function.

[0174] AAV vectors are prepared using the iodixanol gradient method. See Lock, M., et al., Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale. Human Gene Therapy, 2010. 21(10): p.1259-1271. The purified vectors are then subjected to classical qPCR for MPS IIIB using Penn Vector Cores. Then we measure the titer.

[0175] Calcium and magnesium-free davercholic phosphate-buffered saline (dPBS) is used as the control (vehicle control) and diluent for the vector. The test material is diluted with sterile phosphate-buffered saline (PBS) to the appropriate concentration for each dose group. The diluted vector is kept on moist ice and injected into the animals within 4 hours of dilution.

[0176] C. Vector and vehicle administration The MPS IIIB mouse vector dose is 1 × 10⁶ per mouse at an average age of 18 weeks. 10 , 1 x 10 11 , or 5×10 10 This is a single GC. Note that ddPCR (Lock, M., et al., Absolute Determination of Single-Stranded and Self-Complementary Adeno-Associated Viral Vector Genome Titers by Droplet Digital PCR. Human Gene Therapy Methods, 2014. 25(2): p.115-125) shows approximately three times higher titers than the classical qPCR method. The mice are anesthetized with isoflurane. Each anesthetized mouse is firmly grasped by the flaccid skin at the back of the head and injected with a Hamilton syringe fitted with a 27-gauge needle adjusted to be inserted 3 mm deep into the anterior and lateral aspects of the bregma with the free hand.

[0177] D. Neurobehavioral evaluation Coordination and balance will be evaluated using a rocking rotor rod two months after injection (MPS IIIB). Mice will be accustomed to the rotor rod during two 120-second trials at a constant low speed (5 rpm). After a 2-minute rest, the mice will be placed back on the rotor rod and subjected to the rocking paradigm, in which the rod will rotate at a constant speed of 10 rpm, reversing the direction of rotation every other rotation. Three trials will be performed with a 2-minute rest between trials. Results will be expressed as the mean latency until the mouse falls from the rod, with longer latencies indicating better coordination.

[0178] E. Histology Mice are euthanized by cardiac puncture and blood loss under ketamine / xylazine anesthesia, three months after injection. Tissues are rapidly collected; half are flash-frozen on dry ice (enzyme activity), and the other half are immersed and fixed in 10% neutral formalin for histology, then embedded in paraffin. The collected tissues are from the brain, spinal cord, liver, and heart.

[0179] Hematoxylin and eosin (H&E) staining are performed on paraffin sections according to a standard protocol. Histopathology is scored in the brain and spinal cord by a committee-certified veterinary pathologist blinded to treatment. The brain score is the cumulative sum of four grade severity scores for glial vacuolation in the brain, neuronal vacuolation in the cerebral cortex, neuronal vacuolation in the brainstem and hindbrain, and perivascular mononuclear cell infiltration (maximum score of 20). The cumulative score is analyzed by a one-way Anova-Kruskall-Wallis test and a post-hoc Dunn multiple comparison test with an alpha of 0.05.

[0180] F. Enzyme activity and glycosaminoglycan accumulation For enzyme activity assays and GAG content analysis, the proteins were extracted into an acidic solution (0.2% Triton, 0.9% NaCl, pH 4) by mechanical homogenization (Qiagen TissueLizer). The samples were frozen and thawed, and clarified by centrifugation. The proteins were quantified by BCA assay.

[0181] NAGLU activity was measured by adding 10 μL of sample to 0.1 M sodium acetate at pH 3.58. The measurement is performed by incubation with 20 μL of 2 mM 4-MU-2-acetamido-2-deoxy-alpha-D-glucopyranoside (Toronto Research Chemicals) dissolved in 50 mM NaCl and 0.05% Triton X100. After incubation at 37°C for 2 hours, the mixture is diluted in glycine NaOH buffer at pH 10.6, and the freed 4-MU is quantified by fluorescence (excitation at 365 nm, emission at 450 nm), compared to a standard dilution of free 4-MU, and normalized by protein content.

[0182] The GAG ​​content in tissue extracts is measured using a dye-binding method with a commercially available kit (Blyscan Biocolor GAG kit) according to the manufacturer's recommendations.

[0183] G. Anti-transgene antibody Blood for the measurement of serum anti-hNAGLU antibodies is collected by submandibular hemorrhage at several in vivo time points and at terminal autopsy by cardiac puncture. Serum is separated, frozen on dry ice, and stored at -80°C until analysis. Polystyrene plates are coated overnight with 5 μg / mL recombinant human NAGLU (R&D Systems) in PBS titrated to pH 5.8. Plates are washed and blocked for 1 hour in 2% bovine serum albumin (BSA) in neutral PBS. Plates are then incubated with serum samples diluted 1:1000 in PBS. Bound antibodies are detected with horseradish peroxidase (HRP) conjugated goat anti-mouse antibody (Abcam) diluted 1:10,000 in PBS with 2% BSA. The assay is developed using a tetramethylbenzidine substrate, stopped with 2N sulfuric acid, and then the absorbance at 450 nm is measured.

[0184] Pharmacological studies in H. mice Transgene expression This study will evaluate the transgene product expression of AAVhu68.CB7.CI.hNAGLUcoV3.RBG after ICV administration in adult C57BL6 (wild-type) mice and MPS IIIB mice. The goal of this study is to evaluate the cellular transgene product expression in disease-related target tissues in the brain (a key target organ) and peripheral tissues (serum and liver) after ICV administration of AAVhu68.CB7.CI.hNAGLUcoV3.RBG in the MPS IIIB mouse model.

[0185] Wild-type mice accept a single ICV dose of PBS as a control. MPS IIIB mice accept 1 × 10⁶ doses. 10 GCs or 5 × 10 10MPS IIIB mice will receive a single ICV dose of AAVhu68.CB7.CI.hNAGLUcoV3.RBG at individual GC doses. MPS IIIB mice will also receive a single ICV dose of PBS as a control. On day 7, serum will be collected to assess transgene product expression (NAGLU enzyme activity). On day 28, serum will be collected for anti-NAGLU titer. Brain and liver will also be collected at autopsy to assess transgene product expression (NAGLU enzyme activity or NAGLU immunofluorescence [IF]).

[0186] Figures 2A to 2E show low doses (1 × 10⁻⁶). 10 Individual GCs) and high doses (5 × 10) 10 This study demonstrates dose-dependent transgene expression in the brains of MPS IIIB mice administered with AAVhu68.CB7.CI.hNAGLUcoV3.RBG (individual GCs). Low and high dose MPS IIIB mice exhibit statistically significantly higher transgene expression in the brain compared to controls. Anti-NAGLU antibody titers were examined using ELISA.

[0187] Figures 6A and 6B provide a comparison of differently engineered sequences in WT C57BL6 mice after IV administration, based on enzyme activity readout. Figure 6A demonstrates NAGLU activity in the liver, and Figure 6B demonstrates NAGLU activity in plasma. Native cDNA (hNAGLUwt) is compared with three engineered sequences (hNAGLUco, hNAGLUcoV3, and hNAGLUcoV1) to evaluate transgene expression. The wt sequence and the three engineered sequences are compared in 3 × 10⁻¹⁶ samples. 11 The individual GC doses were administered. hNAGLUcoV3 demonstrated the highest enzyme activity and performed superiorly to other constructs and native cDNA.

[0188] Cumulatively, 1 × 10 to MPS IIIB mice 10 GCs or 5 × 10 10ICV administration of AAVhu68.CB7.CI.hNAGLUcoV3.RBG at individual GC doses results in transgene product expression (NAGLU enzyme activity and NAGLU protein expression) in disease-associated target tissues (brain).

[0189] Reduction of lysosomal pathology LAMP-1 IHC was performed to evaluate lysosomal accumulation lesions in the brains of untreated MPS IIIB mice, AAVhu68.CB7.CI.hNAGLUcoV3.RBG-treated MPS IIIB mice, and wild-type controls. An increase in LAMP-1 positive area indicates an increase in lysosomal accumulation. Untreated MPS IIIB mice demonstrated increased LAMP-1 staining in the cortex and hippocampus compared to AAVhu68.CB7.CI.hNAGLUcoV3.RBG-treated MPS IIB mice and wild-type controls (Figures 3A-3C). MPS IIIB mice were given 1 × 10⁶ 10 GCs or 5 × 10 10 AAVhu68.CB7.CI.hNAGLUcoV3.RBG was administered therapeutically at individual GC doses. Figure 3A shows the percentage LAMP-1 area in the cortex, and Figure 3B shows the percentage LAMP-1 area in the hippocampus.

[0190] Quantification of LAMP-1 IHC staining using image analysis software confirms that untreated MPS IIIB mice exhibit greater LAMP-1 positive staining throughout the brain (cortex and hippocampus) (indicated by a larger mean LAMP-1 positive area) compared to MPS IIB mice treated with AAVhu68.CB7.CI.hNAGLUcoV3.RBG and wild-type controls.

[0191] LAMP-1 immunohistochemical staining is performed on deparaffinized paraffin sections. Briefly, antigen recovery is performed by boiling the slides in 10 mM citrate buffer (pH 6.0) at 100°C for 6 minutes. The slides are then incubated with 2% hydrogen peroxide for 15 minutes, blocked with avidin / biotin reagent (Vector Laboratory, catalog number: SP-2001) for 15 minutes each, and incubated with 1% donkey serum in phosphate-buffered saline (PBS) with 0.2% Triton-X for 10 minutes at room temperature. The slides are then incubated with rat anti-mouse LAMP-1 primary antibody (Abcam, catalog number: Ab25245) at 37°C for 1 hour. The slides are washed and then incubated with biotinylated donkey anti-rabbit IgG secondary antibody (Jackson, catalog number: 711-065-152) for 45 minutes at room temperature. The slides are washed and then incubated with Vectastain ABC reagent (Vector Laboratories, catalog number: PK-6100). Colorimetric development is performed using the 3,3'-diaminobenzidine (DAB) kit (Vector Laboratories, catalog number: SK-4100), followed by counterstaining with hematoxylin and coverslipping for evaluation.

[0192] Cumulatively, there is a dose-dependent reduction in LAMP-1 staining in the brain, which is associated with AAVhu. This shows improvement in lysosomal pathology in MPS IIIB mice treated with 68.CB7.CI.hNAGLUcoV3.RBG.

[0193] Substrate reduction Next, a glycosaminoglycan (GAG) (HS) accumulation / reduction assay was performed. GAG (HS) accumulation / reduction was examined using LC-MS / MS quantification of disaccharide degradation products from buanolysis of HS in the brain and liver. The assay was performed in untreated MPS IIIB mice, AAVhu68.CB7.CI.hNAGLUcoV3.RBG-treated MPS IIIB mice, and wild-type controls. In treated MPS IIIB mice, 1 × 10⁶ 10 GCs or 5 × 10 10 AAVhu68.CB7.CI.hNAGLUcoV3.RBG was administered at individual GC doses.

[0194] Figure 4A shows a statistically significant dose-dependent reduction in GAG levels in the brains of MPS IIIB mice treated with AAVhu68.CB7.CI.hNAGLUcoV3.RBG. Figure 4B shows a statistically significant dose-dependent reduction in GAG levels in the livers of MPS IIIB mice treated with AAVhu68.CB7.CI.hNAGLUcoV3.RBG. Figures 4A and 4B show GAG levels plotted as ng of GAG (HS) per 1 mg of protein.

[0195] These results demonstrate a dose-dependent reduction of heparan sulfate (HS), a disease-related biomarker, in brain tissue, indicating targeted involvement.

[0196] Example 2: Determination of the minimum effective dose (MED) in a mouse model of MPSIIIb We will conduct experiments to evaluate the expression, physiological activity, and minimum effective dose (MED) of AAVhu68.CB7.CI.hNAGLUcoV3.RBG, an AAVhu68 vector expressing human N-acetyl-α-D-glucosaminidase (NAGLU), after single intraventricular (ICV) administration in a mouse model of MPSIIIb.

[0197] AAVhu68.CB7.CI.hNAGLUcoV3.RBG was administered to MPS IIIb mice (n=10 per group) with an average age of 4 months via the ICV pathway at four dose levels (determined by qPCR titer measurement of the vector) on day 0, with a 3-month post-injection (pi) observation period. Vehicle-treated MPS IIIB mice and heterozygous littermates served as controls (n=10 per group).

[0198] Bioactivity is assessed by measuring NAGLU activity in the brain, spinal cord, liver, serum, and heart three months after injection. Efficacy and MED are determined by measuring the ability in a rocking rotor rod two months after injection, and brain and spinal cord lysosome accumulation and histopathology three months after injection.

[0199] Example 3: Pharmacological / toxicological studies in rhesus monkeys We will conduct experiments to evaluate the safety of intrathecal administration of three doses of AAVhu68.CB7.CI.hNAGLUcoV3.RBG.

[0200] The control substance was administered via suboccipital puncture to three macaque monkeys (both sexes) in Group 1. The AAVhu68.CB7.CI.hNAGLUcoV3.RBG vector was administered via suboccipital puncture to six rhesus monkeys randomized to Groups 2 and 3. The macaque monkeys in Group 2 received a higher dose (3 × 10⁻¹⁴). 13 In a study where GCs (N=3) accepted the test substance, macaque monkeys in group 3 showed a lower dose (1 × 10⁻¹⁰). 13 Each GC accepts the test sample (N=3). As part of a general safety panel, blood and cerebrospinal fluid are collected. Serum and peripheral blood mononuclear cells (PBMCs) are collected to introduce the capsid and transgene into the body fluids and cells. Investigate the cellular immune response.

[0201] After the pre-mortem phase of these studies was completed 90 ± 3 days after vector administration, the macaque monkeys were sacrificed, and their tissues were collected for comprehensive histopathological examination. Lymphocytes were collected from the spleen and bone marrow to examine the presence of CTLs in these organs at autopsy.

[0202] Example 4: Long-term effects of AAV.hNAGLU administration The experiment will investigate the long-term effects of AAV.hNAGLUcoV3 on MPS IIIb mice. Twenty MPS IIIb mice will be given a high dose of AAV.CB7.CI.hNAGLUcoV3.RBG(9×10) at 2 months of age. 10 Inject one GC (ICV) into each mouse. An additional 20 MPS IIIa mice and 20 wild-type mice receive a PBS control injection. Monitor the mice for 7 months after injection, assigning them a clinical score weekly during the 7 months, and subjecting them to behavioral and cognitive tests.

[0203] A multi-parameter grade classification scale was developed to assess disease correction and response to treatment over the duration of the study. Scores were assigned to individual mice based on assessments of tremor, posture, hair quality, grip, corneal opacity, and gait / mobility combinations. The clinical scoring system was adapted based on previously described methods (e.g., Burkholder et al. Curr Protoc Mouse Biol. June 2012, 2:145-65, Tumpey et al. J Virol. May 1998, 3705-10, and Guyenet et al. J Vis Exp. May) See 2010,39;1787).

[0204] Example 5: Study on the minimum effective dose for long-term survival The experiment will investigate the long-term survival of AAV.hNAGLUcoV3 in MPSIIIb mice using a minimum effective dose study. Five mouse groups will be studied, each containing 10 males and 10 females. The groups will consist of WT control mice, MPSIIIB control mice, and 1.3 × 10⁶ mice. 10GC of individual values, 4.5 × 10 10 GC of a certain number, or 1.3 × 10⁻¹⁰ 11 The study included MPS IIIB mice administered AAVhu68.hNAGLUcoV3 at individual GC doses. All groups were administered at 2–3 months of age. Body weight was measured weekly. An open-field test was performed at day 210. An elevated zero-maze test was performed at day 180. Blood samples were collected at days 7, 30, 60, 90, 150, and at the end of the study. Autopsies were performed at the humane endpoint. The study included analysis of behavioral, biomarker, and survival data, which included clinicopathological data, cerebrospinal fluid and brain GAG levels, as well as serum, brain, and liver NAGLU activity.

[0205] 1.3 × 10 10 GC of individual values, 4.5 × 10 10 GC of a certain number, or 1.3 × 10⁻¹⁰ 11 Figure 7 shows the clinical scores in male and female WT and MPS IIIB mice compared to MPS IIIB mice administered with AAVhu68.hNAGLUcoV3 at doses of 1.3 × 10⁻¹⁰ GC. Higher clinical scores indicate a worse phenotype. All mice treated with AAVhu68.hNAGLUcoV3, regardless of dose, showed similar clinical scores to WT mice. 10 GC of individual values, 4.5 × 10 10 GC of a certain number, or 1.3 × 10⁻¹⁰ 11 Figure 8 compares the survival curves of WT and MPS IIIB mice compared to MPS IIIB mice treated with AAVhu68.hNAGLUcoV3 at doses of GC. The probability of survival is shown. Survival rescue is shown in all MPS IIIB mice treated with AAVhu68.hNAGLUcoV3, regardless of dose, compared to untreated MPS IIIB mice.

[0206] Example 6: Pharmacological and safety evaluation in rhesus monkeys The experiment was conducted, and the result was 3.3 × 10 11The safety of intracisional injection of AAVhu68.CB7.CI.hNAGLUcoV3.RBG at doses of GC / brain g was evaluated. Three animals were administered the treatment. After the completion of the pre-mortem phase of these studies 90 ± 3 days after vector administration, two of the NHPs were necropsied and tissue was collected for examination (Figures 9A-9E). The treatment was well tolerated in these two animals, exhibiting typical mild to moderate DRG pathology. Figure 9A shows brain slice "5" taken from the cortex and periventricular region, and Figure 9B shows brain slice "9" taken from the occipital lobe cortex. Figures 9C-9E show hNAGLU expression by in situ hybridization.

[0207] The third NHP developed neurotoxicity starting 15 days after vector administration. Steroids were administered via intramuscular injection on day 16, after which the animal was switched to oral prednisone. An emergency necropsy was performed on this animal on day 42. The animal exhibited a cytotoxic immune response (non-self response) to hNAGLU and had neurological signs and exacerbated DRG pathology due to a robust T-cell response to the hNAGLU epitope. Further studies were conducted to confirm responses to subpools of interest (Figures 10A-10C, Tables A and B). The predicted immunodominant epitope was LAPEDPIFPI (SEQ ID NO: 33) from a pooled sample containing the following peptides: peptide 55-SEQ ID NO: 34, peptide 56-SEQ ID NO: 35, peptide 57-SEQ ID NO: 36, peptide 58-SEQ ID NO: 37, peptide 59-SEQ ID NO: 38).

[0208] Table A below summarizes the spot-forming units (SFUs) per million cells. DMSO and Pool B were tested in four replicates. An asterisk indicates that the sample was tested as a single-sample replicate. Group Pool B, B.1, B.6, B.7, B.8, B.9, peptide 56, and peptide 57 showed a positive response. Group Pool B, B.1, B.6, B.7, B.8, B.9, and peptides 56, 57, and 58 showed values ​​slightly below three times the DMSO confirmation cutpoint. [Table 2]

[0209] Table B below summarizes the positive subpool based on collected liver lymphocyte data. An asterisk indicates a value slightly below 3 times the DMSO response. [Table 3]

[0210] The response to individual peptides was evaluated within a subpool that generated a positive IFN-γ response. The IFN-γ response to individual peptides identified one immunodominant epitope in peptide pool B. The predicted immunodominant epitope is LAPEDPIFPI (SEQ ID NO: 33). Since no known mutations exist in this region, human patients are considered to be tolerant to this epitope.

[0211] All publications referenced herein are incorporated herein by reference in their entirety. U.S. Provisional Patent Application No. 63 / 507,586, filed June 12, 2023, is incorporated herein by reference in its entirety. Similarly, sequence numbers referenced herein and appearing in the accompanying sequence listings are incorporated herein by reference. While the present invention is described in relation to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the accompanying claims.

Claims

1. A recombinant AAV (rAAV) comprising a vector genome within an AAV capsid, wherein the vector genome is a nucleic acid molecule comprising an expression cassette, the expression cassette comprising a nucleic acid sequence comprising an engineered nucleic acid sequence encoding functional human N-acetyl-alpha-glucosaminidase (hNAGLU) and operably linked to a regulatory sequence for the hNAGLU, wherein the hNAGLU coding sequence is sequence number 1 or a sequence at least 99% identical to sequence number 1 (hNAGLUcoV3).

2. The rAAV according to claim 1, wherein the hNAGLU code sequence is sequence number 1 (hNAGLUcoV3).

3. The rAAV according to claim 1 or 2, wherein the regulatory sequence comprises a hybrid promoter, the hybrid promoter comprising a cytomegalovirus early stage (CMV IE) enhancer, a chicken beta-actin promoter, and a chimeric intron containing a chicken beta-actin intron.

4. The rAAV according to any one of claims 1 to 3, wherein the regulatory sequence includes a promoter element, and the promoter element includes a chicken beta-actin promoter having the sequence of SEQ ID NO: 4 or a sequence that is at least 99.9% identical to SEQ ID NO:

4.

5. The rAAV according to any one of claims 1 to 4, wherein the regulating sequence includes an enhancer element, and the enhancer element includes a CMV IE enhancer having the sequence of sequence number 3 or a sequence that is at least 99.9% identical to sequence number 3.

6. The rAAV according to any one of claims 1 to 5, wherein the regulatory sequence includes an intron, and the intron includes a chicken beta-actin intron having the sequence of SEQ ID NO: 5 or a sequence that is at least 99.9% identical to SEQ ID NO:

5.

7. The rAAV according to any one of claims 1 to 6, wherein the regulatory sequence further comprises rabbit betaglobin polyA having the sequence of sequence number 6 or a sequence identical to sequence number 6 by at least 99.9%.

8. The rAAV according to any one of claims 1 to 7, wherein the expression cassette comprises the sequence of sequence number 7 (CB7.CI.hNAGLUcoV3.RBG).

9. The rAAV according to any one of claims 1 to 8, wherein the vector genome comprises the sequence of SEQ ID NO: 8 (CB7.CI.hNAGLUcoV3.RBG).

10. The rAAV according to any one of claims 1 to 9, wherein the AAV capsid is AAVhu68 capsid.

11. The rAAV according to any one of claims 1 to 9, wherein the AAV capsid is AAVhu95 capsid, AAVhu96 capsid, AAV9 capsid, or AAVrh91 capsid.

12. The rAAV is intended for use in the treatment of mucopolysaccharidosis III B (MPS III B), disorders associated with defects in the hNAGLU, and / or for improving gait or mobility, reducing tremors, reducing spasms, improving posture, or reducing the progression of vision loss in subjects with hNAGLU-related disorders. , the rAAV according to any one of claims 1 to 11.

13. A pharmaceutical composition comprising rAAV according to any one of claims 1 to 11 in a pharmaceutical buffer.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is suitable for co-administration with a functional hNAGLU protein.

15. The pharmaceutical composition according to claim 13 or 14, wherein the pharmaceutical composition is formulated for administration via intraventricular (ICV), intrathecal (IT), intracisional (ICM), or intravenous (IV) injection.

16. The aforementioned pharmaceutical composition contains 1 × 10⁻¹⁶ units per gram of brain mass. ９ 10⁴ genome copies (GC) (GC / g) ~ approximately 1 × 10⁶ １３ A pharmaceutical composition according to any one of claims 13 to 15, which can be administered in a dose of GC / brain mass g.

17. The pharmaceutical composition according to any one of claims 13 to 16, wherein the pharmaceutical composition is formulated to have a pH of 6 to 8.

18. A method for treating a human subject diagnosed with MPS IIIB, a disorder related to a defect in the hNAGLU, and / or for improving gait or motor function, reducing tremor, reducing spasms, improving posture, or reducing the progression of visual loss in a subject having an hNAGLU-related disorder, comprising: administering to the subject a suspension of rAAV according to any one of claims 1 to 11 in a formulation buffer, 1 × 10 ９ Individual GC / brain mass g ~ approximately 1 × 10⁻⁶ １３ A method comprising administering a dose of GC / brain mass g.

19. The method according to claim 18, wherein the suspension is suitable for co-administration with a functional hNAGLU protein.

20. The method according to claim 18 or 19, wherein the suspension is administered to the subject requiring it by intraventricular, intrathecal, intravenous, or intracisional injection.

21. The method according to any one of claims 18 to 20, wherein the suspension is administered via an Omaya device.

22. The method according to any one of claims 18 to 21, wherein the suspension has a pH of 6 to 8.

23. (a) The subject receives enzyme replacement therapy at a reduced dosage or less frequency compared to standard treatment consisting solely of enzyme replacement therapy, and / or (b) The method according to any one of claims 19 to 21, wherein the subject demonstrates improvement of a biomarker related to MPS IIIB.

24. The method according to any one of claims 18 to 23, wherein the rAAV is administered once to the subject in need.

25. The method according to any one of claims 18 to 23, wherein the rAAV is administered two or more times to the subject in need.

26. Recombinant adeno-associated virus (rAAV) according to any one of claims 1 to 11 or a pharmaceutical composition according to any one of claims 13 to 17 for use in the preparation of a pharmacopoeia for the treatment of MPS IIIB, a pharmacopoeia associated with defects in hNAGLU, and / or a pharmacopoeia for improving gait or motor function, reducing tremor, reducing spasms, improving posture, or reducing the progression of vision loss in a subject having hNAGLU-associated disorder.

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